Treasurer for the City of Hampton Locality that, by ordinance, enforces payment of local motor vehicle license fee must issue some form of license upon payment of fee; may prescribe form of license, which must be displayed on vehicle.
City Attorney for the City of Richmond Board for Contractors Regulations do not supplant locality’s ability, under its general police powers, to require licensed plumbers to install backflow prevention devices when such requirement is related directly to protection of locality’s water supply system.
Presumption of openness regarding requests for court records in digital format. Duty of circuit court clerks to furnish copies of records requested by citizens, without distinction between paper and digital formats, provided records are not sealed by court order or otherwise exempt from disclosure by law.
Sheriff for Tazewell County Responsibility of police department of town issuing warrant for fugitive’s arrest to retrieve and return to court fugitive held in another locality. Governor may appoint any agent to retrieve and return to court fugitive located in another state.
Reserving existing or future vacancies in established mobile home park solely for seniors, to satisfy federal and state 80% senior occupancy rate requirement, could be interpreted to discriminate against families with children, in violation of state and federal fair housing laws. If mobile home park eventually reaches senior occupancy rate requirement and does not otherwise violate state and federal fair housing laws based on familial status, it may become eligible to operate and advertise as housing for seniors.
Attorney General defers to determination by Commonwealth Transportation Board that funds set aside in Virginia Transportation Act for Southeast Bypass project are to be spent solely on planning, engineering and construction of bypass ; mayand not on be expended to improvinge roads along bypass corridor and alternatives to bypass.
Member, House of Delegates Hampton Housing Ordinance is unauthorized to extent ordinance applies in areas other than conservation and rehabilitation districts designated by city’s local governing body or blighted areas.
School division coaches and other school personnel may render first aid to students when necessary. Unless such persons are certified athletic trainers, they may not employ physical modalities or tape students’ ankles or wrists in order to prevent or treat injuries or other physical conditions. Certified athletic trainers are responsible for actions of noncertified individuals acting under their supervision and direction; must ensure that such individuals do not perform functions requiring professional judgment or discretion of certified athletic trainers. School board that fails to hire certified athletic trainer is entitled to absolute sovereign immunity. Absent gross negligence, school board employees may be entitled to sovereign immunity for failure to hire certified athletic trainer.
02-107 The Honorable Samuel A. Nixon, Jr.
Member, House of Delegates ‘Living wage’ requirement is matter of general social or economic policy that is unrelated to goods or services sought to be procured under Act. Locality has no authority to require contractors to provide living wage to employees as condition to award of public contract.
for the City of Saunton Parcel of real estate remaining after division of property, resulting in two or more different owners, does not have to be reassessed immediately during first year of biennial real estate assessment cycle; must be reassessed as of January 1 of second year of biennial assessment cycle, taking into consideration value of land as divided. Authority for board of equalization to hear and consider taxpayer complaints in second year of biennial assessment.
Member, House of Delegates Timely issuance of process invokes jurisdiction of circuit court to conduct probation revocation hearing; lack of statutory time period within which to conduct hearing does not violate due process rights of defendant.
City of Staunton Commissioner of revenue may release names and addresses of businesses licensed within his locality for purposes of solicitation. Business is considered trading under assumed or fictitious name when assumed or fictitious name certificate is filed in appropriate clerk’s office.
the City of Hampton No conflict between statutory and regulatory definitions of ‘definite place of business’; reliance on both definitions for purposes of administering BPOL tax. Determination whether ‘visit’ becomes ‘definite place of business’ is question of fact for local taxing official. Application of internal consistency test for purposes of apportioning BPOL taxes to businesses located in and out of state. BPOL taxable gross receipts deductions should be fairly apportioned between activity within jurisdiction and outside Commonwealth.
02-116 The Honorable J. Jack Kennedy, Jr.
Circuit Court Circuit court clerk may not decline to record deed of trust containing grantor’s social security number; may be liable for modifying deed of trust offered for recordation.
Member, Senate of Virginia Parent may consent to drug testing for minor child and obtain results of nondiagnostic drug testing performed on minor child not receiving treatment for substance abuse.
Member, House of Delegates Meaning of ‘religious association’ for purpose of exempting property of Young Life from taxation by classification. Commissioner of revenue, or other local taxing official, is responsible for determining whether property owned by Young Life qualifies for tax exemption.
Commonwealth’s Attorney for Russell County Statute governing admissibility into evidence at trial of written results of hospital-taken blood alcohol tests is procedural. Commonwealth may introduce such test results into evidence at trial conducted after effective date of statute, even though motor vehicle accident giving rise to charge of involuntary manslaughter as result of driving while intoxicated occurred before effective date of statute.
Member, House of Delegates Authority of Governor to suspend licensure requirements of health professionals, enforce quarantines, and control and allocate services and resources under federal and state emergency services programs in response to state of emergency. Requirement that Commonwealth provide just compensation for taking of private resources.
Church property, which is in process of being developed for its intended use and is being used for certain church-related activities, may be tax exempt. Determination depends on question of fact reserved for local commissioner of revenue or other appropriate taxing official.
02-089 The Honorable Vincent F. Callahan, Jr.
Member, House of Delegates Fairfax County School Board has no authority to add sexual orientation as category in its nondiscrimination policy, absent enabling legislation.
Member, House of Delegates Allowance of credit against individual and corporate income tax in amount equal to 50% of fair market value of land or interest in land donated for conservation purposes. Donor of land or interest in land may transfer tax credit to other taxpayers to make full use of credit. Meaning of ‘taxpayer’ for purposes of Act.
Member, Senate of Virginia Authority for Chesapeake School Board, based on population count, to increase annual salary of its members to maximum paid to city council members, and annual salary of its chairman to maximum paid to city mayor, upon passage of motion in 2003 approving specific salaries of $25,000 and $27,000, respectively. July 1, 2004, is earliest date that such salary increases may be effective.
Member, Senate of Virginia Authority for Department of Corrections to employ prisoners to perform roofing work on buildings located on prison grounds; no requirement to procure such services under Acts competitive sealed bidding procedures.
County Attorney for Prince William County Processing fee authorized by ordinance to be collected by clerk on convicted individuals admitted into county, city, or regional jails is reserved solely for use by local sheriff’s office, even though county or city may participate in regional jail.
Member, House of Delegates Authority may pledge funds as collateral for line of credit or loan or grant funds to Dickenson County Industrial Development Authority for purchase of machinery and tools or real estate of medical facility, which qualifies as ‘authority facility’ under Act, provided contemplated use of funds furthers public purpose of Authority; may not make pledge, loan or grant to IDA to fund operating expenses of medical facility.
County Attorney for Northumberland Co. Amendments to Chesapeake Bay Preservation Area Designation and Management Regulations, allowing encroachments into 100-buffer areas for agricultural and silvicultural activities conducted in accordance with best management practices related to improving water quality and protecting Chesapeake Bay and its tributaries, do not violate Equal Protection Clause of U.S. Constitution. Such practices.
Judge, Nineteenth Judicial Circuit of Virginia Circuit court clerk may not accept and hold equity interest in Virginia stock corporation operated by commissioner of accounts, subject to direction of court, when transfer is not part of case or controversy properly before court, without improperly assuming legislative function.
for the City of Harrisonburg Law-enforcement officer conducting lawful stop to investigate alleged criminal activity may not arrest for obstruction of justice suspect who refuses to identify himself to officer. Depending on circumstances, suspect may be detained for purpose of determining his identity.
Commissioner of the Revenue for Page County Authority for local governing body to increase minimum acreage for land classified for open-space use for purpose of special land use taxation; no statutory authority for such increase for land classified for agricultural, horticultural or forest use.
Amended ordinance establishing Elizabeth Lake Estates Service District does not specify Elizabeth Lake Estates Civic Association as entity to develop plan for services to be rendered in service district, receive funds, or provide services; provides for tax levy to be set annually as part of budget process with other tax rates. Amended ordinance does not create long-term unconditional debt obligation, in violation of Constitution, and does not delegate legislative authority of city council.
Juvenile and Domestic Relations Court Statutory authorization for juvenile court to dispose of charges of unlawful possession of alcohol by juveniles.
Circuit Court of the City of Alexandria Clerk may authorize minister or other clergy to perform marriages upon production before circuit court of proof of ordination or licensure, without personally appearing before clerk. Issuance of order authorizing minister or other clergy to perform marriages is discretionary; clerk may compel personal appearance as condition of such order.
Member, House of Delegates Department of Conservation and Recreation has no authority to issue regulation prohibiting, within state parks, carrying of concealed handguns by valid permit holders.
Sheriff for the City of Chesapeake Sheriff is appropriate official to determine reasonableness of levying distress warrant for delinquent taxes and holding public sale of property, proceeds from which will not satisfy secured interests or unpaid taxes. Meaning of ‘priority’ as term relates to payment of sale proceeds toward satisfaction of delinquent taxes or secured interests. Secured party with lien on distressed property must receive notice of distress sale.
02-077 Mr. William G. O’Brien, Chairman, Board of Directors, Virginia Resources Authority Inclusion of Virginia Resources Authority in Commerce and Trace Secretariat is for state government organizational purposes; does not alter independent nature of Authority to govern its affairs according to enabling statutes. Board of Directors has exclusive power over personnel policies of Authority, with exception of appointment of Executive Director.
Member, House of Delegates Transference of authority for teacher licensure from Board of Education to independent licensure board is inconsistent with constitutional mandate charging Board with general supervision of Commonwealth’s school system.
and Domestic Relations Court Juvenile court laws do not allow juvenile court judge to temporarily detain juvenile, pending disposition hearing, who was not detained prior to adjudication hearing at which he was determined delinquent.
Member, House of Delegates 2002 amendment providing that any residential cooperative association under Act shall not be considered business for state and local tax purposes and that tangible personal property of association shall be entitled to exemption from personal property tax on household goods is not declaratory of law existing prior to July 1, 2002, effective date of legislation, and is not retroactive in its application.
Judge, Albemarle General District Ct. Special justice, appointed to preside over commitment proceedings involving persons alleged to mentally retarded or mentally ill and in need of hospitalization, may represent individuals in commitment hearings held within judicial circuit of such justice.
Member, House of Delegates E-911 personnel may access criminal justice information generated by VCIN/NCIC terminals per statutorily authorized agreement with local sheriff’s office, subject to requirements of State Police. Absent specific statutory authorization, sheriff may not enter into agreement binding his successors in office.
02-081 Mr. Thomas J. McCarthy, Jr.
County Attorney for Pulaski Co. Locality may issue more than one vehicle license free of charge to former volunteer rescue squad and fire department members meeting statutory length-of-service requirements; may not issue more than one such license, or additional licenses at discounted rate, to active members.
02-084 The Honorable Fenton L. Bland, Jr.
Member, House of Delegates Hospital authority, organized as political subdivision, may sell its assets to for-profit corporation upon approval by circuit court of authority’s dissolution.
Recordation tax should be collected on deeds of trust where federal government is guarantor or beneficiary.
Commissioner of the Revenue for the City of Hampton Specific statutes relating to the calculation of gross receipts of shareholders in professional corporations and of members in professional limited liability companies control calculation of Business and Professional Occupation License tax over more general statutes authorizing and implementing such tax.
Statutes relating to video programming excise tax, by their own terms, provide for a tax in lieu of the Business and Professional Occupation License tax or exempt the activity from the BPOL tax altogether.
Prior Opinion of the Attorney General determining that the Business and Professional Occupation License tax laws contain no exemption for salaried professionals, and that such professionals are subject to the tax does not conflict with 2000 Business and Professional Occupation License Tax Guidelines promulgated by the Department of Taxation.
Statutes relating to the calculation of gross receipts of shareholders in professional corporations and of members in professional limited liability companies attribute gross receipts only to employees who are also shareholders of the professional corporation or members of the professional limited liability company. Other salaried professional employees who hold state regulatory licenses, but are not shareholders or members, are not subject to local license taxation under those statutes.
Executive Secretary of the Supreme Court of Virginia Juvenile court has no authority to appoint counsel for child who is subject of abuse and neglect, entrustment agreement, or petition for termination of residual parental rights, but must appoint guardian ad litem for such child.
Juvenile court has no authority to appoint guardian ad litem for juvenile defendant, in addition to appointment of legal counsel, to represent child in delinquency, child in need of services, or child in need of supervision proceeding.
Juvenile court has no authority to appoint guardian ad litem, in addition to appointment of legal counsel, to represent parent, guardian or other adult charged with abuse or neglect of child or parent or guardian who would be subjected to loss of residual parental rights.
In cases other than abuse and neglect, entrustment agreements, termination of parental rights, delinquency, or child in need of services or supervision, juvenile court may appoint either counsel or guardian ad litem, but not both.
Senate Minority Leader Although statutorily authorized testing equipment must be used for tailpipe exhaust emissions tests performed under the Federal Clean Air Act, Virginia’s emissions inspections laws authorize the use of other equipment or software for nontailpipe tests or checks that are part of the federal motor vehicle inspection and maintenance program. Virginia law provides that its enhanced emissions inspections program shall include, and be limited to, testing procedures necessary to comply with the Clean Air Act. Virginia must include in its program, nontailpipe exhaust tests that comply with the Federal Act and may exclude tailpipe exhaust tests that the Act no longer requires.
County Attorney for Buchanan County Judges and court personnel carrying out a judge’s order are protected by judicial immunity when participating in court-ordered community service programs. Other public officials and probation officers may be protected by the doctrine of sovereign immunity for discretionary acts.
Superintendent, Department of State Police Uniform Machine Gun Act does not prevent the discharge or firing of a machine gun by a person not exempt from the Act. A person not exempt from the Act may transport a machine gun away from his registered bona fide permanent residence or business address. The meaning of the phrase "immediate vicinity" as used in the Act, requires a determination of fact rather than one of law, it is not an appropriate issue on which to render an official advisory opinion.
Judge, Fifteenth District Juvenile and Domestic Relations Court Under 2002 Appropriation Act courts have no authority to assess fee against guardian of child for whom guardian ad litem has been appointed.
Under 2002 Appropriation Act courts can order reimbursement by parent when appointment of guardian ad litem required.
Commonwealth’s Attorney for the City of Harrisonburg and Rockingham County Police officer may not arrest without a warrant for violation of Virginia Code § 4.1-305 unless offense committed in his presence; offense defined by § 4.1-305 requires dominion and control over alcoholic beverage and is not committed in the officer’s presence if beverage was ingested at an earlier time and place.
02-071 The Honorable Frank D. Hargrove, Sr.
Member, House of Delegates Whether a "bus" is exempt from requirements of child restraint device for transporting children in motor vehicles and whether the interior design of a vehicle or the weight and size of a child makes it "impractical" for the child to be placed in a child restraint device is a factual question. Words are to be given their usual and ordinary meaning. Significantly, the 2002 Session of the General Assembly did not change the types of vehicles that were exempt from child restraint device requirements.
Clerk of the Fairfax County Circuit Court Clerk should assess recordation tax on Security Instrument drawn under Islamic law based on original acquisition balance as defined in instrument and assess separate fees for Assignment Agreement, since document effects two transactions of equal dignity.
Member, House of Delegates Governor has power to direct State Board of Elections to reduce amounts it reimburses localities for salaries of electoral board members and general registrars in order to achieve legislatively mandated budget cuts.
Member, House of Delegates Virginia Code § 63.1-248.3.A(12) imposes a duty on volunteer and professional Boy Scout leaders, who have reason to suspect that a child has been abused or neglected, to report the matter to the Department of Social Services.
Sheriff for Westmoreland County Northern Neck Regional Jail Board has no independent authority to pay bonuses to its employees. Instead, the Board has the authority to request the participating localities of regional jail to adopt ordinance providing for payment of bonuses to employees of Northern Neck Regional Jail.
County Attorney for York County Constitutionality of local ordinance limiting time period within which political signs advocating particular candidate or political party may be displayed on private property, either before or after primary or general election, must be determined on case-by-case basis.
Clerk of the Circuit Court of Fairfax County Clerk of court should collect $10 assessment fee on every deed recorded on or after May 1, 2002, on which state recordation tax is levied. Deed of correction is not subject to $10 fee. Fee is collected only in jurisdiction where instrument is first recorded and is not collected for recordation of same deed for real estate that is located in more than one jurisdiction. Deeds of release and deeds of partition are not subject to state recordation tax or $10 assessment fee. Assessment by circuit court clerk of $10 fee for recordation of deed of easement, deed of dedication or deed of subdivision depends on clerk’s current treatment of such deeds for state recordation tax purposes.
Treasurer for the City of Hampton Ordinance directing City of Hampton to turn over to Elizabeth Lake Estates Civic Association tax revenue collected in service district is unconstitutional. City’s delegation of its legislative authority to Association is unconstitutional. Tax imposed by ordinance is unenforceable.
Rockbridge County & the City of Lexington Attorney General declines to render opinion regarding whether operation of Free Spin machines under described circumstances constitutes illegal gambling.
Member, House of Delegates Disclosure by spouse abuse shelter to law-enforcement officials of presence of resident under described circumstances.
Member, House of Delegates Department of Taxation has authority to promulgate guideline definition of term "contractor" that clarifies and explains which businesses are included in "contractor" classification for purpose of determining applicable maximum BPOL tax rate, provided language is not inconsistent with Commonwealth’s license tax laws.
Member, House of Delegates The Workforce Investment Act precludes the General Assembly from directing the Governor to reallocate Unobligated WIA Funds to a community college located within the same geographic area from which the funds were obtained. Consistent with the historical practices of prior Attorneys General, the Attorney General is unable to comment on any actions colleges may take to seek reallocation of such funds.
Judge, General District Court of Halifax County Order of remittance does not serve as satisfaction of judgment in general district court; does act as satisfaction of judgment entered on previously forfeited bond under applicable statutes pertaining to judgments filed in circuit courts.
Judge, Fifteenth District Juvenile and Domestic Relations Court Decisions of Court of Appeals of Virginia in Commonwealth ex rel. May v. Walker and Mahoney v. Mahoney overrule decision in Avery v. Commonwealth, Department of Social Services, which allows bifurcated appeal in case of father found guilty of civil contempt for failure to pay court-ordered child support. Party in civil contempt proceeding initiated in juvenile court must post appeal bond within 30 days, in amount of support arrearage, in order to perfect appeal to circuit court for trial de novo.
02-029 The Honorable Kenneth R. Plum Member, House of Delegates General Assembly would need to enact legislation authorizing Fairfax County to amend its human rights ordinance to prohibit discrimination based on sexual orientation.
Judge, Twentieth Judicial Circuit Commissioner of accounts is not permitted access to criminal history records of delinquent fiduciaries through Virginia Criminal Information Network, unless such records are released pursuant to circuit court order or rule.
County Attorney for Alleghany County Determination regarding whether locality may impose flat tax on itinerant merchants and peddlers without violating Commerce Clause depends on resolution of questions of fact that may establish genuine administrative burden experienced by locality. Attorney General traditionally has declined to render official opinions when request involves question of fact rather than one of law.
Judge, Clarke County General District Court Impoundment provisions are applicable to arrest for driving during suspension period, provided driver’s suspension or revocation resulted from DUI conviction.
Member, House of Delegates Act does not address question whether developer of retirement community, which controls homeowners’ association of development, may establish budget for association that is not based on fact. Developer must provide to potential buyers disclosure statement reflecting financial status of association. Developer may retain control of association and bill homeowners for association expenses, provided developer retains ownership of majority of lots constituting development.
Clerk, Circuit Court of Amelia County Notary public commissioned by State of Maryland is not authorized to notarize in Commonwealth documents for recordation in Virginia circuit court clerk’s office.
Member, House of Delegates Colonial Heights police department is responsible for transporting arrestee to regional jail. Once at regional jail, jail superintendent is responsible for conveying prisoners to and from court. City council may adopt ordinance requiring sheriff to perform extra transport duties and may compensate sheriff accordingly. Prisoners awaiting further criminal proceedings at courthouse are within control of sheriff, who is responsible for returning them to jail superintendent for transport to regional jail at conclusion of day’s hearings. If it is necessary for prisoner to remain for further court proceedings within same day, prisoner should be kept in courthouse holding cell until proceedings are concluded and then released to jail superintendent for transport to regional jail.
Superintendent, Department of State Police Department of State Police is authorized to provide mental health information maintained in Central Criminal Records Exchange to Federal Bureau of Investigation for use in National Instant Criminal Background Check System Index.
Member, House of Delegates Compensation received by standing trustee in Chapter 13 bankruptcy for performance of his administrative duties associated with such bankruptcy is subject to local business license taxation.
Treasurer for the City of Chesapeake Local elected constitutional officer is "unit of local government" falling within definition of "agency" under Act. Act’s definition of "agency" includes local constitutional officer.
Commonwealth’s Attorney for Chesterfield County Requirement that law-enforcement agency enter into Virginia Criminal Information Network certain information contained on warrant or capias received by agency is directory in nature, and does not otherwise violate affected individual’s constitutional rights.
Sheriff for the City of Chesapeake Propriety of actions of sheriff’s office with regard to storage of personal property removed from residential premises pursuant to unlawful detainer or ejectment.
Clerk of the Circuit Court of Chesterfield County Automated case management systems maintained by clerk of circuit court, whether storage media is on or off premises, are records of clerk’s office under custody of such clerk. Access to such system lies within sound discretion of clerk.
Member, House of Delegates Water authority may include fee for connecting to water system constructed by authority as proportionate part of its construction costs. Statutory use of "shall" appears to be advisory or directory in nature, and permits authority to review periodically and adjust amount of connection fees, as necessary.
Member, House of Delegates Industrial development authorities may use design-build contracts for construction of "authority facilities" or "facilities," as defined in Industrial Development and Revenue Bond Act, without having to engage in competitive bidding and without having to involve Design-Build/Construction Management Review Board.
01-123 Mr. W. Forrest Matthews, Jr.
Director, Virginia Retirement System Appropriation Act provides no authorization for payment of administrative expenses incurred by Virginia Retirement System in oversight of deferred compensation plan; no statutory authorization for Retirement System to charge and recover administrative costs from Department of Accounts. Retirement System may not recover its administrative expenses from employees who participate in cash match plan, but may bill participating employers for expenses. No authority for Retirement System to charge and collect administrative fee from participants of optional retirement plan for political appointees or recover such costs from institutions of higher education. Attorney General is unable to comment regarding method and source by which Virginia Retirement System may recover costs incurred and associated with administration of VolSAP when Fund assets are insufficient, or on action Retirement System should take for optional retirement plan for school superintendents should General Assembly deny request for general fund appropriation.
Mortgage broker may not provide compensated services in same real estate sales transaction in which affiliated real estate broker is providing compensated services. Term "partner" encompasses limited partner in mortgage broker that is organized as limited partnership.
Clerk, Circuit Court of Bedford County Statutory requirement that social security or other control numbers appearing on marriage licenses not be disclosed applies retroactively to licenses filed on and after July 1, 1997. Attorney General declines to comment on practicability of devising method by which marriage licenses processed and imaged since 1997 may be changed to comply with statutory nondisclosure requirements.
01-085 Mr. Henry A. Thompson, Sr.
County Attorney for Sussex County Governance of biosolids activities within Commonwealth resides with Department of Health. Local ordinance requiring applicant to obtain conditional use permit before applying or storing biosolids in locality is preempted by comprehensive state program regulating biosolids use.
Sheriff for Williamsburg-James City County Sheriff maintains custody of individual transported to medical facility for emergency medical evaluation and treatment until individual is delivered to temporary detention facility upon final execution of order.
Director, Department of Housing and Community Development Municipality has no authority to enforce Statewide Fire Prevention Code on state property if agreement has not been entered into with State Fire Marshall, or Director of Department of Housing and Community Development, for enforcement of Code. Localities have no authority to enforce Building Code against Commonwealth through their building inspectors.
Treasurer for the City of Portsmouth Statute of limitations for collection of all local taxes, including food and beverage taxes, begins to run on December 31 of year to which tax is attributable, i.e., year for which tax was assessed.
Sheriff for the City of Chesapeake Sheriff is chief law-enforcement officer of office of sheriff for purposes of issuing concealed weapons permits to retired deputies.
No constitutional prohibition against member of House of Delegates serving as temporary assistant Commonwealth’s attorney.
02-006 The Honorable Thomas C. Wright, Jr.
Member, House of Delegates Cigarette manufacturer’s sweepstakes promotion at retail establishment is prohibited.
Treasurer may not demand payment of business, professional and occupational license taxes that have not been assessed. Taxpayer is under no obligation to remit such tax payments until they are assessed and due. To extent confidential tax information maintained by commissioner of revenue needs to be obtained by treasurer or bank acting as depository, dissemination of otherwise confidential taxpayer information is allowable.
Town Attorney for the Town of Onancock Town may appropriate public funds to support local business and civic association’s annual Regatta, but may not contribute in-kind services of town employees to assist in setting up Regatta and loan of town-owned equipment. No statute requires town to obtain and examine governing documents of organizations to which it makes contributions. Association is not "public body" under Act; documents of Association are not subject to Act’s public records disclosure requirements. Attorney General declines to comment regarding obligation of town manager to take action in response to citizen complaint regarding alleged improper action by town council or mayor. Authority of town to make cash contribution to Central Accomack Little League.
Advanced legal fees remain property of judgment debtor. Judgment creditor may garnish unearned advance fees paid by judgment debtor to attorney. Potential judgment creditor may attach such funds.
Substitute judge may be designated to serve as district judge only when chief district judge has determined that another full-time district judge or retired judge is not reasonably available. Substitute judge not so appointed should not be called upon by hospital seeking judicial consent for medical treatment of minor.
Splitting off of smaller parcels from large tract does not make such tract ineligible for land use assessment and taxation. Question whether taxpayer made material misstatement of facts in revalidation application or that material change in facts has occurred is decision to be made by commissioner of revenue based on all relevant facts. Use of statutory procedures to correct erroneous assessments. No authority for local governing body to determine if property qualifies for special land use assessment and taxation in individual case.
Member, House of Delegates Records’ custodian has discretion to determine whether to release to minor’s parent library records for purpose of identifying books checked out by minor that are overdue.
Sheriff for the City of Hopewell Local sheriff is not required to provide more than one deputy sheriff to general district court for courtroom security in absence of order stating that particular case presents substantial security risk.
City of Radford may amend its Code to provide for $200 fine for violating portion of ordinance concerning permit parking.
Town of Christiansburg Act prohibits collection by agency of personal information that is secret; permits installation of surveillance cameras and audio monitoring equipment in and on premises of recreation center, provided notices advising of such surveillance are clearly visible.
Member, House of Delegates Proposed amendment requesting increase in appropriation for secure confinement does not constitute special legislation and favors no specific organization that provides enhanced faith-based services to inmates or is controlled by church or sectarian society.
County Attorney for New Kent Co. Treasurer is not required to relinquish his office when involuntarily recalled to active military duty. No statute prevents treasurer from continuing to oversee and manage his office via use of Internet, or in person during evening hours and weekends when he is physically present in county. Treasurer may continue to receive compensation for performing duties of his office while involuntarily recalled to active duty.
County Attorney for Franklin Co. Planning commission of Franklin County may not review for compliance with comprehensive plan existing locations of telecommunications towers in areas of county not subject to zoning. Such review may be undertaken only when application for telecommunications is made with county.
Virginia Retirement System Time spent as local elected official does not constitute time spent employed by locality and is not time purchasable as prior service credit.
Member, House of Delegates Boat registered in Virginia county but normally garaged, docked, or parked in North Carolina has acquired taxable situs in both states. No statute requires county to refund or reduce payment of taxes on boat.
County Attorney for Albemarle Co. Determination whether property of Christian Aid Mission may be classified as tax exempt rests within judgment of commissioner of revenue, after careful consideration of attendant facts. Unused and undeveloped parcels are subject to real estate taxation while they remain as such. Any portion of such land used by other entities may be exempt if activities of using entities are charitable, religious or educational. Rent paid by using entity considered source of revenue or profit to Mission is taxable.
01-086 The Honorable K. Mike Fleenor Jr.
Pulaski Co. Pharmacist, prior to filling prescription, may obtain fingerprint from customer as proof of identification, provided pharmacist returns or destroys print within 21 days of transaction’s completion or termination; may provide to law-enforcement officials fingerprint of any customer suspected of prescription fraud.


text:
R. Hiyya b. Abuiah said: He who is intimate with a heathen woman is as though he had entered into marriage relationship with an idol, for it is written, and hath been intimate with the daughter of a strange god:10 hath then a strange god a daughter — But it refers to one who cohabits with a heathen woman.
R. Hisda said: If the zealot comes to take counsel [whether to punish the transgressors enumerated in the Mishnah], we do not instruct him to do so. It has been stated likewise: Rabbah b. Bar Hana said in R. Johanan's name: If he comes to take counsel, we do not instruct him to do so. What is more, had Zimri forsaken his mistress and Phinehas slain him, Phinehas would have been executed on his account;22 and had Zimri turned upon Phinehas and slain him, he would not have been executed, since Phinehas was a pursuer [seeking to take his life].
He did not know what to reply.
Jer. III, 20. The simile shews that the reference is to idolatry.
This is his punishment and the answer to R. Kahana's question.
The meaning of this is given in the following story.
I.e., it would be exposed to this disgrace, of being cast away in the streets, and yet another, viz., burning.
From Palestine; v. p. 390, n. 1.
This is a mnemonic: N = niddah, a menstruous woman; SH = Shifhah, a non-Jewish maidservant; G = goyyah, a heathen woman; and A = esheth, ish, a married woman. He is regarded as having transgressed in respect of all four, and as such will be punished by heaven.
V. p. 544, n. 7.
Zonah = harlot; for the first three v. preceding note.
R. Dimi, who includes this.
I.e., they expect their wives to observe the marriage bond.
For the zealot may slay only when he is engaged in the commission of the offence.
Simeon was Jacob's second son; Levi, to which Moses belonged, the third.
I.e., seeing the profanation of the Divine Name, he did not wait for Moses' ruling.
Since he rose up out of the congregation, i.e., the Sanhedrin, implying that he went out.
and Phinehas waited for his strength to weaken,16 not knowing that [God is] a King, against whom there is no rising up.17 In the Baraitha we learnt: Sixty [time], until he became like an addled egg, whilst she became like a furrow filled with water. R. Kahana said: And her seat was a beth s'eah.18 R. Joseph learned: Her womb opening was a cubit.
R. Johanan said: [Zimri] had five names: Zimri, the son of Salu, Saul, the son of the Canaanitish woman, and Shelumiel, the son of Zurishaddai. Zimri, because he became like an addled egg [beza hamuzereth]; the son of Salu, because he outweighed [hisli]25 the sins of his family;26 Saul, because he lent himself [hish'il fr. sha'al] to sin; the son of the Canaanitish woman, because he acted in a Canaanitish fashion, [i.e., depravedly]; whilst his real name was Shelumiel the son of Zurishaddai.
Had he withdrawn, Phinehas could not have punished him.
Thus showing that he was punishing immorality, and not satisfying a private hate.
So that it should not interfere with the spear as he was carrying them out aloft.
Thereby distracting their attention: otherwise Zimri's partisans would have slain him.
Levi, the first ancestor of his tribe, had shewn zeal for his sister's honour (Gen. XXXIV, 25f.); Aaron, Phinehas' grandfather, had turned away God's wrath on the occasion of Korah's revolt. Num. XVII, 13.
V. Ex. VI. 25: And Eleazar, Aaron's son, took him one of the daughters of Putiel to wife. According to the legend, Putiel was Jethro, so called because as a priest of Midian he had fattened ([H], with which Putiel is here connected) cattle for idolatrous sacrifices.
The numerical value of zarzir [H], whilst cohabitation is understood from 'loins'.
Heb. [H], (weakened) is connected here with [H].
I.e., he need not have waited, for Zimri was already doomed.
I.e., she became very bloated. Beth se'ah is a field requiring one se'ah of seed.
V. 82a; he had instructed her to surrender only to the greatest man in Israel.
A common name for a dissolute woman. [The word is connected with the Arabic denoting 'womb opening', v. MGWJ. LXXIII, p. 398].
I.e., surely she goes to these secluded spots only for immoral purposes.
From [H]. Others: he caused the sins of the family to rise, i.e., became notorious. (Jast.); Rashi (one version) caused his sins to be searched out, probed.
The reading here in our printed texts differs slightly from that of the Mishnah on 81b; the latter has been followed; cp. DS. a.l.
V. supra 56a. May Jose Smite Jose: blasphemy was punished by death.
That verse hints at death.

matches: v. V. v. V. V. v. V.


text:
To improve the enzymatic hydrolysis (saccharification) of lignocellulosic biomass by Trichoderma reesei, a set of genes encoding putative polysaccharide-degrading enzymes were selected from the coprophilic fungus Podospora anserina using comparative genomics. Five hemicellulase-encoding genes were successfully cloned and expressed as secreted functional proteins in the yeast Pichia pastoris. These novel fungal CAZymes belonging to different glycoside hydrolase families (PaMan5A and PaMan26A mannanases, PaXyn11A xylanase, and PaAbf51A and PaAbf62A arabinofuranosidases) were able to break down their predicted cognate substrates. Although PaMan5A and PaMan26A displayed similar specificities toward a range of mannan substrates, they differed in their end products, suggesting differences in substrate binding. The N-terminal CBM35 module of PaMan26A displayed dual binding specificity toward xylan and mannan. PaXyn11A harboring a C-terminal CBM1 module efficiently degraded wheat arabinoxylan, releasing mainly xylobiose as end product. PaAbf51A and PaAbf62A arabinose-debranching enzymes exhibited differences in activity toward arabinose-containing substrates. Further investigation of the contribution made by each P. anserina auxiliary enzyme to the saccharification of wheat straw and spruce demonstrated that the endo-acting hemicellulases (PaXyn11A, PaMan5A, and PaMan26A) individually supplemented the secretome of the industrial T. reesei CL847 strain. The most striking effect was obtained with PaMan5A that improved the release of total sugars by 28% and of glucose by 18%, using spruce as lignocellulosic substrate.
Lignocellulosic biomass is a desirable feedstock for the supply of second-generation bioethanol, being the largest renewable source of carbohydrates, but the recalcitrance of raw materials makes biotechnological conversion complex and costly (34).
The main components of plant cell walls are cellulose, lignin, and hemicellulose, which form a tight complex with varying proportions between plants (for a review, see reference 45). Cellulose microfibrils are formed by crystalline-like combination of linear and highly ordered polymer chains of β-1,4-linked d-glucose residues, and lignin consists of an amorphous aromatic heteropolymer with hydrophobic properties. Hemicellulose is a combined designation of a diverse set of noncrystalline carbohydrate polymers from plant tissues that form closely associated networks with cellulose microfibrils and lignin (6, 24). Typically, the most abundant component of hemicellulose in the cell walls of monocots (e.g., cereals) is β-1,4-xylan, which consists of β-1,4-linked d-xylose residues substituted with l-arabinosyl, 4-O-methyl-glucuronosyl and acetyl side chains (46, 53). Arabinofuranosyl residues of arabinoxylan may also be esterified with hydroxycinnamic acid residues, e.g., ferulic and p-coumaric acids (53). In softwoods (gymnosperms), the main hemicellulose component is β-1,4-mannan, which consists of a backbone formed by β-1,4-linked d-mannose and d-glucose residues substituted with α-1,6-linked d-galactose residues (39). Linear mannan and glucomannan chains containing more than 5% (wt/wt) d-galactose are called galactomannans and galactoglucomannans, respectively (39).
The proportion of monosaccharide residues in plant biomass degradation products depends on the raw material and the hydrolysis procedure, but significantly, pentose sugars such as d-xylose, constitute the second most abundant carbohydrate in plant raw materials after glucose (26). Thus, it is considered now that the use of all sugar monomers is one of the possible strategies to provide cost-efficient biorefinery processes (28), which is partly due to the recent progress made with pentose-fermenting yeast strains (36). Therefore, bioconversion of hemicelluloses into fermentable sugars is essential to enable the commercial production of lignocellulosic ethanol (24).
One of the crucial steps in the bioconversion of LCB to fuel-grade ethanol is the enzymatic degradation of this recalcitrant biomass (28). The ascomycete Trichoderma reesei (teleomorph Hypocrea jecorina) is one of the most widely used industrial fungi, able to produce cellulase-rich enzyme cocktails with up to 100 g of extracellular protein per liter of culture (13). Such high yields enable a wide application of T. reesei enzyme preparations in different fields of white biotechnology, in the bioconversion of biomass to fuel and other useful products (34). Recent genetic and biochemical technologies have improved our knowledge of the full set of T. reesei enzymes. Although analysis of the secretome of T. reesei CL847 industrial strain revealed only 22 secreted enzymes, most of them involved in biomass degradation (27), complete genome sequencing of the QM6a strain (35) identified about 200 genes encoding glycoside hydrolases as defined by the CAZy classification (10). Despite this high number, the T. reesei genome is surprisingly poor in terms of number and diversity of carbohydrate-degrading enzymes, including GHs, polysaccharide lyases, and carbohydrate esterases potentially able to participate in LCB degradation, compared to other fungi known for their carbohydrate-degrading capacity (35). In addition, the T. reesei genome has the smallest number of carbohydrate-binding modules of all of the Sordariomycetes sequenced to date (35). The greatest current challenge facing the development of second generation biofuels is enzyme cost reduction, the main strategy to meet this goal being the improvement of the efficiency of T. reesei enzymatic cocktail for the saccharification of LCB (34).
Possible strategies to circumvent T. reesei weaknesses in LCB hydrolysis include the search for enzymatic activities able to supplement those of T. reesei industrial enzyme preparations. The identification of novel glycosidases that are either distantly related to or absent from the T. reesei genome is a promising option for enhancing enzymatic hydrolysis of lignocellulosic materials. The increasing number of sequenced genomes annotated for their content of genes encoding for CAZymes (i.e., CAZomes) is a potential source of novel enzymes that warrants exploration. Among the fungal genomes already sequenced, that of the coprophilic ascomycete Podospora anserina, a late grower in the droppings of grass herbivores, encodes for a large, highly specialized set of genes involved in the utilization of carbon sources commonly found in its natural biotope, together with the highest number of CBMs of all of the fungal genomes sequenced to date (19). Interestingly, its CAZome has also revealed an increased potential for the digestion of resistant components of hemicellulose, presenting abundant enzyme sets dedicated to the hydrolysis of cross-linked xylan (19), from which no CAZymes have yet been characterized experimentally.
Owing to the heterogeneity of the hemicellulose fraction of LCB, complete hydrolysis requires a broad variety of cooperatively acting enzymes, such as xylanases, xylosidases, arabinofuranosidases, glucuronidases, acetylxylan esterases, ferulic acid esterases, and p-coumaric acid esterases for the xylan fraction and mannanases, galactosidases, glucosidases, and acetylmannan esterases for the mannan fraction. Until now, most of the characterization and applied studies on hemicellulases have focused on xylanases found in CAZy families GH10 and GH11. Accessory enzymes, such as arabinose-debranching enzymes, are found in six families (GH3, GH43, GH51, GH54, GH62, and GH93), whose members display different specificities. Although mannan is the principal hemicellulose component in softwood, studies of mannan-hydrolyzing enzymes found in families GH5 and GH26 have been largely neglected compared to xylanases.
The objective of the present study was to investigate the potential of single enzyme candidates from the hemicellulolytic enzymatic arsenal of P. anserina for improving the degradation of LCB by an industrial strain of T. reesei. By means of genome analysis using CAZy database tools, we identified genes encoding enzymes that degrade carbohydrates distantly related to or absent from T. reesei. We thus selected and cloned a set of genes from P. anserina encoding putative hemicellulases, which were heterologously expressed and purified to give insight into their biochemical characteristics and enzymatic specificities. The T. reesei enzyme cocktail was further used as a reference to investigate the contribution made by each specific P. anserina auxiliary enzyme on wheat straw and/or spruce, taking advantage of the recently developed automated saccharification method (40).
Abbreviations.Abf, α-l-arabinofuranosidase; AFMB, autoclaved fraction of maize bran; AX, arabinoxylan; CAZy, carbohydrate active enzyme; CBM, carbohydrate binding module; CE, carbohydrate esterase; DNS, dinitrosalycilic acid; d.m., dry matter; GH, glycoside hydrolase; HPAEC-PAD, high-performance anion exchange chromatography coupled with pulsed amperometric detection; IEF, isoelectric focusing; LBG, locust bean gum; LCB, lignocellulosic biomass; Man, endo-β-1,4-mannanase; PL, polysaccharide lyase; PX, mixture of pectin, birchwood, and larchwood xylans; Xyn, endo-β-1,4-xylanase.
Bioinformatics-guided sequence selection.Previous analysis of the T. reesei (anamorph of Hypocrea jecorina) strain QM6a genome had revealed weaknesses in hemicellulolytic capacity (35). Candidate hemicellulolytic CAZymes from the P. anserina genome were subjected to BLASTp analysis against all sequences present in CAZy (10) supplemented by T. reesei CAZyme sequences (35). A number of P. anserina candidates that were (i) absent or distant from those found in the T. reesei genome, (ii) distant from known characterized enzymes from other sources, and/or (iii) displayed an unusual combination of catalytic and carbohydrate-binding modules, were selected for cloning and characterization. For each individual target, the enzyme activity was predicted from the sequence comparisons with BLASTp against a specific library of characterized enzyme modules derived from CAZy. As a complement, the signal peptide cleavage sites were predicted using the signalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/).
Cloning of genes encoding hemicellulases.The homokaryotic P. anserina strain S mat+ used in the present study was kindly provided by P. Silar (CNRS, Paris, France). Currently used methods and culture media can be accessed at the P. anserina Genome Project web site (http://podospora.igmors.u-psud.fr). For the induction of enzymes, P. anserina was grown on M2 medium supplemented with AFMB (autoclaved fraction of maize bran at 15 g liter−1) or PX (pectin, birchwood xylan, and larchwood xylan from Sigma-Aldrich (Lyon, France), each at 5 g liter−1. AFMB was prepared as described previously (7). Growth was carried out in baffled flasks at 120 rpm and 27°C. The mycelium was recovered by sterile filtration using miracloth after 3, 4, and 5 days of culture and directly frozen at −80°C. Total RNA was extracted by using RNeasy plant kit (Qiagen, Courtaboeuf, France), and first-strand cDNA synthesis was performed with SuperScript reverse transcriptase (Invitrogen, Cergy-Pontoise, France) and oligo(dT18) primer according to the manufacturer's instructions. DNA was amplified by PCR using Pwo Super yield DNA polymerase (GE Healthcare, Buc, France) and the specific primers (including restriction sites) designated in Table 1 . The amplified fragments were subcloned into pCRII-TOPO vector (Invitrogen) and subjected to sequencing, and the corresponding genes were deposited in GenBank (for accession numbers, see Table 2 and the legend to Fig. 1). Amplified fragments were inserted at the corresponding sites into vector pPICZαA or pPICZαC (Table 1) in frame with both the yeast α-secretion factor and the C-term-His6 tag encoding sequences. The resulting recombinant expression plasmids were linearized with PmeI and transformed into P. pastoris X-33 (Invitrogen) by electroporation. P. pastoris transformants were isolated on plates containing increasing concentrations of zeocin ranging from 100 to 1,000 μg ml−1. Zeocin-resistant P. pastoris transformants were then screened for protein expression in 10 ml of BMGY (in 50-ml tubes) at 30°C in an orbital shaker (200 rpm) for 16 h to an optical density at 600 nm of 2 to 6, and expression was induced by transferring cells into 2 ml of BMMY, followed by growth for another 3 days. Each day the medium was supplemented with 3% (vol/vol) methanol. The supernatant was then analyzed by SDS-PAGE to determine the transformant with the best secretion yield for each enzyme.
Modular representation of hemicellulases used in the present study. GH, glycoside hydrolases; CBM, carbohydrate-binding modules. The corresponding GenBank accession numbers are HM357135 (PaMan5A), HM357136 (PaMan26A), HM357137 (PaXyn11A), HM357138 (PaAbf51A), and HM357139 (PaAbf62A).
Recombinant enzymes production and purification.For each enzyme, the best-producing transformant was grown in 200 ml of BMGY in a 1-liter baffled flask. The cells were then transferred to 40 ml of BMMY in 200-ml flasks stirred at 200 rpm and 30°C. The suspension was recovered after 3 days induction by centrifugation (10 min, 4,000 × g), and the supernatant was concentrated 10 times by using Amicon centrifugal units (10-kDa cutoff, 4,000 × g; Millipore, Molsheim, France). A nickel chelate His-Bind Resin (GE Healthcare) column (0.7 by 5 cm) was connected to a fast-performance liquid chromatography Äkta apparatus (GE Healthcare) and equilibrated with 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, and 10 mM imidazole. The concentrated supernatant was diluted into the equilibration buffer and loaded onto the column at 4°C. The column was washed with equilibration buffer, and the enzyme was eluted with the same buffer containing 150 mM imidazole. The eluate was concentrated in the centrifugal units and repeatedly washed with sodium acetate buffer (50 mM, pH 5).
Biochemical characterization.Protein concentration was determined by using the Bio-Rad protein assay kit with bovine serum albumin as the standard (Bio-Rad, Marnes-la-Coquette, France). SDS-PAGE was performed in 12% (wt/vol) polyacrylamide gel (Bio-Rad) using a Pharmacia LMW electrophoresis calibration kit (GE Healthcare). Native IEF was carried out at 4°C in the Bio-Rad gel system, using pI standards ranging from 4.45 to 8.2 (Bio-Rad). Proteins were visualized with either Coomassie staining or IEF gel staining solution (0.04% [wt/vol] Coomassie blue R250, 0.05% [wt/vol] Cocrein Scarlet, 10% [vol/vol] acetic acid, 27% [vol/vol] isopropanol).
Enzyme assays.Unless otherwise indicated, assay mixtures contained substrate and suitably diluted enzyme in McIlvaine's buffer (pH 5). Reactions were carried out at 37°C for 5 to 30 min, and the enzyme activity was determined spectrophotometrically. The activity of enzymes toward xylan, mannan, or arabinan was measured by using the 3,5-dinitrosalicylic acid method described earlier (2). Briefly, 10 μl of enzyme was mixed with 190 μl of substrate (xylan or mannan at 10 mg ml−1) in McIlvaine's buffer (pH 5) at 37°C. The reaction was terminated by the addition of 300 μl of dinitrosalicylic acid reagent at 1% (wt/vol) and boiled for 5 min. The reaction mixtures were cooled and centrifuged for 5 min at 13,000 rpm, and 200 μl was transferred to a microtiter plate. The absorbance at 550 nm was measured relative to a xylose or mannose standard curve (0 to 180 μg ml−1). One unit of endo-glycosidase activity was defined as the amount of protein that released 1 μmol of sugar monomer per min. Low-viscosity wheat arabinoxylan, konjac glucomannan, carob galactomannan, ivory nut mannan, sugar beet arabinan, and debranched arabinan were purchased from Megazyme International (Wicklow, Ireland). Birchwood xylan, carboxymethyl cellulose, and locust bean gum galactomannan were purchased from Sigma-Aldrich. Activity toward pNP-α-l-arabinofuranoside (Sigma-Aldrich) was determined by measuring the release of 4-nitrophenol from p-nitrophenyl-α-l-arabinofuranoside (pNP-Ara; 10 mM; Sigma-Aldrich) in 50 mM sodium phosphate buffer (pH 5) at 37°C in a reaction volume of 100 μl. The reaction was stopped by adding 900 μl of 1 M sodium carbonate (pH 9), and the release of 4-nitrophenol was quantified at 410 nm with a molar extinction coefficient for 4NP of 18,300 M−1 cm−1. One unit of enzyme activity was defined as the amount of protein that released 1 μmol of arabinose per min.
The apparent optimal pH was estimated by using wheat arabinoxylan or LBG galactomannan at 10 mg ml−1 in McIlvaine's buffer in the pH range from 2.6 to 7.5 (11 pHs tested). The apparent optimal temperature was estimated in the range 30 to 70°C (nine temperatures tested) under the conditions used. For determination of apparent Michaelis-Menten constants, the initial velocities of the enzymes were measured at 37°C in McIlvaine's buffer (pH 5.0), with substrate concentrations ranging from 2 to 20 mg ml−1 for polymeric substrates and from 0.1 to 20 mM for pNP substrates. The molar concentrations of polymeric substrates could not be calculated because of their heterogeneous nature. Consequently, only an apparent value for the Michaelis constant, Km(app), was determined. The kinetic parameters were estimated by using weighted nonlinear squares regression analysis with Grafit software (Erithacus Software, Horley, United Kingdom).
Determination of the PaMan26A N-terminal CBM35 dissociation constant and the carbohydrate binding capacity.For the binding assay, the reaction mixture contained 1 mg of insoluble wheat arabinoxylan or insoluble ivory nut mannan (Megazyme) or Avicel PH101 (Sigma-Aldrich) and 10 to 50 μg of PaMan26A ml−1 in a final volume of 225 μl of sodium citrate buffer (pH 5). For each assay, the mixture was incubated at 4°C for 1 h with vertical rotation (20 rpm). Polysaccharide was removed by centrifugation, and the concentration of unbound protein ([UB], μM) in the supernatant was measured by the Bradford method. The bound protein concentration ([B], μmol per g of polysaccharide) was determined by subtracting [UB] from the total protein concentration. All of the assays were carried out in triplicate. Adsorption parameters were determined as described previously (41) based on typical double-reciprocal plots using the equation [B] = [UB] × [B]max/(Kd + [UB]), where Kd (μM) and [B]max (μmol per g of polysaccharide) are the equilibrium dissociation constant and the maximum amount of protein bound, respectively.
Supplementation of T. reesei.The lignocellulosic raw materials, wheat straw (Triticum aestivum cv. Apache; Aveyron, France) and spruce (Picea abies, Holmen plant; Braviken, Sweden) were kindly prepared by X. Rouau (INRA Montpellier, France). They were reduced to particles of roughly 100 μm by a multistep micronization procedure as previously described (40). Suspensions were made by adding 1 g of wheat straw or 2 g of spruce to 100 ml of 50 mM sodium acetate (pH 5) in the presence of 30 μg of cycloheximide ml−1 and 40 μg of tetracycline ml−1. Using the handling robot (Tecan, Männedorf, Switzerland), 96-well microplates were filled with micronized wheat straw or spruce suspensions, heat sealed, and stored frozen at −20°C until use; then, 30 μg per well of T. reesei industrial xylanase-rich enzyme cocktail (E508) produced from T. reesei CL847 strain (49) was used as a reference in the assay as described previously (40). T. reesei enzyme cocktail contained 0.12 U of filter paper activity, 0.33 U of CMCase, 0.2 U of β-glucosidase, 1.6 U of xylanase, 0.02 U of mannanase, and 0.02 U of arabinofuranosidase per mg of total protein at 37°C (pH 5.0). To investigate the contribution of each specific P. anserina auxiliary enzyme to the saccharification of micronized wheat straw and/or spruce, 30 μg of PaMan5A, 30 μg of PaMan26A, 10 μg of PaXyn11A, 30 μg of PaAbf51A, and 30 μg of PaAbf62A were individually added to the 30 μg of T. reesei enzymatic cocktail. A total of 30 μg of equal amounts of PaMan5A and PaMan26A was also assayed in combination on spruce. The plate was heat sealed and transferred to a shaking incubator set at 40°C. The enzymatic reaction was left to proceed for 24 h, and the plate was filtered. The total reducing sugars were quantified by using the DNS assay as described previously (40). Monosaccharides (glucose, xylose, and mannose) were quantified by HPAEC-PAD (see below). Saccharification results were expressed in μmol of sugar released per mg of d.m.
Analysis of sugar release by HPAEC-PAD.Monosaccharides generated after hydrolysis of micronized LCB were analyzed by HPAEC coupled with PAD (ICS 3000; Dionex, Sunnyvale, CA) equipped with a Carbo-Pac PA-1 analytical column (250 by 4 mm). Enzymatic reactions were stopped by the addition of 50 mM NaOH before injection (10 μl) into the HPAEC system. Elution (1 ml min−1) was carried out in 18 mM NaOH. Calibration curves were plotted using arabinose, glucose, xylose, mannose, and cellobiose standards (Sigma-Aldrich), from which response factors were calculated (Chromeleon program; Dionex) and used to estimate the amount of product released in test incubations. All of the assays were carried out in triplicate. The results are expressed in μmol of sugar released per mg of d.m. End products generated after hydrolysis of polymeric substrates (ivory nut mannan and wheat arabinoxylan) were analyzed as described earlier (4). The reference β-1,4-xylo-oligosaccharides and β-1,4-manno-oligosaccharides were obtained from Megazyme International.
Selection of genes encoding fungal hemicellulases and sequence analysis.Genes encoding polysaccharide-degrading enzymes were selected based on (i) the structural features of lignocellulosic raw materials selected for the present study, i.e., wheat straw and spruce; (ii) BLASTp searches using CAZy database by comparison of T. reesei and P. anserina genomes; (iii) the presence and diversity of CBMs; and (iv) the predicted specificity of putative exo- and endo-acting CAZymes. The search yielded the sequences corresponding to the loci Pa_6_490 (designated PaMan5A following characterization), Pa_5_1950 (PaMan26A), Pa_2_13730 (PaXyn11A), Pa_5_11670 (PaAbf51A), and Pa_0_1370 (PaAbf62A).
One of our main goals was to select targets that would contribute to β-mannan degradation. Sequence comparison showed that PaMan5A exhibited 62% identity to the catalytic module of T. reesei β-mannosidase TrMan5A, a well-characterized enzyme (48). Unlike TrMan5A, which bears a CBM1 module at C-term, PaMan5A bears no additional module. Since genome analysis had revealed that likely orthologs from different Trichoderma bear the C-terminal CBM, while a variety of other fungi bear none (data not shown), PaMan5A was selected as a target. The second selected enzyme degrading β-mannan was PaMan26A, which has no equivalent in the T. reesei genome. Interestingly, only a very reduced number of fungal genomes present closely related catalytic modules (unpublished data). The closest characterized enzyme is the β-mannanase from Humicola insolens (30), with 79% identity at the catalytic module, followed by a number of bacterial β-mannanases. All closely related enzymes have CBM35 modules adjacent to the N-terminal side of the GH26 catalytic module, but the substrate affinity of the corresponding binding modules has never been studied.
The next hemicellulolytic target PaXyn11A was selected, although its catalytic module presented, respectively, 47 and 64% sequence identity to T. reesei xylanases TrXyn1 and TrXyn2, two long-established enzymes. (51). The main singularity of this sequence is the fact that it carries a C-term CBM1 module. The number of characterized GH11 enzymes bearing a C-term CBM1 module is relatively small. They include xylanases from Penicillium funiculosum XynB (9, 25), Neocallimastix patriciarum XynS20 (33), Lentinula edodes Xyn11A (32), and Phanerochaete chrysosporium XynB/XynB-1 (17). Although the substrate specificities of these fungal CBM1-containing enzymes have been described, none was chosen for that feature.
Finally, two enzyme targets for arabinofuranose-containing substrates were considered, PaAbf51A and PaAbf62A. The first has no equivalent in the T. reesei genome, and the closest characterized sequences are those of the α-l-arabinofuranosidases Afq1 from Penicillium chrysogenum (43) and Abf2 from Penicillium purpurogenum (21), both at ca. 50% identity. The second target had 35% identity to a still (to our knowledge) uncharacterized TrAbf2 from T. reesei, although a number of transcription studies have identified its presence upon induction by a number of saccharides (1, 20). The closest characterized α-l-arabinofuranosidase to PaAbf62A is CjAbf62A from Cellvibrio japonicus at 43% identity (31).
Cloning and expression of novel hemicellulases.Five genes (PaMan5A, PaMan26A, PaXyn11A, PaAbf51A, and PaAbf62A) encoding putative CAZymes were cloned by reverse transcription-PCR using mRNA purified from P. anserina grown on different natural inducers (i.e., AFMB or PX). Although both inducers used were found to induce the expression of PaMan5A, PaMan26A, PaXyn11A, and PaAbf51A genes, only AFMB was able to induce the expression of PaAbf62A gene (Table 2). After sequencing, all of the coding sequences were in agreement with those predicted from genomic data except for the PaAbf51A coding sequence (HM357138), which presented an insertion of 54 nucleotides, leading to 18 extra amino acids with no change in the open reading frame. The coding sequence of each gene was inserted into the P. pastoris expression vector in frame with sequences encoding the yeast α-factor secretion peptide and a His6 tag located at the C terminus. Recombinant genes were then introduced into the Pichia genome under the control of the methanol-inducible promoter. Multicopy transformants were screened on small cultures to select transformants that exhibited satisfactory levels of production by the analysis of each culture supernatant by means of SDS-PAGE. All targeted hemicellulases were successfully detected in the supernatant after induction, indicating correct processing of the α-factor signal sequence. Secretion levels were between 0.1 and 1 g liter−1, facilitating further purification of recombinant enzymes taking advantage of His6 tags. In addition, only trace amounts of endogenous proteins were present in the culture supernatant of transformants (data not shown). Purification yields ranged from 1.2 to 17.6 mg of recombinant protein from 40 ml of supernatant after 3 days induction (Table 3).
Characterization and kinetic properties of individual enzymes.First, P. anserina hemicellulolytic CAZymes purified to homogeneity were biochemically characterized in terms of apparent molecular mass on SDS-PAGE and pI on IEF (Fig. 2 and Table 3). The recombinant proteins displayed apparent molecular masses slightly higher that the theoretical ones (Fig. 2A), probably as a result of posttranslational modification (e.g., glycosylation). Experimental pIs were in close agreement with pIs deduced from the amino acid sequences (Fig. 2B; Table 3). Apparent optimum pH and temperature were first determined on each enzyme, together with temperature stability (Table 3). All of the enzymes were active in the pH 4 to 6 range at temperatures up to 60°C and were stable at 40°C for 1 h with >90% residual activity. P. anserina glycosidases were characterized in depth in terms of activity toward various polysaccharides. Kinetic parameters were further determined at pH 5 and 40°C on the best substrates of each enzyme.
SDS-PAGE analysis of P. anserina hemicellulases: 3 μg of purified recombinant was loaded onto a 12% Tris glycine SDS-PAGE, and the proteins were stained with Coomassie blue. PaMan5A (lane 1), PaMan26A (lane 2), PaXyn11A (lane 3), PaAbf51A (lane 4), and PaAbf62A (lane 5) are shown. The molecular masses of marker proteins (M) are indicated on the left.
PaMan5A and PaMan26A enzymes efficiently hydrolyzed structurally different mannan polysaccharides, such as galactomannans, glucomannans, and β-1,4-mannans from different sources with specific activities ranging from 35 to 86 U mg−1 (Table 4). Neither of the mannanases was active on carboxymethyl cellulose or Avicel. Determination of kinetic parameters showed that PaMan5A and PaMan26A displayed the highest specificity toward konjac glucomannan, with Km values of 0.7 and 0.8 mg ml−1 and catalytic efficiencies (kcat/Km) of 3,142 and 5,750 mg−1 min−1 ml, respectively. PaMan5A and PaMan26A enzymes also acted efficiently on unsubstituted mannan from ivory nut with Km values of 1.6 and 1.0 mg.ml−1 and catalytic efficiencies (kcat/Km) of 1,044 and 1,928 mg−1 min−1 ml, respectively. The PaXyn11A enzyme efficiently hydrolyzed xylans from birchwood and wheat with a preference for wheat arabinoxylan (Km = 5.8 mg ml−1 and kcat/Km = 2,443 mg−1 min−1 ml, Table 4). To evaluate the mode of action of these P. anserina glycosidases, the products generated by the action of PaMan5A and PaMan26A on unsubstituted mannan and PaXyn11A on arabinoxylan were analyzed by HPAEC-PAD. All three enzymes tested displayed typical endo-activity against polymeric substrates, i.e., a mixture of oligosaccharides was generated during the initial stages of hydrolysis (not shown). As the reaction continued, the oligosaccharides were progressively hydrolyzed, yielding principally mannobiose and xylobiose toward the end of the reaction for PaMan5A and PaXyn11A, respectively, and a mixture of mannose, mannobiose, and mannotriose for PaMan26A (Fig. 3).
HPAEC analysis of end products released by endo-acting P. anserina hemicellulases. (A) Ivory nut mannan hydrolysis by PaMan5A and PaMan26A: mannose (M1), mannobiose (M2), and mannotriose (M3). (B) Wheat arabinoxylan hydrolysis by PaXyn11A: xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6).
To investigate the specificity of PaAbf51A and PaAbf62A enzymes, their capacity to hydrolyze pNP-α-l-arabinofuranoside, as well as arabinose-containing polysaccharides, was evaluated. Although PaAbf51A and PaAbf62A displayed similar Km values in the micromolar range (1.2 and 6.1 mM, respectively) toward pNP-α-l-arabinofuranoside, PaAbf51A was approximately 60 times more active than PaAbf62A in terms of specific activity (Table 4). These arabinose-releasing enzymes exhibited low activity against polymeric substrates, preventing accurate determination of the individual kinetic parameters. HPAEC analysis of the reaction products generated by PaAbf51A and PaAbf62A revealed that they released only arabinose from polymeric substrates (wheat arabinoxylan, sugar beet arabinan, and debranched arabinan), thus demonstrating that they cleaved terminal units only (not shown).
Substrate-binding capacities of PaMan26A.The ability of CBM35 harbored by PaMan26A to bind various insoluble polysaccharides was tested. Although under our experimental conditions PaMan26A did not bind to Avicel (crystalline cellulose), binding to mannan and xylan was observed. Based on typical double-reciprocal plots, binding parameters of PaMan26A were determined for mannan and xylan, yielding Kd values of 6.25 and 1.85 μM and Bmax values of 19 and 12 mg of PaMan26A per g of mannan and xylan, respectively (Table 5).
Supplementation of T. reesei enzyme cocktail with P. anserina hemicellulases.Each enzyme was added to the T. reesei enzymatic cocktail, E508, for the saccharification of micronized wheat straw and spruce. The release of both reducing sugars and individual sugar monomers was quantified by using the DNS assay and HPAEC-PAD, respectively, and compared to E508 (Fig. 4). The addition of PaXyn11A resulted in an increase in reducing sugars, glucose, and xylose of ca. 10% from micronized wheat straw. However, the addition of exo-acting enzymes did not affect the release of sugar monomers compared to T. reesei alone. The combination of PaAbf51A and PaAbf62A exo-acting enzymes with PaXyn11A did not improve the results obtained with the xylanase alone on micronized wheat straw (data not shown). Using spruce as lignocellulosic substrate, a 15% increase in total sugars was observed when PaXyn11A was added, with no effect on the glucose release. Supplementation of E508 with either PaMan5A or PaMan26A significantly increased the release of total sugars by 28 and 17%, respectively, and the release of glucose by 18 and 10%, respectively. Supplementation of E508 with a combination of PaMan5A and PaMan26A improved the release of total sugars by 34% and the release of glucose by 23%. Also, the addition of mannanase alone or in combination improved the release of xylose and mannose by 40 to 50% (Fig. 4).
Saccharification of lignocellulosic micronized substrates (wheat straw and spruce) by T. reesei enzymatic cocktail (E508) supplemented with P. anserina hemicellulases. Saccharification was carried out on wheat straw (A) and spruce (B) for 24 h at 40°C as described in Materials and Methods. The release of sugars was expressed in μmol per mg of dry matter (d.m.). White bars indicate the release of reducing sugars determined by using the DNS assay. Black bars indicate the release of glucose measured using HPAEC. Gray bars indicate the release of xylose from wheat straw (A) and the release of xylose plus mannose from spruce (B) measured using HPAEC. Values above each bar are means of sextuplicate measures, and error bars represent standard errors of the mean.
In the present study, we characterized five novel hemicellulases from different glycoside hydrolase families from P. anserina and assessed their individual contributions to the saccharification of LCB. P. pastoris was used as a heterologous expression system to express the set of P. anserina hemicellulases, since it is capable of secreting large amounts of correctly folded fungal proteins into the medium, with very few additional proteins (16), making it ideal for the production of recombinant fungal enzymes. It was recently used successfully to express a suite of polysaccharide-degrading enzymes from Aspergillus (3). Using this expression host, we efficiently produced and easily purified five fungal hemicellulases in one step, thus allowing their in-depth characterization.
PaMan5A and PaMan26A enzymes were both active on structurally different mannans. Determination of the kinetic parameters enabled us to demonstrate that their best substrate was the unsubstituted glucomannan in terms of affinity and catalytic efficiency, suggesting a negative impact of d-galactosyl side groups with hydrolysis for the galactomannan substrates as described for T. reesei mannanase (50). Although PaMan5A and PaMan26A displayed similar specificities toward a range of mannan substrates, they differed in terms of end product formation. PaMan26A released mannobiose and mannotriose from mannan polymer (ivory nut mannan), as reported for the mannanase from T. reesei (47). PaMan5A released mainly mannobiose without accumulation of mannotriose, as described for the recently characterized Aspergillus niger GH5 mannanase (18). These results indicate differences in the mode of action of substrate binding within the active sites of the two characterized P. anserina mannanases.
Our data strongly suggest that the N-terminal CBM35 module harbored by PaMan26A displays specificity toward hemicellulose polymers, mannan and xylan, with a similar constant Kd in the μM range. A CBM35 module is conserved in three Cellvibrio xylan-degrading enzymes and has been shown to bind uronic acid of xylan with a calcium-dependent interaction (38), while a module from a Cellvibrio mannanase binds to decorated soluble mannans and manno-oligosaccharides (52). Subtle changes in amino acid chemistry and conformation within the binding site of these bacterial CBM35s have radical impacts on their specificity (14). In fungi, only one CBM35 module binding to β-galactan has been characterized to date in a Phanerochaete chrysosporium galactan β-1,3-galactosidase (29).
Although family GH11 glycoside hydrolases are one of the best-characterized CAZy families, containing exclusively endo-xylanases, PaXyn11A was selected because it harbors a CBM1 module. This family module, formerly designated cellulose-binding domain family 1 (CBD1), is a well-characterized module specific to cellulose and exclusively in fungi (12). PaXyn11A xylanase exhibited an “endo” mode of action on polymeric substrates, releasing mainly xylobiose as major end product with a high catalytic efficiency, a common characteristic of fungal GH11 xylanases (4, 11).
Fungal species have been found to produce several arabinofuranosidase isoenzymes with different modes of action and substrate specificity (21). The PaAbf51A and PaAbf62A arabinofuranosidases characterized in the present study followed Michaelis-Menten kinetics toward pNP-Ara. Since the activity of these enzymes is of the “exo” type, very low activity was observed toward natural substrates. The kinetic properties of PaAbf51A toward pNP-Ara were comparable to the GH51 fungal enzymes characterized to date with a Km value in the mM range, an optimum pH around 5, and an optimum temperature above 50°C (21). P. anserina PaAbf62A displayed a 400-fold decrease in catalytic efficiency compared to its PaAbf51A arabinofuranosidase counterpart, mainly due to a low kcat value. This observed difference suggests that these two enzymes may act on different substitutions.
Genome sequencing of T. reesei has revealed the relatively small number of genes encoding enzymes involved in the breakdown of LCB in terms of diversity compared to other fungi. With only 16 genes encoding putative hemicellulases, T. reesei has the smallest set of hemicellulases of all of the saprophytic fungi analyzed (35). Among ascomycetes, the genome of P. anserina revealed the potential of this coprophilic fungus to hydrolyze recalcitrant lignocellulosic residues (19). It contains a significantly higher number of putative cellulases and xylanases compared to A. niger, A. nidulans and A. oryzae, reflected by its growth profile, which is better than Aspergillus species on cellulose and xylan (15). These observations fit well with the natural biotope of P. anserina, which is a late colonizer of herbivore dung, in which lignocellulose is the main remaining organic component. To our knowledge, none of the numerous putative glycoside hydrolases from different CAZy families encoded by the P. anserina genome have been characterized to date.
Since the T. reesei cocktail is xylanase-rich, the positive effect observed by adding PaXyn11A enzyme on the increase of both reducing sugars and glucose from wheat straw could be due to the presence of a CBM1 module linked to the enzyme that may contribute to the overall xylanase efficiency. To our knowledge, the T. reesei genome contains no gene encoding a GH11 xylanase harboring a CBM1. A possible explanation for the capacity of CBMs to increase the activity of glycoside hydrolases against insoluble substrates is that they reduce the “accessibility problem” by bringing the appended catalytic modules into intimate, prolonged association with their target substrate, thereby enhancing catalytic efficiency (23). Another hypothesis is that xylanases drastically change the structure and physicochemical properties of the AX population. Xylanases in the same GH family, i.e., with similar specificities, differ substantially in substrate selectivity, and the classification of GH family alone is not sufficient to predict the breakdown of insoluble AXs by xylanases (8). Although mechanistic information on the catalytic and substrate specificity of xylanases is helpful for comparing xylanase efficiencies, other factors, such as their degree of selectivity toward soluble versus insoluble substrate, may play a role in determining the functionality of these enzymes in the degradation of hemicellulose from raw materials (5). Although T. reesei has the smallest set of arabinofuranosidases (two GH43, no GH51, one GH62, and no GH93) of all plant cell wall-degrading ascomycetes (35), PaAbf51A and PaAbf62A did not potentiate the action of T. reesei enzymes in our experimental conditions.
In the present study, a positive effect on the release of reducing sugars and glucose from spruce was observed when T. reesei was supplemented with individual and combined addition of PaMan5A and PaMan26A mannanases. Galactoglucomannans are the main hemicellulose components in softwood (10 to 15% dry mass), with concentrations varying with the type of softwood and the location in the plant cell wall (39). Glucomannans can be found incorporated into aggregates of cellulose, as well as parallel to the cellulose fibrils, to which they are tightly connected (44). Our hypothesis is that PaMan5A and PaMan26A mannanases can facilitate the action of the T. reesei enzymatic cocktail by removing mannans that are found around cellulose fibrils. Also, T. reesei mannanase (TrMan5a) is expressed at low levels of secretion with only ∼0.02 U mg−1 detected in the E508 enzymatic cocktail. Although PaMan5A does not carry a CBM, the supplementation effect was higher than that found for PaMan26A. Further fusion of PaMan5A to a CBM specific for cellulose (CBM1) could be way to further enhance the positive effect observed in the present study. We recently demonstrated that the fusion of the Aspergillus niger mannanase (AnMan5) to a CBM1 module potentiates the action of the enzyme toward mannan-containing lignocellulosic substrates (42).
Helper enzymes can loosen up the lignocellulosic structure via degradation of hemicellulosic substrates (mannan/xylan), thus increasing the surface area of cellulose attacked by T. reesei cellulases. Because of the heterogeneity of hemicellulose between lignocellulosic substrates, enzymes with different characteristics are required in industrial applications, but the choice of the enzyme is still largely empirical (5). In the present study, hemicellulose-acting enzymes selected using comparative genomics brought a clearly marked improvement in both the liquefaction and the solubilization of LCB and the release of glucose and xylose/mannose when supplemented to T. reesei. The fact that this effect was more striking with the addition of mannanase might be due to the close association of softwood glucomannan with cellulose microfibrils, whereas xylan is interconnected to lignin. Since pretreatement of lignocellulose is also responsible for the high process cost of bioethanol production from LCB, helper enzymes (e.g., hemicellulases) could be an alternative to chemical and/or physical pretreatments. With addition of xylan- or mannan-acting enzymes to the actual cocktail, we could consider lowering the dosage of the T. reesei cocktail to achieve the same glucose yield (22, 37), since cellulose would be more accessible to cellulases. Tailor-made enzymatic cocktails need to be developed according to the lignocellulosic structure of each biomass for optimal hydrolysis.
The analysis of P. anserina genome has recently revealed a large and highly specialized set of genes involved in utilization of natural carbon sources (e.g., lignocellulose) commonly found in its natural biotope (19). The present findings pave the way for further studies to give more insights into P. anserina's enzymatic machinery. Among this unexpected unique enzymatic equipment, its genome presents a number of other interesting targets potentially acting on the recalcitrant components of plant cell wall. Finding complementary auxiliary enzymes displaying novel specificities will be required to deconstruct efficiently LCB for the production of biofuels and high-value products.
This study was funded by the French National Research Agency (ANR, program E-TRICEL ANR-07-BIOE-006).
We thank X. Rouau and G. Ghizzi for the preparation of micronized substrates, P. Silar for advice on P. anserina culturing methods, and D. Navarro for helpful discussions.
Accepted manuscript posted online 29 October 2010.
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