TECHNICAL FIELD

This disclosure relates to a method of producing cellulase by membrane separation and concentration of a cellulase component derived from filamentous fungi and a device therefor.

BACKGROUND

Polysaccharides such as starch or cellulose are available as biomass resources. A method of producing monosaccharides such as glucose or xylose by enzymatically hydrolyzing such polysaccharides has been known. The sugar liquid obtained by breaking down such polysaccharides is widely used as carbon sources in microorganism culture. In particular, as an enzyme that hydrolyzes cellulose, cellulase is known. As microorganisms that produce this cellulase, filamentous fungi are known. The filamentous fungi produce a large amount of cellulases as extracellular enzymes by setting up culture conditions to inductively produce the cellulase. As these filamentous fungi, available are filamentous fungi such as the genusAspergillus, the genusHumicola, the genusMucor, the genusTrichoderma, and the genusAcremonium, which are widely used in production of cellulase.

Enzyme components that exhibit various substrate specificities are present in cellulase derived from filamentous fungi. Specifically, present are cellobiohydrolase that acts on the crystalline region of cellulose, endoglucanase that acts from within cellulose molecular chain to reduce the molecular weight thereof, β-glucosidase that acts on water-soluble oligosaccharides or cellobiose, and hemicellulase that acts on hemicellulose. It has been known that each of these enzyme components with varied substrate specificity acts synergistically on cellulose fibers and thereby efficient degradation of cellulose is taken place (see Encyclopedia of Cellulose, Chapter 6, Biodegradation of Cellulose (edited by The Cellulose Society of Japan, 2000)). It has also been known that, in such a cellulase derived from filamentous fungi, each of the enzyme components whose molecular weight varies in size from small to large is present, wherein the molecular weight ranges from 20 to 100 kDa.

With regard to cellulase derived from filamentous fungi, methods of membrane separation and concentration using an ultrafiltration membrane in the same manner as common enzymes are known. For instance, methods of separation and recovery of cellulases using an ultrafiltration membrane have been known, which method comprises membrane separation and concentration of cellulases from a culture of microorganisms such as filamentous fungi or a cellulose hydrolysate obtained by using the cellulase (see WO 2007/034999 and Japanese Patent Application Laid-Open Publication Nos. 2010-221136 and 2009-240167). As a treatment prior to the step of the membrane separation and concentration through such an ultrafiltration membrane, methods have been suggested, which method comprises removing in advance solids contained in an aqueous solution of cellulase from a microorganism culture or cellulose hydrolysate by use of, for example, a ceramic spin filter (see JP '136) or a non-woven fabric with a pore diameter of 20 to 200 μm (see JP '167). Yet, for the membrane separation and concentration of each of the cellulase components, it has been common to use an ultrafiltration membrane having a fine pore diameter or molecular weight cut off that sufficiently blocks molecular weight corresponding to the cellulase component to be recovered.

In the membrane separation and concentration of filamentous fungi cellulase using a conventional ultrafiltration membrane, problems have included fouling of the ultrafiltration membrane and inactivation or aggregation of the cellulase component. These problems have caused a filtration treatment speed of the ultrafiltration membrane to become slower or have caused a cellulase activity obtained by the ultrafiltration membrane to become lower, which has been also problematic.

In view of this, it could be helpful to provide a method of efficient membrane separation and concentration of cellulase components derived fromTrichodermaby using an ultrafiltration membrane and to provide a device therefor.

SUMMARY

We thus provide:[1] A method of producing cellulase comprising the following steps of (1) to (3):(1) the step of subjecting an aqueous solution of cellulase derived from filamentous fungi to filtration through an ultrafiltration membrane with a molecular weight cut off of 100,000 to 200,000 to obtain a filtrate and to concurrently obtain a concentrated enzyme liquid as a retentate;(2) the step of further subjecting the filtrate obtained in step (1) to filtration through a second ultrafiltration membrane with a molecular weight cut off of 5,000 to 50,000 to obtain a second concentrated enzyme liquid as a retentate; and(3) the step of mixing the concentrated enzyme liquid obtained in steps (1) and (2) to obtain cellulase derived from filamentous fungi.[2] The method of producing cellulase according to [1], wherein the filamentous fungi isTrichoderma.[3] The method of producing cellulase according to [1] or [2], wherein the aqueous solution of cellulase derived from filamentous fungi is adjusted to pH 2.6 to 5.4 or pH 8.6 to 9.4 in step (1).[4] The method of producing cellulase according to any of [1] to [3], wherein the aqueous solution of cellulase derived from filamentous fungi is a filamentous fungi culture liquid or a hydrolysate of biomass containing cellulose obtained by using the cellulase derived from filamentous fungi.[5] The method of producing cellulase according to any of [1] to [4], wherein the aqueous solution of cellulase derived from filamentous fungi comprises one or more enzyme components selected from the group consisting of cellobiohydrolase, endoglucanase, and xylanase.[6] The method of producing cellulase according to any of [1] to [5], wherein a temperature of the aqueous solution of cellulase derived from filamentous fungi is in a temperature range of 15 to 35° C.[7] A device of producing cellulase comprising a tank for an aqueous solution of cellulase, an ultrafiltration pump that is connected therewith, and at least one of an ultrafiltration membrane with a molecular weight cut off of 100,000 to 200,000 that are connected in series or in parallel, a holding tank for a filtrate of the above-mentioned ultrafiltration membrane, a second ultrafiltration membrane with a molecular weight cut off of 5,000 to 50,000, and a second holding tank for a concentrated enzyme liquid.[8] The device of producing cellulase according to [7], wherein the tank for an aqueous solution of cellulase comprises a pH sensor and is further connected with a pH adjuster tank.

It is possible to efficiently carry out membrane separation and concentration of cellulase components from an aqueous solution of cellulase derived from filamentous fungi. Specifically, part of cellulase components derived from filamentous fungi can be separated and recovered in step (1) and further the remaining cellulase component derived from filamentous fungi contained in a filtrate can be recovered in step (2), thereby recovering a notably high Avicel degrading activity.

