Actinomycetes: A Source of Lignocellulolytic Enzymes

Lignocellulose is the most abundant biomass on earth. Agricultural, forest, and agroindustrial activities generate tons of lignocellulosic wastes annually, which present readily procurable, economically affordable, and renewable feedstock for various lignocelluloses based applications. Lignocelluloses are the focus of present decade researchers globally, in an attempt to develop technologies based on natural biomass for reducing dependence on expensive and exhaustible substrates. Lignocellulolytic enzymes, that is, cellulases, hemicellulases, and lignolytic enzymes, play very important role in the processing of lignocelluloses which is prerequisite for their utilization in various processes. These enzymes are obtained from microorganisms distributed in both prokaryotic and eukaryotic domains including bacteria, fungi, and actinomycetes. Actinomycetes are an attractive microbial group for production of lignocellulose degrading enzymes. Various studies have evaluated the lignocellulose degrading ability of actinomycetes, which can be potentially implemented in the production of different value added products. This paper is an overview of the diversity of cellulolytic, hemicellulolytic, and lignolytic actinomycetes along with brief discussion of their hydrolytic enzyme systems involved in biomass modification.


Introduction
Actinomycetes, a separate taxonomic group within domain bacteria, are members of the order Actinomycetales [1]. They are Gram positive bacteria, primarily aerobic and spore formers, with high G+C content [2]. As their name reflects (in Greek, "atkis" means ray and "mykes" means fungus), they share some morphological features with fungi [3]. They show filamentous growth, producing aerial or substrate mycelium. Actinomycetes are responsible for earthy smell of the soil [1]. They are ubiquitous in nature, found both in terrestrial and aquatic habitats [1,4], including mangroves and sea sediments [5]. They belong to both mesophilic and thermophilic groups [6], which broaden the range of habitats inhabited by them. Actinomycetes are known to produce an extensive range of bioactive compounds including various enzymes having multiple biotechnological applications.
Lignocellulolytic enzymes, one of the potent enzymes produced by actinomycetes, can be exploited widely in various lignocelluloses based industries [7]. Lignocellulases   tapped for production of lignocellulases [7,[17][18][19][20]. In this review, the diversity and applications of lignocellulolytic actinomycetes have been discussed along with description of their lignocellulases enzyme systems involved in biomass degradation.

Lignocellulose: Structure and Uses
Lignocellulose is comprised of three main components, that is, cellulose, hemicellulose, and lignin [21] (Figure 1). Cellulose is the high molecular weight linear polymer of Dglucopyranose units linked together by -(l → 4)-glycosidic bonds, with cellobiose dimer being the repeating unit. The cellulose chains are hydrogen bonded to each other, making a bundle of microfibrils, which further aggregate together to make cellulose fibrils [22]. The structure shows variations from amorphous to crystalline regions. The cellulose fibrils are packed in the cell wall in a matrix of hemicelluloses and lignin [23]. Hemicelluloses are linear or branched heteropolysaccharides composed of D-xylose, L-arabinose, D-galactose, D-glucose, or D-mannose sugars with or without different uronic acids and may include xylans, mannans, glucans, glucuronoxylans, arabinoxylans, glucomannans, galactomannans, galactoglucomannans, -glucans, and xyloglucans [10,24]. The branching and composition vary among different plant sources. Xylan, the polymer of xylose, is the most abundant hemicellulosic component [10]. Lignin is a very complex polyphenolic heteropolymer primarily made up of three phenyl propionic alcohol monomers, that is, pcoumaryl alcohol, coniferyl alcohol, and sinapyl alcohol [25].
Enzyme Research 3 Celluloses, hemicelluloses, and lignin are packed closely in a crisscross network and glued with the help of a variety of noncovalent and covalent linkages. Lignin acts as a cementing agent and provides structural support. It also provides impermeable barrier to the enzymes, making whole structure robust and resistant [22]. Cellulose is the most abundant polymer of plant cell walls (35-50%), followed with hemicellulose (20-35%) and lignin (10-25%) [10].
Cellulose is used for many purposes such as in manufacturing of paper and textile fabric, in production of biofuel (from fermentable sugar glucose), as inert packing and insulating material, and in food and drugs as base and stabilizer [26]. Hemicelluloses are utilized in a range of applications. Biodegradability and nontoxicity of hemicelluloses enable it to be used as dietary fibre [27] and as edible coating over foodstuffs for their stabilization. Mannans have cholesterol lowering properties [28]. Xyloglucans and -glucans are used in ice-creams and other foods due to their gelling, stabilizing, and thickening effects. Adhesive properties are used in paper making. Hemicelluloses are rich source of xylose sugars, which are fermented to ethanol fuel [29]. Characteristics of lignin also make it an important component used in the production of a wide range of value added products [30]. Lignin can be used as a source of activated carbon and phenol useful in the synthesis of wide array of chemicals [31] and to produce biocomposites [32] or polymers [33] which can replace plastic based products either partially or completely. It can be used as binder in small scale processes and as filler in place of oil based carbon fibres. Biosorption properties of lignin [34] are also foreseen as a potent application. Furthermore, lignin is a rich source of energy [35]. Thus, lignocellulose is a repertoire of industrially useful components. But the structural characteristics of the cell wall or lignocellulose are such that the extraction of individual component to homogeneity and purity is a difficult task requiring specific chemical or biological procedures. Also many applications are based on degradation products of the cellulose, hemicellulose, or lignin making hydrolysis an essential requirement. Hydrolysis can be mediated both chemically and enzymatically. Chemical methods involve use of expensive chemicals, harsh conditions and have certain other limitations. Enzymatic methods are, therefore, preferred and lignocellulolytic enzymes play very important role in these applications. Presently biomass utilization is executed through biorefinery, which aims at significant utilization of all components generating least possible amount of waste. When one constituent of biomass is utilized, others are simultaneously used for production of valuable products. During biofuel production, sugars from hemicellulose and cellulose components are used in ethanol formation and lignin residue left is used for power generation or in other uses of lignin. Lignocellulases perform important functions in biorefinery processes also.  [36,37]. Exoglucanases act on reducing or nonreducing ends of cellulose chains releasing glucose units, whereas endoglucanases hydrolyse -1,4-glycosidic bonds randomly inside the cellulose chains releasing dextrans of variable lengths [38]. Cellobiohydrolases cleave glycosidic bonds at nonreducing ends and release cellobiose units [39]. These enzymes are particularly important in hydrolysing crystalline cellulose because of their processivity [36]. -glucosidases enzymes take part in hydrolysis of cellobiose units to monomeric glucose [38]. Complete hydrolysis of cellulose involves synergistic effect of all these enzymes, showing synergy between endoglucanases and exoglucanases (endo-exo synergy), exoglucanases acting on the reducing and nonreducing ends (exo-exo synergy), between cellobiohydrolases and -glucosidases, and between catalytic and carbohydrate binding domains [39]. Figure 2 shows schematic presentation of enzymatic hydrolysis of cellulose polymer.

Lignocellulolytic Enzyme Systems in Actinomycetes
Microbial cellulase systems are either complexed or noncomplexed [39]. Complexed systems, known as cellulosomes, are characteristics of anaerobic bacteria, consisting of multienzyme complex protuberances from cell surface stabilized by dockerin and adhesion proteins. In aerobic bacteria, including most of the actinomycetes, cellulases are noncomplexed or free and are secreted extracellularly using specific secretion pathways.
Among cellulase producing actinomycetes, Cellulomonas fimi, Microbispora bispora, and Thermobifida fusca have been studied extensively [6,39]. Thermobifida fusca is a thermophilic, spore forming actinomycete [6]. The genome of T. fusca consists of 3.6 billion bp in a single circular chromosome, with 3117 coding sequences, and has 67.5% G+C content which stabilizes DNA in extreme temperature conditions [40]. The genome encodes for 36 glycoside hydrolases distributed in 22 GH (glycoside hydrolases) families [40]. Cellulase system of T. fusca is comprised of six extracellular cellulases (4 endocellulases and 2 exocellulases) and one intracellular -glucosidase [6,[40][41][42]. Each enzyme has a separate catalytic and carbohydrate binding domain, both linked together with a linker peptide [36,41]. Carbohydrate binding domains in all six cellulases belong to the same family, that is, 2CBD [41]. The catalytic domains, however, are different in all enzymes belonging to different families, with Cel5A and Cel5B from GH family 5, Cel6A and Cel6B from GH family 6, Cel9A and Cel9B from GH family 9, and Cel48A from family 48 [43]. Cel5A, Cel5B, Cel6A, and Cel9B are endocellulases and do not show processivity [36,40,41]. Cel6B and Cel48A are processive exocellulases which act at nonreducing and reducing ends, respectively. Cel9A is a novel processive cellulase with both exo-and endocellulase actions, starting exohydrolysis from the nonreducing end [36]. The position of CBD varies in different cellulases, which is Nterminus in Cel5A, Cel6B, and Cel48A and C-terminus in Cel6A, Cel9A, and Cel9B [41]. Sequence studies of catalytic domains have revealed less than 31% similarity between cellulases from taxonomically similar as well as dissimilar microbes, which indicates development of cellulase system as a result of horizontal gene transfer compared to the gene duplication [44]. The three-dimensional structure of Cel6A shows -barrel with a deep active site cleft formed by one shorter and one turned loop, consisting of four conserved Asp residues [42,45]. Active site of GH48 is also in a cleft [42]. Structural characteristics of exocellulases allow them to bind processively, whereas open active sites of endocellulases enable them to bind cellulose internally at random sites [42]. Structural elucidation of Cel9A has shown that it gives activity between exo-and endocellulases because its weak binding domain 3c CBD is aligned with the active site in the catalytic domain, allowing processive hydrolysis by the enzyme [42]. T. fusca also produces cellulose and chitin binding proteins, E7 and E8, the CBM33 proteins which improve cellulose hydrolysis mediated through exoglucanases [36,40,42]. Cellulases from T. bifida have also been found showing synergism with endocellulases and Trichoderma reesei CBHI [6]. Cellulomonas fimi is a facultative anaerobe, but it does not consist of cellulosomes of cellulolytic anaerobes; rather it produces free cellulases [46]. Similarly, facultatively anaerobic Cellulomonas flavigena also secrets free cellulases [46]. Both carry out efficient hydrolysis of celluloses and hemicelluloses.
The expression of cellulolytic genes in T. bifida is induced by cellobiose, whereas easily utilizable sugar glucose shows catabolite repression as in most of the other cellulolytic microbes [49]. The regulation of cellulolytic genes is mediated by the CelR repressor, which binds to a 9-14 bp palindrome Enzyme Research 5 (5 -TGGGAGCGCTCCCA-3 ) in region 5 -upstream of the cellulase genes in the absence of cellobiose or cellulose [50]. During induction, cellulose is hydrolysed to cellobiose by constitutive cellulases. Cellobiose binds to CelR releasing it from the promoter, allowing transcription of the downstream genes [47]. In addition to the presence of glucose, cellulase genes expression is also regulated by cAMP levels.
The extracellular cellulases are secreted by actinomycetes using either one or both of the common bacterial systems for secretion of extracellular proteins, that is, sec general secretion system and sec independent twin-arginine translocation (TAT) systems. The general Secretion route catalyses transmembrane translocation of proteins in their unfolded conformation, whereas Twin-arginine (TAT) system translocates secretory proteins in their native folded state. In T. bifida both of these systems were discovered, whereas S. coelicolor mainly utilizes TAT systems for protein export [40].

Lignolytic Enzymes.
Lignin degradation is mediated by a complex of enzymes containing three principal enzymes laccases (EC 1.10.3.2), manganese peroxidases (MnP, EC 1.11.1.13), and lignin peroxidases (LiP, EC 1.11.1.14) [55,56]. Laccases are the oxidoreductases which degrade polyphenol, the principal recalcitrant component in the lignocellulose [15,57]. They are extracellular inducible enzymes which employ simple oxygen as an oxidizing agent as well as cofactor. They are multicopper oxidases having four copper atoms in their active sites, taking part in oxygen reduction [58]. Low substrate specificity of laccases enables them to degrade wide variety of compounds. Manganese and lignin peroxidases are together known as heme peroxidases containing protoporphyrin IX as a prosthetic group. Lignin peroxidases can specifically degrade high redox potential compounds and are known to oxidize phenolic as well as nonphenolic aromatic rings, which make up around 90% of the lignin polymer. They require H 2 O 2 for their activity. Veratryl alcohol is an attractive substrate for LiP, which oxidises other substrates by acting as the redox mediator for indirect oxidation. Manganese peroxidases are low redox potential heme peroxidases requiring H 2 O 2 for their activity. They can be manganese dependent or versatile peroxidases [56,58].

Genetic Engineering
The genes from several lignocellulolytic actinomycetes have been successfully cloned to show heterologous expression in different microbes. GH1 and GH3 enzymes of C. fimi ATCC 484 expressed in E. coli have shown efficient hydrolysis of celluloses and xylanases [65]. CelStrep gene from cellulolytic Streptomyces sp. G12 cloned and expressed in E. coli was found to belong to GH12 family and catalysed hydrolysis of carboxymethylcellulose following a Michaelis-Menten kinetics with a of 9.13 mg/mL and a max of 3469 M min −1 [66]. Streptomyces reticuli consists of Cel1 gene encoding for avicelase enzyme which alone can hydrolyse crystalline cellulose effectively [67]. When this gene was cloned and expressed in E. coli, Bacillus subtilis, and Streptomyces spp., enzyme was produced but in lower amounts probably due to the absence of genes encoding for essential regulatory factors [68]. From xylanolytic Actinomadura sp strain FC7 two genes, xylI and xylII, have been cloned, expressed, and well characterized in Streptomyces lividans [69]. Xylanase gene xylBS27 belonging to GH11 from Streptomyces sp. S27 has been successfully cloned and expressed in Pichia pastoris, hydrolysing xylan to xylobiose [70]. Similarly, expression of laccase gene from Streptomyces coelicolor (SLAC) in Streptomyces lividans produced large amount of high purity laccase (350 mg L −1 ) [71]. The gene for a thermostable laccase from Streptomyces lavendulae REN-7 was successfully cloned and expressed in E. coli [72]. Cloning of a lignin peroxidase from Streptomyces viridosporus T7A into Streptomyces lividans TK64 has resulted in better lignocellulose degradation by genetically engineered S. lividans compared to S. lividans TK64 [73]. Thus, genetic engineering techniques can be and are being used for constructing industrially valuable strains with potent applications based on actinomycetes lignocellulolytic enzymes.

Cellulolytic Actinomycetes.
Cellulolytic potential of actinomycetes has been explored since inspection of other microorganisms for cellulase production. Various research studies support high cellulose degradation potential of microbes from actinomycetales. Table 1 represents the diversity of actinomycetes producing cellulase enzymes.

Hemicellulolytic Actinomycetes.
Diverse types of actinomycetes belonging to wide range of habitats and active in different environmental conditions are known to produce hemicellulolytic enzymes. Streptomyces have been found to be the most abundant hemicellulases producer among actinomycetes. In a study by Boroujeni et al. [74], all of the isolated hemicellulolytic actinomycetes were found to belong to Streptomyces genus. Xylanase has been successfully purified from Streptomyces sp. E-86 and characterized for its xylanolytic activity [75]. Optimization studies were carried out for endoxylanase production by Streptomyces sp. F2621 isolated from Turkey [76]. -xylosidase activity of Streptomyces has been used in saccharification of ball milled wheat straw [77]. A thermostable xylanase was obtained from Streptomyces sp. QG-11-3, which has shown biobleaching effects in eucalyptus kraft pulp [78]. Xylanase has also been produced from other strains of Streptomyces sp. such as Streptomyces sp. strain C1-3 [79], Streptomyces sp. CD3 [80], Streptomyces sp. 7b [81], Streptomyces sp PC22, Streptomyces sp 234P-16I, SWU-10, Streptomyces sp. MDS [82], and Streptomyces sp. [83]. In Streptomyces rochei and Streptomyces chromofuscus, xylanase production has been achieved using treated Papyrus and cotton stalk pulp. The obtained xylanase when used for studying bleaching effects has shown enhanced brightness in the presence of EDTA [84]. Xylanases have also been obtained from Streptomyces albus and Streptomyces hygroscopicus and have shown successful production of biogas using oil cake and straw waste [85].

Lignolytic Actinomycetes.
Lignolytic activity is exhibited by diverse range of actinomycetes, which play important role in biodegradation processes in the environment. Search is in progress for more actinomycetes with high lignolytic potential, using advanced techniques combined with conventional methods. A study by Fernandes et al. [111] have used specifically designed primers for detection of laccase-like genes within actinomycetes and has identified gene fragments undetectable by known primers, which corresponded to superfamilies I and K based on laccase and multicopper oxidase engineering database. Arias et al. [112] have shown production of laccase by Streptomyces cyaneus CECT 3335 using soya flour. Laccase was purified and characterized and was found to show increase in brightness of eucalyptus kraft pulp in biobleaching studies. The enzyme was able to oxidize veratryl alcohol suggesting potential of the strain in industrial applications. Veratryl alcohol oxidation and other lignolytic activities have also been demonstrated in Streptomyces viridosporus [113,114].

Reference
Streptomyces sp.

Extracellular cellulase
High efficiency and bioactivity observed during compositing [168] Enzyme  [118]. Laccase and lignin peroxidase activities have also been observed in Streptomyces cinnamomeus [119]. Laccase enzyme studies were carried out including their structural elucidation in Streptomyces lavendulae, Streptomyces psammoticus, Streptomyces ipomoeae, and Streptomyces sviceus [61]. In a study by Escudero et al. [120], Tsukamurella and Cellulosimicrobium actinomycetes showed ABTS (2,2 -azinobis-3-ethylbenzothiazoline-6-sulphonic acid) oxidation rate of 108 U/L and 0.56 U/L, respectively [120]. Submerged fermentation in marine actinomycete Streptomyces lydicus, isolated from Egypt Red Sea, grown in medium supplemented with peanut shell, produced 1.625 U/mL laccase under optimized conditions [121]. Thermoalkali stable laccase from Thermobifida fusca has shown oxidation of several dye intermediates including 2,6-dimethylphenylalanine and paminophenol [64]. Streptomyces sviceus was also found to be showing lignolytic activity [63]. Streptomyces psammoticus has shown enhanced production of laccase under solid state fermentation conditions in the presence of pyrogallol inducer, which was taken to the level of scale-up studies using a packed bed bioreactor [122]. Study by Niladevi and Prema [123] has shown production of all three enzymes, that is, laccase, manganese peroxidase, and lignin peroxidase, by Streptomyces psammoticus. Actinomycete Rhodococcus ruber has shown oxidation and degradation of polyethylene as a result of laccase production by it [124]. A study by Aoyama et al. [125] demonstrated laccase production by Streptomyces atratus. In search of the genes involved in lignocellulose degradation during composting of agricultural wastes, twodomain laccase-like multicopper oxidase genes were identified in Streptomyces violaceusniger [62]. Rhodococcus jostii was found to produce lignin peroxidases capable of modifying lignin [126]. Several other studies have shown lignin degradation ability in many other actinomycetes including Streptomyces flavovirens [127], Streptomyces setoni [128], Actinomadura spp. [55], and Streptomyces thermoviolaceus [129].

Future Prospects
Owing to the abundance and renewability of lignocellulosic biomass, it is considered as most appropriate and economical feedstock for production of various industrially useful products. Lignocellulases enzymes are, therefore, critical in processes associated with bioconversion of lignocelluloses. Presently most of the commercially exploited lignocellulases rely on fungal or bacterial microorganisms. Actinomycetes are relatively less explored for their biomass hydrolysis potential. The studies can be elaborated in search of new actinomycetes producing lignocellulose degrading enzyme systems. Different feedstock shows variation in their chemical composition. The production of enzymes needs to be optimized for different biomass. The production of lignocellulases from all microbial sources is still quite expensive. Efforts can be made for reducing the cost of production of these enzymes using high potency actinomycete enzyme systems with broader range of tolerance and active at diverse environmental conditions. Genetic engineering techniques can be used to construct enzyme systems with desirable characteristics. Also the studies can be expanded gradually to scale up to the industrial levels for their subsequent adoption in commercial processes.

Conclusion
Actinomycetes are an important source of lignocellulose hydrolysing enzymes. They constitute considerable proportion of the soil or aquatic microflora responsible for biomass degradation in nature. The research studies on search of lignocellulose hydrolysing actinomycetes revealed the abundance and diversity of these microbes in different ecological niches. The genetic and protein studies on their hydrolytic enzymes lead to the elucidation of structural and mechanism details of enzymes and their relatedness with other known lignocellulose producers and their enzyme systems. Relatively scanty information is available on lignocellulolytic actinomycetes. The research studies, therefore, need to be elaborated in view of utilization of lignocellulolytic potential of actinomycetes applicable in different industrial sectors.