Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara.

BACKGROUND: Cellulases play a key role in the enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. Thus, the discovery of novel cellulases from exotic biological niches is of great interest as they may present properties that are valuable in the biorefining of lignocellulosic biomass. RESULTS: We have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multi-domain enzyme from the unusual gill endosymbiont Teredinibacter waterburyi of the wood-digesting shipworm Psiloteredo megotara. The catalytic GH5 domain, was cloned and recombinantly produced with or without a C-terminal family 10 carbohydrate-binding module (CBM). Both variants showed hydrolytic endo-activity on soluble substrates such as ß-glucan, carboxymethylcellulose and konjac glucomannan, respectively. However, low activity was observed towards the crystalline form of cellulose. Interestingly, when co-incubated with a cellulose-active LPMO, a clear synergy was observed that boosted the overall hydrolysis of crystalline cellulose. The crystal structure of the GH5 catalytic domain was solved to 1.0 Å resolution and revealed a substrate binding cleft extension containing a putative + 3 subsite, which is uncommon in this enzyme family. The enzyme was active in a wide range of pH, temperatures and showed high tolerance for NaCl. CONCLUSIONS: This study provides significant knowledge in the discovery of new enzymes from shipworm gill endosymbionts and sheds new light on biochemical and structural characterization of cellulolytic cellulase. Study demonstrated a boost in the hydrolytic activity of cellulase on crystalline cellulose when co-incubated with cellulose-active LPMO. These findings will be relevant for the development of future enzyme cocktails that may be useful for the biotechnological conversion of lignocellulose.

Sugar oxidoreductases and LPMOs - two sides of the same polysaccharide degradation story?

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides such as chitin and cellulose and their discovery has revolutionized our understanding of enzymatic biomass conversion. The discovery of LPMOs raises interesting new questions regarding the roles of other oxidoreductases and abiotic redox processes in biomass conversion. LPMOs need reducing power and an oxygen co-substrate and biomass degrading ecosystems contain a multitude of redox enzymes that affect the availability of both. For example, biomass degrading fungi produce multiple sugar oxidoreductases whose biological functions so far have remained somewhat enigmatic. It is now conceivable that these redox enzymes, in particular H2O2-producing sugar oxidases, could play a role in fueling and controlling LPMO reactions. Here, we shortly review contemporary issues in the LPMO field, paying particular attention to the possible roles of sugar oxidoreductases.

Identification of a Novel Thermostable Alkaline Protease from Bacillus megaterium-TK1 for the Detergent and Leather Industry.

An increased need by the green industry for enzymes that can be exploited for eco-friendly industrial applications led us to isolate and identify a unique protease obtained from a proteolytic Bacillus megaterium-TK1 strain from a seawater source. The extracellular thermostable serine protease was processed by multiple chromatography steps. The isolated protease displayed a relative molecular weight (MW) of 33 kDa (confirmed by zymography), optimal enzyme performance at pH 8.0, and maximum enzyme performance at 70 °C with 100% substrate specificity towards casein. The proteolytic action was blocked by phenylmethylsulfonyl fluoride (PMSF), a serine hydrolase inactivator. Protease performance was augmented by several bivalent metal cations. The protease tolerance was studied under stringent conditions with different industrial dispersants and found to be stable with Surf Excel, Tide, or Rin detergents. Moreover, this protease could clean blood-stained fabrics and showed dehairing activity for cow skin with significantly reduced pollution loads. Our results suggest that this serine protease is a promising additive for various eco-friendly usages in both the detergent and leather industries.

Improvement of Saccharification and Delignification Efficiency of Trichoderma reesei Rut-C30 by Genetic Bioengineering.

Trichoderma reesei produces various saccharification enzymes required for biomass degradation. However, the lack of an effective lignin-degrading enzyme system reduces the species' efficiency in producing fermentable sugars and increases the pre-treatment costs for biofuel production. In this study, we heterologously expressed the Ganoderma lucidum RMK1 versatile peroxidase gene (vp1) in the Rut-C30 strain of T. reesei. The expression of purified 6×His-tag-containing recombinant G. lucidum-derived protein (rVP1) was confirmed through western blot, which exhibited a single band with a relative molecular weight of 39 kDa. In saccharification and delignification studies using rice straw, the transformant (tVP7, T. reesei Rut-C30 expressing G. lucidum-derived rVP1) showed significant improvement in the yield of total reducing sugar and delignification, compared with that of the parent T. reesei Rut-C30 strain. Scanning electron microscopy (SEM) of tVP7-treated paddy straw showed extensive degradation of several layers of its surface compared with the parent strain due to the presence of G. lucidum-derived rVP1. Our results suggest that the expression of ligninolytic enzymes in cellulase hyperproducing systems helps to integrate the pre-treatment and saccharification steps that may ultimately reduce the costs of bioethanol production.

Characterization of a solvent, surfactant and temperature-tolerant laccase from Pleurotus sp. MAK-II and its dye decolorizing property.

OBJECTIVE: Laccase is industrially important but a major challenge is the production of an ideal laccase with suitable physicochemical properties to tolerate temperature, surfactants, metal ions and solvents towards its potential application in bioremediation. RESULTS: A laccase with a molecular mass of 43 kDa was purified from Pleurotus sp. MAK-II. It was optimally active at pH 4.5 and 60 °C using ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) as substrate. The laccase was susceptible to NaN3 and NaCN. Activity was strongly enhanced by Cu(2+), Mg(2+) and Ca(2+). The purified laccase showed stability towards various surfactants and solvents and decolorized, in the presence of violuric acid as redox mediator, the diazo dye Congo Red and the anthraquinone dye Remazol Brilliant Blue R to the extent of 96 and 72 %, respectively. CONCLUSION: The ideal physicochemical properties of Pleurotus sp. MAK-II-derived laccase suggest that it could be effectively used in the textile dye industry.

Characterization of a novel endoglucanase from Ganoderma lucidum.

We evaluated the production and characterization of endoglucanase from Ganoderma lucidum using different lignocellulose biomasses. We purified a novel carboxymethyl cellulose (CMC) hydrolyzing endoglucanase from the white-rot fungus G. lucidum when the medium was supplemented with 1% (w/v) wheat bran. Endoglucanase was purified 12.5-fold via ammonium sulfate fractionation, Sephadex G-100, and Q-Sepharose column chromatography with a final yield of 15%. SDS-PAGE analysis revealed that the endoglucanase had a molecular mass of 64.0 kDa. The optimal activity of purified endoglucanase was at pH 5.0 and 35 °C, though it was stable between pH 4.0-7.0 and temperatures of 30-60 °C. The purified enzyme was specific to CMC as a suitable substrate. The metal ions Hg(2+), Fe(2+), and Cr(2+) inhibited enzyme activity, while Ca(2+), Mg(2+), and Mn(2+) enhanced enzyme activity. The endoglucanase showed high activity and stability in the presence of different surfactants and non-polar hydrophobic organic solvents. This endoglucanase is tolerant to high temperature, metal ions, surfactants, and solvents, suggesting that it is appropriate for use in biomass conversion for biofuel production under harsh environmental conditions.

Characterization of lignocellulolytic enzymes from white-rot fungi.

The development of alternative energy sources by applying lignocellulose-based biofuel technology is critically important because of the depletion of fossil fuel resources, rising fossil fuel prices, security issues regarding the fossil fuel supply, and environmental issues. White-rot fungi have received much attention in recent years for their valuable enzyme systems that effectively degrade lignocellulosic biomasses. These fungi have powerful extracellular oxidative and hydrolytic enzymes that degrade lignin and cellulose biopolymers, respectively. Lignocellulosic biomasses from either agricultural or forestry wastes are abundant, low-cost feedstock alternatives in nature but require hydrolysis into simple sugars for biofuel production. This review provides a complete overview of the different lignocellulose biomasses and their chemical compositions. In addition, a complete list of the white-rot fungi-derived lignocellulolytic enzymes that have been identified and their molecular structures, mechanism of action in lignocellulose hydrolysis, and biochemical properties is summarized in detail. These enzymes include ligninolytic enzymes (laccase, manganese peroxidase, lignin peroxidase, and versatile peroxidase) and cellulolytic enzymes (endo-glucanase, cellobiohydrolase, and beta-glucosidase). The use of these fungi for low-cost lignocellulolytic enzyme production might be attractive for biofuel production.

Secretome analysis of Ganoderma lucidum cultivated in sugarcane bagasse.

Harmful environmental issues of fossil-fuels and concerns about petroleum supplies have spurred the search for renewable alternative fuels such as biofuel. Agricultural crop residues represent an abundant renewable resource for future biofuel. To be a viable alternative, a biofuel should provide a net energy gain, have environmental benefits, be economically feasible, and should also be producible in large quantities without reducing food supplies. We used these criteria to evaluate the white rot basidiomycota-derived fungus Ganoderma lucidum that secretes substantial amounts of hydrolytic and oxidative enzymes useful for the degradation of lignocellulosic biomass that were not described hitherto. The current bottleneck of lignocellulosic biofuel production is the hydrolysis of biomass to sugar. To understand the enzymatic hydrolysis of complex biomasses, we cultured G. lucidum with sugarcane bagasse as substrate and qualitatively analyzed the entire secretome. The secreted lignocellulolytic enzymes were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and diverse enzymes were found, of which several were novel lignocellulosic biomass hydrolyzing enzymes. We further explored G. lucidum-derived cellulose, hemicellulose and lignin degrading enzymes as valuable enzymes for the second generation of biofuel obtained from a lignocellulose substrate such as sugarcane bagasse.