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Using chromatography on different matrixes, three beta-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) beta-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (beta-glucan from barley and laminarin); ii) beta-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).

Homogeneous beta-xylosidases with molecular mass values 120 and 80 kDa (as shown by SDS-PAGE), belonging to the third family of glycosyl hydrolases, were isolated by anion-exchange, hydrophobic, and gel-penetrating chromatography from enzyme preparations based on the fungi Aspergillus japonicus and Trichoderma reesei, respectively. The enzymes exhibit maximal activity in acidic media (pH 3.5-4.0), and temperature activity optimum was 70 degrees C for the beta-xylosidase of A. japonicus and 60 degrees C for the beta-xylosidase of T. reesei. Kinetic parameters of p-nitrophenyl beta-xylopyranoside and xylooligosaccharide hydrolysis by the purified enzymes were determined, which showed that beta-xylosidase of A. japonicus was more specific towards low molecular weight substrates, while beta-xylosidase of T. reesei preferred high molecular weight substrates. The competitive type of inhibition by reaction product (xylose) was found for both enzymes. The interaction of the enzymes of different specificity upon hydrolysis of glucurono- and arabinoxylans was found. The beta-xylosidases exhibit synergism with endoxylanase upon hydrolysis of glucuronoxylan as well as with alpha-L-arabinofuranosidase and endoxylanase upon hydrolysis of arabinoxylan. Addition of beta-xylosidases increased efficiency of hydrolysis of plant raw materials with high hemicellulose content (maize cobs) by the enzymic preparation Celloviridine G20x depleted of its own beta-xylosidase.

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Pectin lyase A (molecular weight 38 kD by SDS-PAGE, pI 6.7) was purified to homogeneity from culture broth of the mycelial fungus Penicillium canescens using chromatographic techniques. During genomic library screening, the gene encoding pectin lyase A from P. canescens (pelA) was isolated and sequenced, and the amino acid sequence was generated by applying the multiple alignment procedure (360 residues). A theoretical model for the three dimensional structure of the protein molecule was also proposed. Different properties of pectin lyase A were investigated: substrate specificity, pH- and temperature optimum of activity, stability under different pH and temperature conditions, and the effect of Ca2+ on enzyme activity. In the course of the laboratory trials, it was demonstrated that pectin lyase A from P. canescens could be successfully applied to production and clarification of juice.

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Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K(m) and k(cat) values are 0.4 mM and 15 sec(-1) for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galactomannans (GM) of various structures. The K(m) and k(cat) values are 1.3 mg/ml and 67 sec(-1) for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K(m) and k(cat) values are 0.08 mM and 35 sec(-1) for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.

This study provides computational‐assisted engineering of the cellobiohydrolase I (CBH‐I) from Penicillium verruculosum with simultaneous enhanced thermostability and tolerance in ionic liquids, deep eutectic solvent, and concentrated seawater without affecting its wild‐type activity. Engineered triple variant CBH‐I R1 (A65R‐G415R‐S181F) showed 2.48‐fold higher thermostability in terms of relative activity at 65°C after 1 h of incubation when compared with CBH‐I wild type. CBH‐I R1 exhibited 1.87‐fold, 1.36‐fold, and 1.57‐fold higher specific activities compared with CBH‐I wild type in [Bmim]Cl (50 g/L), [Ch]Cl (50 g/L), and two‐fold concentrated seawater, respectively. In the multicellulases mixture, CBH‐I R1 showed higher hydrolytic efficiency to hydrolyze aspen wood compared with CBH‐I wild type in the buffer, [Bmim]Cl (50 g/L), and two‐fold concentrated seawater, respectively. Structural analysis revealed a molecular basis for the higher stability of the CBH‐I structure in which A65R and G415R substitutions form salt bridges (D64 … R65, E411 … R415) and S181F forms π–π interaction (Y155 … F181), leading to stabilize surface‐exposed flexible α‐helixes and loop in the multidomain β‐jelly roll fold structure, respectively. In conclusion, the variant CBH‐I R1 could enable efficient lignocellulosic biomass degradation as a cost‐effective alternative for the sustainable production of biofuels and value‐added chemicals.