Two new β-glucosidases from ethanol-fermenting fungus Mucor circinelloides NBRC 4572: enzyme purification, functional characterization, and molecular cloning of the gene

Two β-glucosidases (BGLs 1 and 2) were purified to homogeneity from the extracellular enzyme preparations of the ethanol-fermenting Mucor circinelloides NBRC 4572 statically grown on rice straw. BGLs 1 and 2 are monomeric glycoproteins whose apparent molecular masses (Ms) are around 78 kDa, which decreased by approximately 10 kDa upon enzymatic deglycosylation. Both BGLs showed similar enzyme characteristics in optimal temperature and pH, stability, and inhibitors. They were active against a wide range of aryl-β-glucosides and β-linked glucose oligosaccharides. Their amino acid sequences shared 81 % identity and exhibited less than 60 % identity with the known family-3 BGLs. Considering properties such as reduced inhibition by ethanol, glucose, and cellobiose, low transglucosylation activity, wider substrate range, less binding affinity to lignocellulosic materials, and abundant expression, BGL1 is likely to be more suitable for bioethanol production than BGL2 via simultaneous saccharification and fermentation of rice straw with M. circinelloides.     

Expression and characterization of GH3 β-Glucosidase from Aspergillus niger NL-1 with high specific activity, glucose inhibition and solvent tolerance

A β-glucosidase gene bglI from Aspergillus niger NL-1 was cloned and expressed in Pichia pastoris. The bglI gene consists of a 2583 bp open reading frame encoding 861 amino acids; the enzyme was classified into glycoside hydrolases 3. To improve the expression level of recombinant BGL in P. pastoris, fermentation conditions were optimized by the single-factor experiments. The optimal fermentation conditions were obtained: initial pH 5.0, methanol concentration 0.5% added into the culture every 24 h, and initial cell density (OD₆₀₀) of 10 for induction. The activity of BGL was increased from 4 U/mL to 45 U/mL in optimal conditions. The BGL was purified by ultrafiltration and (NH₄)₂SO₄ precipitation showing a single band on SDS-PAGE. The optimal activity was at pH 4.0 and 60°C. The recombinant enzyme was stable over a pH range of 3.0–7.0 and retained more than 85% activity after incubation at 60°C for 30 min. The kinetic experiments revealed K _m and V _max for p-nitrophenyl-β-D-glucoside of 0.64 mM and 370 U/mg, for cellobiose 8.59 mM and 1480 U/mg. The activity of BGL was not or only a little affected by many metal ions and EDTA and was enhanced by methanol or n-butyl alcohol. The BGL had a K _i of 48 mM for glucose and retained 76% activity in the presence of 50 mM glucose. The favorable properties of BGL offer the potential for industrial application.     

β-Glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four β-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance

From Aspergillus tubingensis CBS 643.92 four distinct beta-glucosidases (I-IV) were purified by a four-step purification procedure. SDS-PAGE revealed molecular masses of 131, 126, 54 and 54 kDa, respectively, and their isoelectric points were determined to be 4.2, 3.9, 3.7 and 3.6, respectively. The beta-glucosidases exhibited high diversity with respect to pH and temperature optima and stability, as well as to substrate specificity and glucose tolerance. The major beta-glucosidase (I) preferentially hydrolysed oligosaccharides. The acid-stable and heat-tolerant beta-glucosidase II hydrolysed aryl and terpenyl beta-D-glucosides as well as 1-O-trans-cinnamoyl beta-D-glucoside. In contrast to beta-glucosidases I and II, the minor beta-glucosidases III and IV were found to be glucose-tolerant; inhibition constants of 470 and 600 mM, respectively, were determined.