Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid beta-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-beta-1,4-D-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml(-1). The enzyme was acidophilic, with highest activity at pH 1.0-1.5, lower than any known mannanases, and optimal temperature for activity was 65 degrees C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K(m), and V(max) for locust bean gum substrate was 3,373 U mg(-1), 1.56 mg ml(-1), and 6,587.6 micromol min(-1) mg(-1), respectively. The enzymatic activity was not significantly affected by ions such as Ca(2+), Cr(3+), Co(2+), Zn(2+), Na(+), K(+), and Mg(2+) and enhanced by Ni(2+), Fe(3+), Mn(2+) and Ag(+). These favorable properties make MAN5A a potential candidate for use in various industrial applications.

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Backgroundβ-Glucosidase is an important member of the biomass-degrading enzyme system, and plays vital roles in enzymatic saccharification for biofuels production. Candidates with high activity and great stability over high temperature and varied pHs are always preferred in industrial practice. To achieve cost-effective biomass conversion, exploring natural enzymes, developing high level expression systems and engineering superior mutants are effective approaches commonly used.ResultsA newly identified β-glucosidase of GH3, Bgl3A, from Talaromyces leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank. The purified enzyme exhibited outstanding enzymatic properties, including favorable temperature and pH optima (75 °C and pH 4.5), good thermostability (maintaining stable at 60 °C), and high catalytic performance (with a specific activity and catalytic efficiency of 905 U/mg and 9096/s/mM on pNPG, respectively). However, the narrow stability of Bgl3A at pH 4.0–5.0 would limit its industrial applications. Further site-directed mutagenesis indicated the role of excessive O-glycosylation in pH liability. By removing the potential O-glycosylation sites, two mutants showed improved pH stability over a broader pH range (3.0–10.0). Besides, with better stability under pH 5.0 and 50 °C compared with wild type Bgl3A, saccharification efficiency of mutant M1 was improved substantially cooperating with cellulase Celluclast 1.5L. And mutant M1 reached approximately equivalent saccharification performance to commercial β-glucosidase Novozyme 188 with identical β-glucosidase activity, suggesting its great prospect in biofuels production.ConclusionsIn this study, we overexpressed a novel β-glucosidase Bgl3A with high specific activity and high catalytic efficiency in P. pastoris. We further proved the negative effect of excessive O-glycosylation on the pH stability of Bgl3A, and enhanced the pH stability by reducing the O-glycosylation. And the enhanced mutants showed much better application prospect with substantially improved saccharification efficiency on cellulosic materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0560-8) contains supplementary material, which is available to authorized users.

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bThe genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical Cterminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley ␤-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/␤-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages. P lant cell wall polysaccharides (PCWPs), composed mainly of cellulose and hemicellulose, are a promising rich resource for renewable biofuel development (1). The complete deconstruction of PCWPs into fermentable, simple mono-or oligosaccharides requires the concerted action of a complex array of glycoside hydrolases (GHs), including cellulases and hemicellulases (2, 3). The genomes of Gram-positive bacteria of the genus Caldicellulosiruptor encode an arsenal of thermophilic plant cell wall polysaccharide-degrading enzymes (3-5), which are appealing candidates in the design of novel, robust enzyme cocktails for PCWP deconstruction.In the genome of Caldicellulosiruptor bescii, there is a gene cluster containing three genes which encode proteins harboring two tandemly linked CBM3b modules and a GH family 48 (GH48) cellobiohydrolase in the C terminus. These CBM3b and GH48 modules, as well as their linker sequences, are extremely similar at the level of the amino acid sequence (see Fig. S1 in the supplemental material). Notably, however, the three multimodular enzymes differ much in their N termini: CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) ha...

We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-beta-D-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 degrees C, maintained more than 60% of maximal activity when assayed at pH 1.5-4.0, and had good thermal stability at 60 degrees C and remained stable at pH 1.0-6.0. The enzyme activity was enhanced in the presence of Ni(2+) and beta-mercaptoethanol and inhibited by some metal irons (Hg(2+), Cu(2+), Pb(2+), Mn(2+), Li(+), and Fe(3+)) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-D-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg(-1), respectively. The apparent K (m) and V (max) values for beechwood xylan were 5.6 mg ml(-1) and 3,622 micromol min(-1) mg(-1), respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.