•This is the first study on the improvement of N-acetyl-chitobiose on diabetes-related metabolism disorders.•N-Acetyl-chitobiose lowered blood glucose and improved glucose intolerance and insulin sensitivity.•N-Acetyl-chitobiose inhibited MAPK signaling pathways in type 2 diabetes model mice.The effects of N-acetyl-chitobiose [(GlcNAc)2] on diabetes-related metabolic disorders, along with its regulation mechanism of mitogen-activated protein kinase (MAPK) signaling pathway were investigated on type 2 diabetes (T2D) model mice. Treatment with (GlcNAc)2 improved significantly glucose and lipid metabolism by decrease of blood glucose (∼20%), total cholesterol (∼26.5%) and triglyceride (∼16.1%), increase of HDL-cholesterol (∼107.2%), and reversal of insulin resistance in T2D model mice. Moreover, (GlcNAc)2 reduced lipid peroxidation and inflammatory factors in pancreas with increased activity of superoxide dismutase (∼57%), reduced malondialdehyde equivalent (∼22%) and lowered levels of TNF-α, IL-1β and NF-κB. (GlcNAc)2 had also significantly attenuated MAPK signaling pathways especially though IL-1β-Erk/p38-Histone H3 pathway in T2D model mice. It can be concluded that (GlcNAc)2 has potential as a new functional food ingredient to improve T2D-related metabolic disorders.

To identify novel pullulanases from microorganisms and to investigate their biochemical characterizations.A novel pullulanase gene (BmPul) from Bacillus megaterium WW1210 was cloned and heterologously expressed in Escherichia coli. The gene has an ORF of 2814 bp encoding 937 amino acids. The recombinant pullulanase (BmPul) was purified to homogeneity and biochemically characterized. BmPul has an MW of approx. 112 kDa as indicated by SDS-PAGE. Optimum conditions were at 55 °C and pH 6.5. The enzyme was stable below 40 °C and from pH 6.5−8.5. The Km values of BmPul towards pullulan and amylopectin were 3.3 and 3.6 mg/ml, respectively. BmPul hydrolyzed pullulan to yield mainly maltotriose, indicating that it should be a type I pullulanase.A novel type I pullulanase from Bacillus megaterium was identified, heterologously expressed and biochemically characterized. Its properties makes this enzyme as a good candidate for the food industry.

β-1,3-Glucan is one of the most abundant polysaccharides in fungi. Recognition of β-1,3-glucan occurs in both hydrolysis by glycoside hydrolases and immunological recognition. Our study provides a novel structural account of how glycoside hydrolase recognizes and hydrolyzes substrates in a triple-helical form and presents a general structural basis of β-1,3-glucan recognition.

•A novel β-1,3-1,4-glucanase gene (PbBglu16A) from Paenibacillus barengoltzii was expressed.•PbBglu16A was purified and biochemically characterized.•PbBglu16A was an endo-β-1,3-1,4-glucanase with strict substrate specificity.•PbBglu16A has great potential in β-gluco-oligosaccharides production.A novel endo-β-1,3-1,4-glucanase gene (PbBglu16A) was cloned from Paenibacillus barengoltzii and heterogeneously expressed in Escherichia coli. The recombinant β-1,3-1,4-glucanase (PbBglu16A) was purified to homogeneity with a recovery yield of 78.6% and a specific activity of 431.8 U mg−1. The molecular mass of PbBglu16A was estimated to be 44.0 kDa by SDS-PAGE. The optimal pH and temperature of PbBglu16A were 6.0 and 55 °C, respectively. The enzyme was stable within pH 3.5–9.0 and up to 55 °C. PbBglu16A exhibited high substrate specificity towards barley β-glucan, oat β-glucan and lichenin. PbBglu16A showed an endo-type cleavage pattern and hydrolyzed endogenous enzyme-deactivated oat bran into β-gluco-oligosaccharides with a yield of 7.0%, which mainly consisted of trioligosaccharide and tetraoligosaccharide. Further, PbBglu16A could promote mashing with a reduced filtration time (14.0%) and viscosity (3.4%). Thus, PbBglu16A might be a promising candidate for the production of β-gluco-oligosaccharides and in brewing industry.

β-Mannanase randomly cleaves the β-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of β-mannanase have tremendously expanded in recent years, the wild-type β-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 β-mannanase (RmMan5A) from Rhizomucor miehei shows many outstanding properties, such as high specific activity and hydrolysis property. However, owing to the low catalytic activity in acidic and thermophilic conditions, the application of RmMan5A to the biorefinery of mannan biomasses is severely limited.To overcome the limitation, RmMan5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated β-mannanase (mRmMan5A) with high catalytic activity in acidic and thermophilic conditions was obtained, and then characterized. The mutant displayed maximal activity at pH 4.5 and 65 °C, corresponding to acidic shift of 2.5 units in optimal pH and increase by 10 °C in optimal temperature. The catalytic efficiencies (kcat/Km) of mRmMan5A towards many mannan substrates were enhanced more than threefold in acidic and thermophilic conditions. Meanwhile, the high specific activity and excellent hydrolysis property of RmMan5A were inherited by the mutant mRmMan5A after directed evolution. According to the result of sequence analysis, three amino acid residues were substituted in mRmMan5A, namely Tyr233His, Lys264Met, and Asn343Ser. To identify the function of each substitution, four site-directed mutations (Tyr233His, Lys264Met, Asn343Ser, and Tyr233His/Lys264Met) were subsequently generated, and the substitutions at Tyr233 and Lys264 were found to be the main reason for the changes of mRmMan5A.Through directed evolution of RmMan5A, two key amino acid residues that controlled its catalytic efficiency under acidic and thermophilic conditions were identified. Information about the structure–function relationship of GH family 5 β-mannanase was acquired, which could be used for modifying β-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a β-mannanase from zygomycete engineered by directed evolution.

•Heterologous expression of l-phenylalanine oxidase from mushroom Coprinus cinereus.•The enzyme exhibited specific activity towards phenylalanine over other 19 amino acids.•Resolution of racemic of null,l-phenylalanine mixture by the enzyme.•The enzyme exhibited higher enzymatic activity, and phenylpyruvic acid titer.A novel l-phenylalanine oxidase gene from a species of mushroom Coprinopsis cinereus was cloned. With l-amino acid oxidase from Hebeloma cylindrosporum, which is the closest one, it shared only 30.6% sequence identity. This recombinant protein was expressed in Escherichia coli, purified and biochemically characterized. It contained 778 amino acids and was quite different compared with all previously studied enzymes. This enzyme exhibited highest specific activity of 6.04 U/mg towards l-phenylalanine and the optimal pH, temperature of the enzyme catalyzed reaction were 8.5 and 45 °C. The enzyme was stable up to 55 °C within pH range 7.0–9.5. It could oxidize l-phenylalanine to phenylpyruvic acid at high titer (8.1 ± 0.1 g/L), conversion ratio (97.4 ± 0.2%) and productivity (1.02 ± 0.01 g/L h) within 8 h. More importantly, it specifically catalyzed the oxidation of l-phenylalanine with racemic null,l-phenylalanine mixture as substrate. In general, these properties rendered it a useful catalyst in several industrial manufacturers.Download high-res image (144KB)Download full-size image

•The actinidin from cultivar Xuxiang showed high protease and milk-clotting activity.•The actinidin showed favorable tenderization effect on both pork and rabbit muscle.•The actinidin could be a promising protease for production of ACE-inhibitory peptides.Protease activity, milk-clotting activity and protein pattern were compared among seven Chinese kiwifruit cultivars. The actinidin from cultivar Xuxiang exhibited highest protease activity and milk-clotting activity. The purified actinidin from cultivar Xuxiang was a cysteine protease with an optimal pH and temperature of 3.5 and 40 °C, respectively. Improved tenderness was observed with pork and rabbit muscle after actinidin treatment. The shear force was reduced by more than half with pork and rabbit muscle using the purified actinidin at a dosage of 0.5 mg/100 g muscle. Furthermore, angiotensin I-converting enzyme (ACE) inhibitory peptides were produced from five plant-derived proteins using actinidin. The yield of peptides varied from 7.2% to 14.2%, and the ACE inhibitory rates ranged from 71.1% to 88.3%. The results indicate that actinidin could be used as a promising protease for meat tenderization and ACE inhibitory peptides production.

•A novel chitinase gene from Paenicibacillus barengoltzii was cloned and expressed.•The recombinant chitinase was purified and biochemically characterized.•The hydrolysis properties of chitinase on chitin materials were evaluated.•The chitinase hydrolyzed colloidal chitin to yield mainly N-acetyl chitobiose.•The enzyme has great potential in N-acetyl chitobiose production.A novel chitinase gene (PbChi70) from a marine bacterium Paenicibacillus barengoltzii was cloned and functionally expressed in Escherichia coli. The recombinant enzyme (PbChi70) was purified to homogeneity with a recovery yield of 51.9%. The molecular mass of purified enzyme was estimated to be 70.0 kDa by SDS–PAGE. PbChi70 displayed maximal activity at pH 5.5 and 55 °C, respectively. It exhibited strict substrate specificity for colloidal chitin, glycol chitin, powdery chitin, and N-acetyl chitooligosaccharides with degrees of polymerization above three. The enzyme exhibited an endo-type cleavage pattern and hydrolyzed colloidal chitin to yield mainly (GlcNAc)2. Furthermore, colloidal chitin was hydrolyzed by PbChi70 to produce 21.6 mg mL−1 (GlcNAc)2 with the highest conversion yield of 89.5% (w/w). (GlcNAc)2 was further separated by an active charcoal column with a purity of 99% and a final yield of 61%. The unique enzymatic properties of the chitinase may make it a good candidate for (GlcNAc)2 production.

•90 plant resources were investigated for the distribution of protease activities.•Proteases of various types may be developed from kiwifruit, broccoli, ginger and leek.•Novel proteases may be discovered from red pepper.In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10 U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8 U/g), broccoli (16.9 U/g), ginger (16.6 U/g), leek (32.7 U/g) and red pepper (15.8 U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.

A novel chitinase gene (RmChi44) from Rhizomucor miehei was cloned and expressed in Escherichia coli as an intracellular soluble and active protein. The recombinant chitinase (RmChi44) was purified to homogeneity and biochemically characterized. The molecular mass of RmChi44 was estimated to be 44.6 kDa on SDS-PAGE. RmChi44 displayed an acidic pH optimum of 4.5 and was stable within pH 4.5–9.0. The optimal temperature of RmChi44 was found to be 50 °C. The Km values of RmChi44 for colloidal chitin and glycol chitin were 4.02 and 1.55 mg/mL, respectively. RmChi44 hydrolyzed colloidal chitin to yield mainly N-acetyl chitobiose, exhibiting an exotype cleavage pattern. Moreover, the enzyme displayed β-N-acetylglucosaminidase activity, splitting N-acetyl COSs with degree of polymerization (DP) 2–5 into their monomer. In addition, RmChi44 showed antifungal activity against some phytopathogenic fungi. This is the first report on an exochitinase showing β-N-acetylglucosaminidase activity and antifungal activity from Rhizomucor species.

A glycoside hydrolase (GH) family 17 β-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other β-1,3-glucanases, despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH family 17 β-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a β-1,3-glucanase. Mutant E158A exhibited high hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold higher than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-β-1,3-glucanase. Our study provides novel insight into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.

•Rice residue protein was hydrolyzed by multiple proteases to produce antioxidant peptides.•The synergetic effect of antioxidant peptides was investigated and proved to be significant.•The synthetic peptides showed excellent stability against simulated gastrointestinal proteases.•The synthetic peptides may be potential candidates for oxidative stress therapy.Multiple proteases were optimized to hydrolyze the rice residue protein (RRP) to produce novel antioxidant peptides. An antioxidant peptide fraction (RRPB3) with IC50 of 0.25 mg/ml was purified from the RRP hydrolysate using membrane ultrafiltration followed by size exclusion chromatography and reversed-phase FPLC. RRPB3 was found to include four peptides (RRPB3 I–IV) and their amino acid sequences were RPNYTDA (835.9 Da), TSQLLSDQ (891.0 Da), TRTGDPFF (940.0 Da) and NFHPQ (641.7 Da), respectively. Furthermore, four peptides were chemically synthesized and their antioxidant activities were assessed by DPPH radical scavenging, ABTS radical scavenging assay and FRAP-Fe3+ reducing assay, respectively. Both RRPB3 I and III showed synergistic antioxidant activity compared to each of them used alone. All four synthetic peptides showed excellent stability against simulated gastrointestinal proteases. Therefore, the peptides isolated from RRP may be used as potential antioxidants in the food and drug industries.

•A novel acidic cutinase from Thielavia terristris was purified and characterized.•The enzyme exhibited good thermal and pH stability.•The enzyme displayed strong stability in the presence of organic solvents.•The enzyme efficiently synthesized flavor ester butyl butyrate under non-aqueous conditions.•The enzyme is a potential candidate in flavor esters-producing industries.An acidic cutinase (TtcutB) from Thielavia terrestris CAU709 was purified to apparent homogeneity with 983 U mg−1 specific activity. The molecular mass of the enzyme was estimated to be 27.3 and 27.9 kDa by SDS–PAGE and gel filtration, respectively. A peptide sequence homology search revealed no homologous cutinases from T. terrestris, except for one putative cutinase gene (XP003656017.1), indicating that TtcutB is a novel enzyme. TtcutB exhibited an acidic pH optimum of 4.0, and stability at pH 2.5–10.5. Optimal activity was at 55 °C, it was stable up to 65 °C, and retained over 30% activity at 0 °C. Km values toward p-nitrophenyl (pNP) acetate, pNP-butyrate and pNP-caproate were 8.3, 1.1 and 0.88 mM, respectively. The cutinase exhibited strong synthetic activity on flavor ester butyl butyrate under non-aqueous environment, and the highest esterification efficiency of 95% was observed under the optimized reaction conditions. The enzyme’s unique biochemical properties suggest great potential in flavor esters-producing industries.

•Novel β-glucosidase gene RmBglu3B cloned from Rhizomucor miehei and expressed in E. coli.•Optimal pH and temperature of purified RmBglu3B: 5.0 and 50 °C, respectively.•RmBglu3B exhibits a broad range of substrate specificity.•RmBglu3B with exo-β-1,3-glucanase activity preferentially hydrolyzes β-1,3-linked glucosides.•RmBglu3B exhibits high transglycosylation activity, and can produce gentiobiose.A novel β-glucosidase gene, designated RmBglu3B, was cloned from the thermophilic fungus, Rhizomucor miehei CAU432. Its 2196-bp open reading frame encoded 731 amino acids. Its deduced amino-acid sequence showed highest identity (66%) with a glycoside hydrolase family 3 β-glucosidase from R. miehei NRRL5382. RmBglu3B was successfully expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity with 18.2-fold purification and 59% recovery yield. Molecular masses of 76.5 kDa, by SDS–PAGE, and 66.4 kDa, by gel filtration, suggested that it is a monomer. Optimal pH and temperature of the purified enzyme were 5.0 and 50 °C, respectively. RmBglu3B exhibited a broad range of substrate specificity, catalyzing the cleavage of β-1,2, β-1,3, β-1,4 and β-1,6 linkages, in various oligosaccharides, to liberate glucose. RmBglu3B also showed relatively high activity (19.1 U/mg) toward laminaran and transglycosylation activity, enabling gentiobiose production. This enzyme is a potential candidate for several industrial applications.

A novel exochitinase gene (Echi47) was directly cloned from the pig fecal environment DNA using the genomic walking PCR technique and expressed in Escherichia coli BL21 (DE3). Echi47 has an open reading frame (ORF) of 1,161 bp encoding 386 amino acids. The amino acid sequence of Echi47 showed 36% identity with that of chitinase from Coprinellus congregatus. The recombinant exochitinase was purified with specific activity toward colloidal chitin of 6.84 U/mg. Echi47 was optimally active at pH 5.0 and 40 °C, respectively. When colloidal chitin was used as substrate, N-acetylchitobiose [(GlcNAc)2] was mostly produced at the initial stage, suggesting that it is an exochitinase. Echi47 exhibited excellent resistance to pepsin, trypsin, proteinase K, and flavor protease. Under simulated alimentary tract conditions, Echi47 was stable and active, releasing 21.1 mg of N-acetylchitooligosaccharides from 80 mg of colloidal chitin. These properties make Echi47 a potential additive in the food and feed industries.

A novel gene (designated as RmArase) encoding endo-1,5-α-l-arabinanase from a thermophilic fungus Rhizomucor miehei was cloned and expressed in Escherichia coli. The gene had an open reading frame (ORF) of 930 base pairs (bp) encoding 309 amino acids. The amino acid sequence shared highest identity (56%) with a glycoside hydrolase (GH) family 43 endo-1,5-α-l-arabinase from Bacillus subtilis and low identity (35%) with the endo-1,5-α-l-arabinase from Aspergillus niger. The recombinant endo-1,5-α-l-arabinase (RmArase) was purified to homogeneity with a molecular mass of 40.6 kDa. The purified enzyme had a specific activity of 109 units/mg. The optimal temperature and pH of RmArase were determined to be 55 °C and 5.5, respectively. It was stable up to 45 °C and within pH 5.0–8.5. The Km values of RmArase toward debranched arabinan and sugar beet arabinan were 5.8 and 27.5 mg/mL, respectively. RmArase efficiently degraded arabinans to yield and arabinobiose and arabinose as major end products, which was different from most other endo-1,5-α-l-arabinases. The synergistic action of RmArase and the pectinase could significantly improve the degradation of sugar beet pulp. These properties make RmArase useful in several industries.

To characterize a novel α-glucosidase from the thermophilic fungus Malbranchea cinnamomea.The enzyme was purified to homogeneity with purification fold of 40 and a recovery of 7.2 %. It was a monomer with molecular mass of 65.7 kDa on SDS-PAGE. It was optimally active at pH 6 and 50 °C (measured over 10 min) and exhibited a wide range of substrate specificity with the highest specific activity of 47.4 U mg−1 for p-nitrophenyl α-d-glucopyranoside (pNPGlu) followed by isomaltose, panose and sucrose, suggesting that the enzyme belongs to the type I α-glucosidases. The Km values of the α-glucosidase for pNPGlu and isomaltose were 1.1 and 19.3 mM, respectively.Because of its unique properties, the α-glucosidase may have a potential in several industrial applications.

Bacillus subtilis LM 4–2, a Gram-positive bacterium was isolated from a molybdenum mine in Luoyang city. Due to its strong resistance to molybdate and potential utilization in bioremediation of molybdate-polluted area, we describe the features of this organism, as well as its complete genome sequence and annotation. The genome was composed of a circular 4,069,266 bp chromosome with average GC content of 43.83 %, which included 4149 predicted ORFs and 116 RNA genes. Additionally, 687 transporter-coding and 116 redox protein-coding genes were identified in the strain LM 4–2 genome.

•A fungal glycogen branching enzyme from Rhizomucor miehei was cloned and expressed.•The enzyme was characterised and displayed excellent cold adaptation.•The enzyme exhibited ten times higher activity toward amylose than amylopectin.•The enzyme was found to improve bread quality and increase its shelf life.A gene (RmGBE) encoding a glycogen branching enzyme from Rhizomucor miehei was cloned into the pET28a (+) vector and expressed in Escherichia coli, and biochemically analysed. RmGBE had an open reading frame of 2097 bp encoding 698 amino acid residues. The purified enzyme was a monomer of 78.1 kDa. RmGBE was optimally active at 25 °C and pH 7.5. It displayed excellent cold adaptation over a low temperature range of 10–30 °C, retaining over 85% of its relative activity. RmGBE showed the highest specificity to amylose, about ten times higher than to amylopectin. Addition of RmGBE to wheat bread resulted in a 26% increase in specific volume and a 38% decrease in crumb firmness in comparison with the control. Besides, the retrogradation of bread was significantly retarded along with the enzyme reaction. These properties make RmGBE highly useful in the food and starch industries.

A novel β-N-acetylglucosaminidase gene (RmNag) from Rhizomucor miehei was cloned and expressed in Escherichia coli. RmNag shares the highest identity of 37% with a putative β-N-acetylglucosaminidase from Aspergillus clavatus. The recombinant enzyme was purified to homogeneity. The optimal pH and temperature of RmNag were pH 6.5 and 50 °C, respectively. It was stable in the pH range 6.0–8.0 and at temperatures below 45 °C. RmNag exhibited strict substrate specificity for p-nitrophenyl β-N-acetylglucosaminide (pNP-GlcNAc) and N-acetyl chitooligosaccharides. The apparent Km of RmNag toward pNP-GlcNAc was 0.13 mM. The purified enzyme displayed an exo-type manner as it released the only end product of GlcNAc from all the tested N-acetyl chitooligosaccharides. Besides, RmNag exhibited relatively high N-acetyl-β-d-glucosaminide tolerance with an inhibition constant Ki value of 9.68 mM. The excellent properties may give the enzyme great potential in industries. This is the first report on a glycoside hydrolyase family 3 β-N-acetylglucosaminidase from a fungus.

Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis were investigated in the present study. A newly isolated R. microsporus var. rhizopodiformis F518 produced a high level of milk-clotting activity (1,001 SU/mL). A chymosin from the fungus was purified 3.66-fold with a recovery yield of 33.2 %. The enzyme appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 37.0 kDa. It was optimally active at 60 °C and was stable up to 40 °C. The purified enzyme was an acid protease with an optimum pH of 5.2 and retained 80 % of residual activity within pH 2.0–8.0. The inhibition of 96 and 100 % by pepstatin A at 0.01 and 0.02 mM, respectively, revealed that the enzyme is an aspartic protease. Thus, high milk-clotting activity of the chymosin with good stability will strengthen the potential use of the chymosin as a substitute for calf rennet in cheese manufacturing.

A novel alkaline β-1,3-1,4-glucanase (McLic1) from a thermophilic fungus, Malbranchea cinnamomea, was purified and biochemically characterized. McLic1 was purified to homogeneity with a purification fold of 3.1 and a recovery yield of 3.7 %. The purified enzyme was most active at pH 10.0 and 55 °C, and exhibited a wide range of pH stability (pH 4.0–10.0). McLic1 displayed strict substrate specificity for barley β-glucan, oat β-glucan and lichenan, but did not show activity towards other tested polysaccharides and synthetic p-nitrophenyl derivates, suggesting that it is a specific β-1,3-1,4-glucanase. The Km values for barley β-glucan, oat β-glucan and lichenan were determined to be 0.69, 1.11 and 0.63 mg mL−1, respectively. Moreover, the enzyme was stable in various non ionic surfactants, oxidizing agents and several commercial detergents. Thus, the alkaline β-1,3-1,4-glucanase may have potential in industrial applications, such as detergent, paper and pulp industries.

N-acetyl-β-D-glucosamine (GlcNAc) is widely used as a valuable pharmacological agent and a functional food additive. The traditional chemical process for GlcNAc production has some problems such as high production cost, low yield, and acidic pollution. Hence, to identify a novel chitinase that is suitable for bioconversion of chitin to GlcNAc is of great value.A novel chitinase gene (PbChi74) from Paenibacillus barengoltzii was cloned and heterologously expressed in Escherichia coli as an intracellular soluble protein. The gene has an open reading frame (ORF) of 2,163 bp encoding 720 amino acids. The recombinant chitinase (PbChi74) was purified to apparent homogeneity with a purification fold of 2.2 and a recovery yield of 57.9%. The molecular mass of the purified enzyme was estimated to be 74.6 kDa and 74.3 kDa by SDS-PAGE and gel filtration, respectively. PbChi74 displayed an acidic pH optimum of 4.5 and a temperature optimum of 65°C. The enzyme showed high activity toward colloidal chitin, glycol chitin, N-acetyl chitooligosaccharides, and p-nitrophenyl N-acetyl β-glucosaminide. PbChi74 hydrolyzed colloidal chitin to yield N- acetyl chitobiose [(GlcNAc)2] at the initial stage, which was further converted to its monomer N-acetyl glucosamine (GlcNAc), suggesting that it is an exochitinase with β-N-acetylglucosaminidase activity. The purified PbChi74 coupled with RmNAG (β-N-acetylglucosaminidase from Rhizomucor miehei) was used to convert colloidal chitin to GlcNAc, and GlcNAc was the sole end product at a concentration of 27.8 mg mL-1 with a conversion yield of 92.6%. These results suggest that PbChi74 may have great potential in chitin conversion.The excellent thermostability and hydrolytic properties may give the exochitinase great potential in GlcNAc production from chitin. This is the first report on an exochitinase with N-acetyl-β-D-glucosaminidase activity from Paenibacillus species.

A novel chitinase from the persimmon fruit was isolated, purified and characterised in this report. The Diospyros kaki chitinase (DKC) was found to be a monomer with a molecular mass of 29 kDa. It exhibited optimal activity at pH 4.5 with broad pH stability from pH 4.0–9.0. It has an optimal temperature of 60 °C and thermostable up to 60 °C when incubated for 30 min. The internal peptide sequences of DKC showed similarity with other reported plant chitinases. It has the ability to hydrolyse colloidal chitin into chito-oligomers such as chitotriose, chitobiose and into its monomer N-acetylglucosamine. It can be used to degrade chitin waste into useful products such as chito-oligosacchaarides. DKC exhibited antifungal activity towards pathogenic fungus Trichoderma viride. Chitinases with antifungal property can be used as biocontrol agents replacing chemical fungicides.Highlights► The Diospyros kaki chitinase (DKC) is a monomer with a molecular mass of 29 kDa. ► It has optimal activity at pH 4.5 and 60 °C with broad pH and thermostability. ► Internal peptide sequences of DKC showed similarity with other plant chitinases. ► DKC can hydrolyse colloidal chitin into chito-oligosacchaarides. ► DKC exhibited antifungal activity towards pathogenic fungus Trichoderma viride.

An endo-1,4-β-mannanase gene (RmMan5A) was cloned from the thermophilic fungus Rhizomucor miehei for the first time and expressed in Escherichia coli. The gene had an open reading frame of 1330 bp encoding 378 amino acids and contained four introns. It displayed the highest amino acid sequence identity (42%) with the endo-1,4-β-mannanases from glycoside hydrolase family 5. The purified enzyme was a monomer of 43 kDa. RmMan5A displayed maximum activity at 55 °C and an optimal pH of 7.0. It was thermostable up to 55 °C and alkali-tolerant, displaying excellent stability over a broad pH range of 4.0–10.0, when incubated for 30 min without substrate. The enzyme displayed the highest specificity for locust bean gum (Km = 3.78 mg mL–1), followed by guar gum (Km = 7.75 mg mL–1) and konjac powder (Km = 22.7 mg mL–1). RmMan5A hydrolyzed locust bean gum and konjac powder yielding mannobiose, mannotriose, and a mixture of various mannose-linked oligosaccharides. It was confirmed to be a true endo-acting β-1,4-mannanase, which showed requirement of four mannose residues for hydrolysis, and was also capable of catalyzing transglycosylation reactions. These properties make RmMan5A highly useful in the food/feed, paper and pulp, and detergent industries.

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH 6.5 and 65 °C, RmXEG12B at pH 5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U mg−1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β-d-Glc residue, X is a xylosylated β-d-Glc residue, and L is a β-d-Glc residue substituted by xylosyl-galactose).

A low molecular mass cutinase (designated TtcutA) from Thielavia terrestris was purified and biochemically characterized. The thermophilic fungus T. terrestris CAU709 secreted a highly active cutinase (90.4 U ml−1) in fermentation broth containing wheat bran as the carbon source. The cutinase was purified 19-fold with a recovery yield of 4.8 %. The molecular mass of the purified TtcutA was determined as 25.3 and 22.8 kDa using SDS-PAGE and gel filtration, respectively. TtcutA displayed optimal activity at pH 4.0 and 50 °C. It was highly stable up to 65 °C and in the broad pH range 2.5–10.5. Extreme stability in high concentrations (80 %, v/v) of solvents such as methanol, ethanol, acetone, acetonitrile, isopropanol, and dimethyl sulfoxide was observed for the enzyme. The Km values for this enzyme towards p-nitrophenyl (pNP) acetate, pNP butyrate, and pNP caproate were 7.7, 1.0, and 0.52 mM, respectively. TtcutA was able to efficiently degrade various ester polymers, including cutin, polyethylene terephthalate (PET), polycaprolactone (PCL), and poly(butylene succinate) (PBS) at hydrolytic rates of 3 μmol h−1 mg−1 protein, 1.1 mg h−1 mg−1 protein, 203.6 mg h−1 mg−1 protein, and 56.4 mg h−1 mg−1 protein, respectively. Because of these unique biochemical properties, TtcutA of T.terrestris may be useful in various industrial applications in the future.

A novel α-amylase (McAmyA) from the thermophilic fungus, Malbranchea cinnamomea was purified, characterized and crystallized in the present study. McAmyA was purified to apparent homogeneity with a molecular mass of 60.3 kDa on SDS-PAGE. The enzyme exhibited maximal activity at pH 6.5 and was stable within pH 5.0–10.0. It was most active at 65 °C and was stable up to 50 °C. McAmyA was capable of hydrolyzing amylose, starch, amylopectin, pullulan, cyclodextrins and maltooligosaccharides. The full-length cDNA of an α-amylase gene (McAmyA) from the strain was cloned. McAmyA consisted of a 1,476-bp open reading frame encoding 492 amino acids. It displayed the highest amino acid sequence homology (less than 60 %) with the reported α-amylases. The crystal structure of McAmyA was solved at a resolution of 2.25 Å (PDB code 3VM7). The overall structure of McAmyA reveals three domains with ten α helices and 14 β strands, and the putative catalytic residues are positioned at domain A with somewhat different secondary structural circumstances compared with typical α-amylases.

A novel β-mannanase gene (CsMan5A) was cloned from Chaetomium sp. CQ31 and expressed in Pichia pastoris. It had an open reading frame of 1251 bp encoding 416 amino acids and contained two introns. The deduced amino acid sequence shared the highest similarity (73%) with the β-mannanase from Emericella nidulans and belongs to glycosyl hydrolase family 5. The recombinant β-mannanase (CsMan5A) was secreted at extremely high levels of 50,030 U mL−1 and 6.1 mg mL−1 in high cell density fermentor. The purified enzyme was optimally active at pH 5.0 and 65 °C and displayed broad pH stability (pH 5.0–11.0) and exhibited specificity towards locust bean gum (Km = 3.1 mg mL−1), guar gum (Km = 9.3 mg mL−1) and konjac powder (Km = 10.5 mg mL−1). It efficiently degraded mannan polysaccharides into mannose and mannooligosacccharides, and also hydrolyzed mannotriose and mannotetraose. These properties make CsMan5A highly useful in food, feed and paper/pulp industries.Highlights► A novel β-mannanase gene (CsMan5A) was cloned from Chaetomium species. ► CsMan5A was expressed extracellularly in Pichia pastoris at very high levels. ► CsMan5A displayed good thermostability (55 °C) and pH stability (5.0–11.0). ► CsMan5A could efficiently degrade mannan polymers and mannooligosaccharides. ► CsMan5A is suitable for application in the food, feed and paper/pulp industries.

Production, purification, and characterization of a novel β-1,3–1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3–1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The Km values of purified β-1,3–1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3–1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3–1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.

A novel chitinase gene (PtChiA) from the thermophilic fungus Paecilomyces thermophila was cloned and expressed in Escherichia coli as an intracellular soluble protein. The gene sequence alignment indicates that PtChiA belongs to glycoside hydrolase (GH) family 18 and has an open reading frame comprising of 1473 bp nucleotide sequences with five introns. PtChiA encodes 400 amino acids without any predicted signal peptide. PtChiA was purified by Ni-IDA chromatography. It displayed an acidic optimum pH of 4.5 and broad pH stability (pH 4.0–10.5). The enzyme exhibited an optimal temperature of 50 °C and was stable up to 40 °C. PtChiA was strongly inhibited by anionic detergent SDS, and also by metal ions Hg2+ and Mn2+. It did not exhibit any antifungal activity against pathogenic fungi. It has the ability to hydrolyze colloidal chitin into chito-oligomers suggesting its use in conversion of chitin waste into chito-oligosaccharides.Graphical abstractHighlights► PtChiA, a new chitinase gene from Paecilomyces thermophila was cloned. ► PtChiA belongs to GH family 18, comprises of 1473 bp and encodes 400 amino acids. ► PtChiA had an acidic optimum pH of 4.5 and broad pH stability of pH 4.0–10.5. ► It had an optimal temperature of 50 °C and was stable up to 40 °C. ► It has hydrolyzed colloidal chitin into chito-oligomers and can be used in industry.

A xylanase gene from Paecilomyces thermophila was functionally expressed in Pichia pastoris. The recombinant xylanase (xynA) was predominantly extracellular; in a 5 l fermentor culture, the total extracellular protein was 8.1 g l−1 with an activity of 52,940 U ml−1. The enzyme was purified to homogeneity with a recovery of 48 %. The recombinant xynA was optimally active at 75 °C, as measured over 10 min, and at pH 7. The enzyme was stable up to 80 °C for 30 min. It hydrolyzed birchwood xylan, beechwood xylan and xylooligosaccharides to produce xylobiose and xylotriose as the main products.

A novel chitinase was isolated and purified to its homogeneity from pomegranate juice by a combination of ammonium sulphate precipitation and ion-exchange chromatography. The pomegranate juice chitinase (PJC) was purified to specific activity of 14.5 U/mg and a recovery of 34%. The monomeric protein migrated on SDS–PAGE at 29 kDa. The enzyme was found to be glycosylated (7.2%). It exhibited optimal activity at pH 4.5 and 70 °C. The enzyme was stable in the pH range 3.0–9.0 and up to 65 °C. The internal peptide sequence results suggest that the purified PJC shared high homology with class III chitinases of other known plant chitinases. The purified enzyme could hydrolyse colloidal chitin to its oligomers. It did not exhibit any antifungal activity.Research highlights► A novel, monomeric chitinase was purified and characterised from pomegranate juice. ► It was glycosylated, with an optimal pH (pH 4.5) and optimal temperature (70 °C). ► The enzyme was thermostable and exhibited stability in a broad pH range. ► Internal peptide sequence shows high homology to GH family 18 class III chitinases. ► It possesses the ability to hydrolyse colloidal chitin into its oligomers.

A β-galactosidase gene (TM_1195) of Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant β-galactosidase (BgalC), belonging to glycosyl hydrolase (GH) family 42, was purified to homogeneity with 23.4-fold purification and a recovery of 36.6%. Its molecular mass was estimated to be 78 kDa by SDS–PAGE. BgalC exhibited maximum activity at an optimal pH of 5.5 and an optimum temperature of 80 °C. The enzyme displayed important properties, such as stability over a broad pH range of 5.0–9.0 and thermostability up to 75 °C. Km values of BgalC for p-nitrophenyl-β-galactopyranoside (pNPGal), o-nitrophenyl-β-galactopyranoside (oNPGal) and lactose were 1.21, 7.31 and 6.5 mM, respectively. BgalC was efficient in complete removal of lactose from milk. BgalC is significantly one of the few β-galactosidases from family 42 displaying significant hydrolysis of lactose. These properties make BgalC an ideal candidate for commercial use, in the production of lactose-free milk.

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent Km values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.

A novel lectin (AML) was isolated from the roots of a Chinese herb Astragalus membranaceus (Fisch.) Bunge, using a combination of ammonium sulphate fractionation and affinity chromatography. AML was found to be a monomeric protein with a molecular mass of 31.5 kDa. Biochemical characterisation revealed that it is a glycoprotein containing 10.7% neutral sugars. The N-terminus of AML was blocked but amino acid sequences of internal tryptic peptides showed moderate sequence identities with some other known lectins. Amongst the various carbohydrates tested, the lectin was best inhibited by d-galactose and its derivatives with pronounced preference for o-nitrophenyl-β-d-galactopyranoside (8.3 mM). The lectin was stable in the pH range of pH 5.0–12.0 and temperatures up to 55 °C for 30 min. AML inhibited the proliferation of HeLa and K562 cell lines. Thus, the lectin displays a high potential for antitumour activity.

A novel thermophilic xylanase-producing fungus, Chaetomium sp. CQ31 produced 131 U ml−1 of xylanase when grown on a medium containing corncob (3.5%, w/v) at 37 °C for 6 days. A low molecular xylanase was purified 6.5-fold to homogeneity with a recovery yield of 17.5%. Its molecular mass was estimated to be 25.1 kDa by SDS–PAGE. The xylanase had an optimum pH of 7.5, and its optimal temperature was 65 °C. Apparent Km values of the xylanase for birchwood, beechwood and oat-spelt xylan were 1.3, 0.86 and 4.4 mg/ml, respectively. The influence of this xylanase on the quality of Chinese steamed bread (CSB) was further studied. Addition of xylanase in the range 2.5–5.0 ppm caused a 20–24.5% increase in specific volume over the control and remarkable decrease (8.9–24.2%) in firmness was also noticed. This is the first report on the purification, characterisation and application of a xylanase from Chaetomium sp. CQ31.

The aim of this study is to investigate production of l-lactic acid from sucrose and corncob hydrolysate by the newly isolated R. oryzae GY18. R. oryzae GY18 was capable of utilizing sucrose as a sole source, producing 97.5 g l−1l-lactic acid from 120 g l−1 sucrose. In addition, the strain was also efficiently able to utilize glucose and/or xylose to produce high yields of l-lactic acid. It was capable of producing up to 115 and 54.2 g l−1 lactic acid with yields of up to 0.81 g g−1 glucose and 0.90 g g−1 xylose, respectively. Corncob hydrolysates obtained by dilute acid hydrolysis and enzymatic hydrolysis of the cellulose-enriched residue were used for lactic acid production by R. oryzae GY18. A yield of 355 g lactic acid per kg corncobs was obtained after 72 h incubation. Therefore, sucrose and corncobs could serve as potential sources of raw materials for efficient production of lactic acid by R. oryzae GY18.

In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l−1 with an activity of 55,300 U ml−1 in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS–PAGE. It was optimally active at pH 7.0 and a temperature of 70°C. Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp.

The β-glucosidase from Paecilomyces thermophila J18 was found to be capable of hydrolysing daidzin and genistin in a previous study. This report further evaluated the thermostability and hydrolysis of soybean isoflavone glycosides. The enzyme was found to be very stable at 50 °C, and retained more than 95% of its initial activity after 8 h at 50 °C. It converted isoflavone glycosides, in soybean flour extract and soybean embryo extract, to their aglycones, resulting in more than 93% of hydrolysis of three isoflavone glycosides (namely, daidzin, genistin and glycitin) after 4 h of incubation. Also, addition of the β-glucosidase greatly increased the contents of isoflavone aglycones in the suspended soybean flour and soymilk. The results indicate that the thermostable β-glucosidase may be used to increase the isoflavone aglycones in soy products. This is the first report on the potential application of fungal β-glucosidases for converting isoflavone glycosides to their aglycones in soy products.

A β-galactosidase gene (TM_0310) of Thermotoga maritima MSB8 was expressed in Escherichia coli. The recombinant β-galactosidase (designated BgalB) was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. BgalB belongs to the glycoside hydrolase family 42. Its molecular mass was estimated to be 78 kDa and 76 kDa by SDS–PAGE and gel filtration, respectively. The enzyme was optimum at pH 5.5, and it was quite stable over the pH range 5.0–11.4 at 70 °C. It was optimally active at 80 °C and was stable up to 75 °C. Besides, BgalB exhibited broad substrate specificity with a preference for p-nitrophenyl-β-galactopyranoside (pNPGal). Km values of the purified enzyme for pNPGal, o-nitrophenyl-β-galactopyranoside (oNPGal) and pNP-β-fucopyranoside were 2.7 mM, 12.5 mM and 1.4 mM, respectively. These properties make this enzyme an interesting candidate for biotechnological applications. This is the first report of the family 42 β-galactosidases from T. maritima.

Cells of the thermophilic Bacillus subtilis WY34 were immobilized on various formaldehyde-activated polymer membranes and the immobilized cells were used for the production of thermostable mannanase in flasks. The results showed that polyethersulfone membranes (PES) and nylon-6 membranes were the most suitable supports for cell immobilization to produce the mannanase. Moreover, PES and nylon-6 membranes immobilized cells provided 1.78- and 1.74-fold higher mannanase activity compared to the control after 4 days of cultivation, respectively. The immobilized cells on PES and nylon-6 membranes had good stability and retained 131.5 and 114.3% of ability of enzyme production even after six cycles of repeated batch fermentation, respectively. Active cell growth was observed by scanning electron microscopy (SEM) after 16 days (four cycles) repeated batch cultivation. Therefore, the membrane-immobilized cells of B. subtilis WY34 can be proposed as an effective biocatalyst for repeated usage for production of the thermostable mannanase.

The purification and characterization of a novel extracellular β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila J18 were studied. The strain produced the maximum level of extracellular β-glucanase (135.6 U mL−1) when grown in a medium containing corncob (5%, w/v) at 50 °C for 4 days. The crude enzyme solution was purified by 122.5-fold with an apparent homogeneity and a recovery yield of 8.9%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 38.6 kDa. The molecular masses were 34.6 kDa and 31692.9 Da when detected by gel filtration and mass spectrometry, respectively, suggesting that it is a monomeric protein. The enzyme was a glycoprotein with a carbohydrate content of 19.0% (w/w). Its N-terminal sequence of 10 amino acid residues was determined as H2N−A(?)GYVSNIVVN. The purified enzyme was optimally active at pH 7.0 and 70 °C. It was stable within pH range 4.0−10.0 and up to 65 °C, respectively. Substrate specificity studies revealed that the enzyme is a true β-1,3-1,4-d-glucanase. The Km values determined for barley β-d-glucan and lichenan were 2.46 and 1.82 mg mL−1, respectively. The enzyme hydrolyzed barley β-d-glucan and lichenan to yield bisaccharide, trisaccharide, and tetrasaccharide as the main products. Circular dichroism studies indicated that the protein contains 28% α-helix, 24% β-sheet, and 48% random coil. Circular dichroism spectroscopy is also used to investigate the thermostability of the purified enzyme. This is the first report on the purification and characterization of a β-1,3-1,4-glucanase from Paecilomyces sp. These properties make the enzyme highly suitable for industrial applications.

The purification and characterization of a novel extracellular β-glucosidase from Paecilomyces thermophila J18 was studied. The β-glucosidase was purified to 105-fold apparent homogeneity with a recovery yield of 21.7% by DEAE 52 and Sephacryl S-200 chromatographies. Its molecular masses were 116 and 197 kDa when detected by SDS-PAGE and gel filtration, respectively. It was a homodimeric glycoprotein with a carbohydrate content of 82.3%. The purified enzyme exhibited an optimal activity at 75 °C and pH 6.2. It was stable up to 65 °C and in the pH range of 5.0–8.5. The enzyme exhibited a broad substrate specificity and significantly hydrolyzed p-nitrophenyl-β-d-glucopyranoside (pNPG), cellobiose, gentiobiose, sophorose, amygdalin, salicin, daidzin, and genistin. Moreover, it displayed substantial activity on β-glucans such as laminarin and lichenan, indicating that the enzyme has some exoglucanase activity. The rate of glucose released by the purified enzyme from cellooligosaccharides with a degree of polymerization (DP ) ranging between 2 and 5 decreased with increasing chain length. Glucose and glucono-δ-lactone inhibited the β-glucosidase competitively with Ki values of 73 and 0.49 mM, respectively. The β-glucosidase hydrolyzed pNPG, cellobiose, gentiobiose, sophorose, salicin, and amygdalin, exhibiting apparent Km values of 0.26, 0.65, 0.77, 1.06, 1.39, and 1.45 mM, respectively. Besides, the enzyme showed transglycosylation activity, producing oligosaccharides with higher DP than the substrates when cellooligosaccharides were hydrolyzed. These properties make this β-glucosidase useful for various biotechnological applications.Keywords: Characterization; exoglucanase; Paecilomyces thermophila; purification; thermostable; transglycosylation; β-glucosidase;

A β-mannosidase gene (TM1624) from Thermotoga maritima MSB8, the hyperthermophilic bacterium was expressed as a soluble C-terminal His-tagged protein in E. coli. Heat treatment of cell lysate followed by metal affinity- and anion-exchange chromatographic techniques the recombinant β-mannosidase was purified to apparent homogeneity. The recovery of the purified protein from the crude lysate was 23%. Results of SDS-PAGE analysis (96.8 kDa) and gel permeation chromatography (93.2 kDa) indicated monomeric nature of the β-mannosidase protein. The enzyme displayed its maximal activity at pH 7.0 with pH stability over a range of pH 5.0–9.0. Similarly, the optimum temperature for maximal activity was found to be 95 °C and thermostability of up to 85 °C. The substrate specificity and kinetics of the enzyme was studied using different mannooligosaccharides and pNP-β-d-mannopyranoside. The Km value of the purified enzyme for pNPM was 0.49 mM. Different mannooligosaccharides tested as enzyme substrates were hydrolysed in an exo-wise manner when checked by thin-layer chromatography (TLC). The enzyme also exhibited transglycosidase activity when the reaction was carried out with 10% (w/v) of mannobiose in the presence of alcohols or galactose. Because of extreme thermostability and transglyocosylation properties of β-mannosidase from T. maritima, the enzyme may be of industrial applications in future. This is the first report on the purification and characterization of a β-mannosidase from T. maritima.

•A novel esterase gene from Rhizomucor miehei was cloned and expressed in E. coli.•The recombinant esterase was purified and biochemically characterized.•The enzyme was highly stable in high concentrations of organic solvents.•The esterase efficiently hydrolyzed sterically hindered esters of tertiary alcohols.•The study presents a novel fungal HSL family esterase with potential applications.A novel esterase gene (designated RmEstB) from the thermophilic fungus Rhizomucor miehei was cloned and functionally expressed in Escherichia coli. Sequence analysis revealed a 960-bp open reading frame encoding a protein of 319 amino acids. The deduced protein sequence contained an HGGG motif, suggesting that the enzyme is a hormone-sensitive lipase (HSL) family esterase. It showed highest identity of 52% with the esterase from Pseudomonas mandelii. The recombinant esterase was purified to homogeneity at 5.1-fold purification with a recovery yield of 85%. The molecular mass of RmEstB was estimated to be 37 kDa by SDS-PAGE. RmEstB was most active at pH 7.5 and 50 °C. The enzyme was highly stable in the presence of 30% ethanol, methanol, acetone, isopropanol, dimethyl sulfoxide and acetonitrile. RmEstB showed a broad range of substrate specificities toward various p-nitrophenol (pNP) esters (C2–C10) and triglycerides (C2–C6), with the highest specific activities obtained for pNP acetate (255 U/mg) and triacetin (1330 U/mg), respectively. In addition, RmEstB efficiently catalyzed the hydrolysis of sterically hindered esters of tertiary alcohols. This study presents a novel fungal HSL family esterase with potential for some industrial applications.Download full-size image

A β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris under the control of the AOX1 promoter. The coding region of 3036 bp encoded a protein of 1011 amino acids with a deduced molecular mass of 108.7 kDa. The PaGalA without the signal peptide was cloned into a vector pPIC9K and was expressed successfully in P. pastoris as active extracellular β-galactosidase. The recombinant β-galactosidase (PaGalA) was secreted into the medium at an extremely high levels of 22 mg ml−1 having an activity of 9500 U ml−1 from high density fermentation culture, which is by far the highest yield obtained for a β-galactosidase. The purified enzyme with a high specific activity of 820 U mg−1 had a molecular mass of 120 kDa on SDS-PAGE. PaGalA was optimally active at pH 4.5 and a temperature of 60 °C. The recombinant β-galactosidase was able to hydrolyze lactose efficiently at pH 5.0 and 50 °C. It also possessed transglycosylation activities at high concentrations of lactose. PaGalA exhibited better lactose hydrolysis efficiency in whey than two other widely used commercial lactases. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.Graphical abstractA β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.Download full-size imageResearch highlights▶ High-level expression of a β-galactosidase gene was achieved in Pichia pastoris. ▶ The β-galactosidase was optimally active at pH 4.5 and a temperature of 60 °C. ▶ The enzyme efficiently hydrolyzed lactose lactose present in acid- and sweet-whey.

The effect of the recombinantly produced xylanase B (XynB) from Thermotoga maritima MSB8 on the quality of frozen partially baked bread (FPBB) was investigated. Addition of XynB to wheat flour dough resulted in a significant increase in dough extensibility (L), swelling (G), and a decrease in dough resistance to deformation (P), configuration. Bread crumb characteristics were studied by differential scanning calorimeter (DSC) and dynamic-mechanical analysis (DMA). The results show that addition of XynB leads to improvements in the bread quality of FPBB and retards bread staling compared to the control. The greatest improvements were obtained in specific volume (+35.2%) and crumb firmness (−40.0%). The control FPBB was significantly firmer in texture and higher in amylopectin recrystallization than the bread with XynB. During frozen storage of FPBB with and without XynB for 8 weeks, the crumb firmness increased gradually and the specific volume slightly decreased with the frozen storage time. The ΔH values of freezable water (FW) endothermic transitions increased with frozen storage time for all samples. However, addition of XynB lowered the ΔH values indicating a decrease in FW. Therefore, XynB is useful in improving the quality of FPBB. DMA was also used to monitor the shrinking behavior of the samples. Addition of XynB increased the contraction during chilling but significantly diminished the total shrinking and frozen-state shrinking of the bread crumb during the freezing process.

The second xylanase gene (xynB) from the hyperthermophilic Thermotoga maritima was optimized according to the codon usage of Pichia pastoris and expressed in P. pastoris. The optimized gene (xynBop) shared 77.8% of nucleotide sequence identity with that of native gene. A total of 232 nucleotides were changed and the G + C ratio was simultaneously increased from 42.7% to 43.1%. The recombinant xylanase (XynBop) was secreted into the culture medium that reached a total extracellular protein concentration of 10.1 g l−1 with an activity of 40,020 U ml−1 in 5-l fermentor culture. The recombinant enzyme was optimally active at pH 5.5 and at 100 °C, respectively. The secreted expression level makes the enzyme a good candidate for hyperthermostable xylanase production.Graphical abstractDownload full-size imageHighlights► Hyperthermostable xylanase was expressed in Pichia pastoris by optimization of codon. ► A protein concentration of 10.1 g l−1 was secreted with activity of 40,020 U ml−1. ► The xylanase displayed optimal activity at 100 °C and pH 5.5. ► The P. pastoris is a good candidate for hyperthermostable xylanase production.

A lectin (AMML) from the roots of Astragalus mongholicus was extracted and purified by affinity chromatographic technique. Human cervical carcinoma cell line (HeLa), human osteoblast-like cell line (MG63) and human leukemia cell line (K562) were used to check the effects of AMML on cell proliferation, apoptosis and cell cycle. Maximum growth inhibition (92%) was observed with HeLa cells, followed by K562 cells (84%) and MG63 (48%) cells. Morphological observation showed that AMML-treated HeLa cells displayed outstanding apoptosis characteristics, such as nuclear fragmentation and appearance of membrane-enclosed apoptotic bodies. The apoptosis of HeLa cells was confirmed by flow cytometry using Annexin V/FITC and propidium iodide (PI) staining technique. For the first time we also report a significant cell cycle arrest at S phase of HeLa cells by AMML. Therefore, the present investigation may lead to the possible therapeutic use of Astragalus mongholicus lectin.

An extracellular xylanase produced by Streptomyces matensis DW67 was purified from the culture supernatant by ammonium sulfate precipitation, ion exchange and gel filtration chromatography and characterized. The xylanase was purified to 14.5-fold to homogeneity with a recovery yield of 14.1%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of 21.2 kDa. However, it had a very low apparent molecular mass of 3.3 kDa as determined by gel filtration chromatography. The N-terminal sequence of first 15 amino acid residues was determined as ATTITTNQTGYDGMY. The optimal temperature and pH for purified xylanase was 65 °C and pH 7.0, respectively. The enzyme was stable within the pH range of 4.5–8.0 and was up to 55 °C. The xylanase showed specific activity towards different xylans and no activity towards other substrates tested. Hydrolysis of birchwood xylan by the xylanase yielded xylobiose and xylotriose as principal products. The enzyme hardly hydrolyzed xylobiose and xylotriose, but it could hydrolyze xylotetraose and xylopentaose to produce mainly xylobiose and xylotriose through transglycosylation. These unique properties of the purified xylanase make this enzyme attractive for biotechnological applications, such as bioblenching in paper and pulp industries, production of xylooligosaccharides. This is the first report of the xylanase from S. matensis.

A novel β-glucosidase gene (designated PtBglu3) from Paecilomyces thermophila was cloned and sequenced. PtBglu3 has an open reading frame of 2,557 bp, encoding 858 amino acids with a calculated molecular mass of 90.9 kDa. The amino acid sequence of the mature polypeptide shared the highest identity (70%) to a glycoside hydrolase (GH) family 3 characterized β-glucosidase from Penicillium purpurogenum. PtBglu3 without the signal peptides was cloned into pPIC9K vector and successfully expressed in Pichia pastoris as an active extracellular β-glucosidase (PtBglu3). High activity of 274.4 U/ml was obtained by high cell-density fermentation, which is by far the highest reported yield for β-glucosidase. The recombinant enzyme was purified to homogeneity with 3.3-fold purification and a recovery of 68.5%. The molecular mass of the enzyme was estimated to be 116 kDa by SDS–PAGE, and 198.2 kDa by gel filtration, indicating that it was a dimer. Optimal activity of the purified enzyme was observed at pH 6.0 and 65 °C, and it was stable up to 60 °C. The enzyme exhibited high specific activity toward pNP-β-d-glucopyranoside, cellooligosaccharides, gentiobiose, amygdalin and salicin, and relatively lower activity against lichenan and laminarin. The present results should contribute to improving industrial production of β-glucosidase.Highlights► A novel glycoside hydrolyase family 3 glucosidase gene was cloned and sequenced. ► PtBglu3 was high-level expressed in Pichia pastoris using high cell-density fermentation. ► The enzyme was purified to homogeneity and the purified enzyme was characterized. ► The enzyme exhibited broad substrate specificity.

⿢First β-mannosidase (AcMan5) from Absidia corymbifera was cloned and characterized.⿢AcMan5 is a fungal β-mannosidase belonging to GH family 5.⿢Optimal pH and temperature of AcMan5 were pH 5.5 and 60 °C, respectively.⿢AcMan5 displays high transglycosylation activity for potential applications.A novel glycoside hydrolase family 5 β-mannosidase gene (AcMan5) from the filamentous fungus Absidia corymbifera D1 was cloned and expressed in Escherichia coli. AcMan5 had an open reading frame of 1311 bp encoding 436 amino acids. The β-mannosidase (AcMan5) shared highest amino acid sequence identity (73%) with the β-mannosidase from Rhizomucor miehei. The purified β-mannosidase was a monomer of 51.9 kDa. Its optimal pH and temperature were pH 5.5 and 60 °C, respectively. The enzyme was stable up to 60 °C and within a pH range of 4.0⿿10.5. It was found to be a typical exo-β-mannosidase acting on the non-reducing end of manno-oligosaccharides. In addition, AcMan5 displayed high transglycosylation activity. It could transfer mannose residue to six acceptors ⿿ glucose, galactose, fructose, gentiobiose, laminaribiose and sucrose ⿿ among 20 candidates at low donor concentration in a short time. Due to its hydrolytic and transglycosylation properties, AcMan5 may have potential in several industrial applications. This is the first report on a β-mannosidase from Absidia species.Download high-res image (95KB)Download full-size image

To fulfill the need for acid-tolerant and thermostable β-1,3-1,4-glucanases, an error-prone PCR and DNA-shuffling approach was employed to enhance the activity of thermostable β-1,3-1,4-glucanases from Paecilomyces thermophila (PtLic16A) at acidic pH. Mutant PtLic16AM2 was selected and characterized, and showed optimal activity at pH 5.0, corresponding to an acidic shift of 2.0 pH units relative to the wild-type enzyme. Other properties of PtLic16A such as temperature optimum and substrate specificity that are beneficial for industrial applications did not change. Based on the substituted residues of PtLic16AM2, three site-directed mutations, D56G, D221G and C263S, were designed to study these residues’ roles. The amino acid residues at positions 56 and 263 were found to be important in determining optimal pH activity. Activity of the D221G variant showed no significant difference from the wild-type. Thus, it appears that the change in optimal pH for PtLic16AM2 was mainly caused by the combination of substitutions D56G and C263S. This study provides a β-1,3-1,4-glucanase (PtLic16AM2) with high potential for industrial applications.Highlights► The activity of thermostable β-1,3-1,4-glucanases was enhanced at acidic pH. ► The error-prone PCR and DNA shuffling approach was employed for directed evolution. ► The optimal pH of mutant PtLic16AM2 was shifted from 7.0 to 5.0. ► The change of optimal pH of PtLic16AM2 was mainly caused by D56G and C263S together.

Immobilization of XynB from the hyperthermophilic Thermotoga maritima on nickel-chelate IDA (iminodiacetic acid disodium salt monohydrate)-Eupergit C 250L (Ni-E, nickel-chelate Eupergit C 250L) resulted in high immobilization yield (76.3%), catalytic efficiency (77.9%) and bond protein (96.6%). Thermostability of Ni-E XynB was significantly improved by 1 mol l−1 phosphate buffer (pH 7.0) treatment. Chemical surface modifications were followed by FT-NIR (Fourier transform near infrared) spectroscopy, strongly indicated that a chemical reaction between the amines or some other groups and epoxy groups took place in Eupergit C 250L during immobilization. Characterization of the immobilized XynB was further evaluated. The optimum pH was not affected by immobilization, but the optimum pH range of the immobilized XynB was pH 4.6–6.6 while that of the free XynB was pH 5.6–7.5. The free XynB had an optimum temperature of 90 °C, whereas that of the immobilized XynB was shifted to 100 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Autohydrolysis liquor of corncob (ALC) was used as the substrate for enzymatic hydrolysis by the immobilized XynB in a packed-bed enzyme reactor. The major hydrolysis product is xylobiose. The immobilization procedure developed provides a promising solution for application of xylanases in continuous hydrolysis of ALC.

•A novel thermostable feruloyl esterase was purified and characterized.•The enzyme was stable over a broad pH range and exhibited good thermostability.•The enzyme exhibited strict substrate specificity for methyl ferulate.•The feruloyl esterase gene was cloned and sequenced.A feruloyl esterase from Chaetomium sp. CQ31 was purified and biochemically characterized. The purified feruloyl esterase had a specific activity of 38.6 U/mg. The molecular mass of the enzyme was estimated to be 30.2 kDa by SDS-PAGE, and 29.6 kDa by gel filtration, indicating that the enzyme was a monomer. The optimum pH and temperature of the enzyme were pH 7.5 and 60 °C, respectively. It was stable over a broad pH range of 4.0–10.0, and also exhibited good thermostability. The enzyme displayed strict substrate specificity. The Km and Vmax values for methyl ferulate were 0.98 μmol/min/mg and 42.6 U/mg, respectively. Furthermore, the feruloyl esterase gene was cloned and sequenced. Open reading frame (ORF) of the feruloyl esterase gene (879-bp) encodes 274 amino acids. The deduced amino acid sequence of the feruloyl esterase gene exhibited the highest identity (79%) with that of type B feruloyl esterase from Magnaporthe oryzae.Download full-size image

•A glyoxylate reductase (PtGR) from a fungus was biochemically and structurally characterized.•PtGR forms a homodimer and has a high specificity toward glyoxylate.•PtGR displayed broad pH stability (pH 4.5–10) and an optimal temperature of 50 °C.•PtGR folds with a typical crystal structure of d-2-hydroxy-acid dehydrogenase.•A cationic cluster help PtGR to prefer NADPH rather than NADH as coenzyme.A glyoxylate reductase gene (PtGR) from the fungus Paecilomyces thermophila was cloned and expressed in Escherichia coli. PtGR was biochemically and structurally characterized. PtGR has an open reading frame of 993 bp encoding 330 amino acids. The deduced amino acid sequence has low similarities to the reported glyoxylate reductases. The purified PtGR forms a homodimer. PtGR displayed an optimum pH of 7.5 and broad pH stability (pH 4.5–10). It exhibited an optimal temperature of 50 °C and was stable up to 50 °C. PtGR was found to be highly specific for glyoxylate, but it showed no detectable activity with 4-methyl-2-oxopentanoate, phenylglyoxylate, pyruvate, oxaloacetate and α-ketoglutarate. PtGR prefered NADPH rather than NADH as an electron donor. Moreover, the crystal structure of PtGR was determined at 1.75 Å resolution. The overall structure of apo-PtGR monomer adopts the typical d-2-hydroxy-acid dehydrogenase fold with a “closed” conformation unexpectedly. The coenzyme specificity is provided by a cationic cluster consisting of N184, R185, and N186 structurally. These structural observations could explain its different coenzyme and substrate specificity.