Molecular cloning and anti-fungal effect of endo-β-1,3-glucanase from Thermotoga maritima

A gene encoding an endo-β-1,3-glucanase from Thermotoga maritima MSB8 (TmβG) was cloned and expressed in Escherichia coli. The purified enzyme produced various β-1,3-glucooligosaccharides from soluble laminarin, and mainly β-1,3-glucooligosaccharides smaller than laminaritetrose from insoluble curdlan. The optimum pH and temperature of the enzyme were 5.0 and 80°C, respectively. TmβG inhibited the growth of Candida albicans, which indicates that the enzyme could potentially be used as an anti-fungal agent to control invasive infections.     

Molecular cloning and characterization of a β-glucanase from Piromyces rhizinflatus

Cellulose is the most abundant renewable polysaccharide with a high potential for degradation to useful end products. In nature, most cellulose is produced as crystalline cellulose. Therefore, cellulases with high hydrolytic activity against crystalline cellulose are of great interest. In this study, a crystalline cellulose degradation enzyme was investigated. The cDNA encoding a β-glucanase, CbhYW23-2, was cloned from the ruminal fungus Piromyces rhizinflatus. To examine the enzyme activities, CbhYW23-2 was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis was then employed for the planned statistical optimization of the β-glucanase activities of CbhYW23-2. The optimal conditions for the highest β-glucanase activity of CbhYW23-2 were observed at 46.4°C and pH 6.0. The results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity of CbhYW23-2. CbhYW23-2 also showed hydrolytic activities toward Avicel, carboxymethyl cellulose (CMC), lichenan, and pachyman. The results also proved that the high activity of CbhYW23-2 on crystalline cellulose makes it a promising candidate enzyme for biotechnological and industrial applications.     

Molecular cloning and characterization of a thermostable α-amylase exhibiting an unusually high activity

An α-amylase gene was cloned from the thermophilic bacterium Bacillus subtilis isolated from Indonesian oil palm shell waste. The gene expressed an extracellular enzyme. Optimal hydrolysis conditions for the enzyme were 70°C and pH 6.0. The specific activity of the enzyme was 16.0 kU per mg of protein, which was higher than for other thermostable amylases. Hydrolytic products of the enzyme using starch and glycogen were mainly maltohexaose and maltopentaose. The enzyme had a K _m value of 0.099 mg/mL for amylopectin, more than 10 times lower than for amylose. The catalytic efficiency of the enzyme using amylopectin was 39,200 mL/mg·s and was 3,270 mL/mg·s using amylose. The enzyme liquefied corn starch at pH 5.0, which was successfully converted to glucose using commercial glucoamylase and pullulanase without pH adjustment. The enzyme has advantages for industrial applications.     

Expression and characterization of β-1,3-1,4-glucanase of Aspergillus usamii in Escherichia coli and its application in sourdough bread making

A β-1,3-1,4-glucanase gene (Auglu12A) from Aspergillus usamii was successfully expressed in Escherichia coli BL21(DE3). The recombinant enzyme, reAuglu12A was efficiently purified using the one-step nickel-nitrilotriacetic acid affinity chromatography. The specific activity of reAuglu12A was 694.8 U/mg, with an optimal temperature of 55°C and pH of 5.0. The reAuglu12A exhibited stability at temperatures up to 60°C and within the pH range of 4.0–5.5. The reAuglu12A hydrolytic activity was increased in the presence of metal ions, especially K⁺ and Na⁺, whereas it exhibited a K_m and V_max of 8.35 mg/mL and 1254.02 µmol/min/mg, respectively, toward barley β-glucan at pH 5.0 and 55°C. The addition of reAuglu12A significantly increased the specific volume (p < 0.05) and reduced crumb firmness and chewiness (p < 0.05) of wheat–barley sourdough bread during a 7-day storage period compared to the control. Overall, the quality of wheat–barley sourdough bread was improved after incorporation of reAuglu12A (especially at 3000 U/300 g). These changes were attributed to the synergistic effect of acidification by sourdough and its metabolites which provided a conducive environment for the optimal action of reAuglu12A in the degradation of β-glucans of barley flour in sourdough. This stabilized the dough structure, thereby enhancing the quality, texture, and shelf life of the bread. These findings suggest that reAuglu12A holds promise as a candidate for β-glucanase application in the baking industry.     

Molecular structure of large-scale extracted β-glucan from barley and oat: Identification of a significantly changed block structure in a high β-glucan barley mutant

Health effects of β-glucan are typically related to dose, size and viscosity without taking the specific molecular structure into account. High β-glucan mutant barley, mother barley and oat β-glucans were large-scale extracted by comparable protocols using hot water, enzyme assisted hydrolysis and ethanol precipitation leading to similar molecular masses (200–300 kDa). Multivariate data analysis on all compositional, structural and functional features demonstrated that the main variance among the samples was primarily explained by block structural differences as determined by HPSEC–PAD. In particular the barley high β-glucan mutant proved to exhibit a unique block structure with DP3 and DP4 contributions of: 78.9% and 16.7% as compared to the barley mother (72.1% and 21.4%) and oat (66.1% and 29.1%). This unique block structure was further confirmed by the ¹H NMR determination of the β-1,4 to β-1,3 linkage ratio. Low solubility of the barley samples was potentially an effect of substructures consisting of longer repetitive cellotriosyl sequences. FT-Raman and NMR spectroscopy were useful in measuring sample impurities of α-glucans and prediction of β-linkage characteristics.     

Purification and characterization of a β-glucosidase capable of hydrolyzing soybean isoflavone glycosides from Pichia guilliermondii K123-1

A β-glucosidase, efficiently hydrolyzing isoflavone glycoside to isoflavone aglycone, was purified from Pichia guilliermondii K123-1, isolated from Korean soybean paste by ammonium sulfate precipitation, ion exchange column chromatography, gel filtration, and fast protein liquid chromatogram (FPLC). The molecular mass of purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The optimum temperature for enzyme activity was 45°c and it decreased dramatically above 50°c. The maximal activity was at pH 4.5 and more than 80% of the activity was retained for 24 hr in the pH range from 4.0 to 8.0 at 4°C. The N-terminal amino acid sequence of the enzyme was determined to be GLNWDYDNDK. Based on its substrate specificity and catalytic properties, the activity of the purified β-glucosidase was more effective when the sugar moiety of the glycoside was glucose and the size of the aglycone similar to that of the isoflavones. The purified β-glucosidase efficiently converts genistin and daidzin to genistein and daidzein 1.96 and 1.75 times more than almond meal β-glucosidase.     

Purification, characterization, and production of β-mannanase from Bacillus subtilis TJ-102 and its application in gluco-mannooligosaccharides preparation

A novel β-mannanase-producing strain, Bacillus subtilis TJ-102, was identified and characterized. Response surface method was applied to improving and enhancing the enzyme production. The optimized media components were obtained: 45.25 g/L konjac, 9.29 g/L Na₂HPO₄·12H₂O, 2.60 g/L CaCO₃, 1.0 g/L (NH₄)₂SO₄, 0.3 g/L KH₂PO₄, 1.0 g/L NaCl, 1.0 g/L MgCl₂·6H₂O, and 0.01 g/L FeSO₄. Under these conditions, the β-mannanase activity could achieve 205.3 U/mL in a 7-L fermentor. Then, β-mannanase was 7.39-fold purified by salting out, ultrafiltration, anion-exchange, and size-exclusion preparative chromatography with a recovery of 21.41 % and a specificity of 125.36 U/mg proteins. β-Mannanase was stable below 65 °C and pH 5.0–8.0, which exhibited excellently enzymatic efficiency in the preparation of gluco-mannooligosaccharides (GMOS) by hydrolyzing konjac flour. The GMOS yield of 57.76 % has been achieved with 8.71 % of mannose and 14.49 % of glucose, demonstrating the potential use of β-mannanase in food industry.     

Novel low-temperature-active, salt-tolerant and proteases-resistant endo-1,4-β-mannanase from a new Sphingomonas strain

Sphingomonas sp. JB13, isolated from slag of a >20-year-old phosphate rock-stacking site, showed the highest 16S rDNA (1343bp) identity of 97.2% with Sphingomonas sp. ERB1-3 (FJ948169) and <97% identities with other identified Sphingomonas strains. A mannanase-coding gene (1191bp) was cloned and encodes a 396-residue polypeptide (ManAJB13) showing the highest amino acid sequence identities of 56.2% with the putative glycosyl hydrolase (GH) family 26 endo-1,4-β-mannanase from Rhodothermus marinus (YP_004824245), and 44.2% with the identified GH 26 endo-1,4-β-mannanase from Cellvibrio japonicus (2VX5_A). The recombinant ManAJB13 (rManAJB13) was expressed in Escherichia coli BL21 (DE3). Purified rManAJB13 displayed the typical characteristics of low-temperature-active enzymes: showing apparent optimal at 40°C, ~55% of the maximum activity at 20°C and ~20% at 10°C, and thermolability at 45°C (~15min half-life). The potential mechanism for low-temperature-activity of GH 26 endo-1,4-β-mannanases might be ascribed to the more hydrophobic residues (AILFWV) and less polar residues (NCQSTY) compared with typical thermophilic and mesophilic counterparts. The purified rManAJB13 exhibited >85% mannanase activity at the concentration of 0-4.0M NaCl. No loss of enzyme activity was observed after incubating the enzyme with 1M or 2M NaCl, or trypsin or proteinase K at 37°C and pH 6.5 for 1h. The K(m), V(max) and k(cat) values were 5.0mgml(-1), 277.8μmol min(-1)mg(-1), and 211.9s(-1), respectively, using locust bean gum as the substrate.     

Purification,characterization, and gene cloning of a new cold-adapted β-galactosidase from Erwinia sp. E602 isolated in northeast China

β-Galactosidases are widely used in industry for elimination of lactose from milk products. A new β-galactosidase was obtained from bacterial strain Erwinia sp. E602, newly isolated in northeast China. The enzyme was purified with the methods of ammonium sulfate fractionation, ion exchange, and gel filtration chromatography for further study of the enzymatic characteristics. The purified enzyme had a molecular weight of near 110 kDa. The optimum reaction temperature and pH of this enzyme was determined to be 40°C and 7.0, respectively, indicating that this enzyme was a mesophilic neutral β-galactosidase. Furthermore, the enzyme retained near 10% of the activity at 0°C, which also suggested its cold-adapted property. Kinetics of the β-galactosidase was studied, and the K_m (Michaelis constant) and V_max (maximum enzymatic reaction rate) of this enzyme were 0.21 mmol/L and 263.16 µmol/mg per minute, respectively. The effects of metal ions on the enzymatic activity and the lactose hydrolysis efficiency in milk, as well as its trans-glycosylation activity, were studied in this work. The β-galactosidase coding gene was cloned to be a 3-kb length fragment, which shared at most 81% of identity with the published sequences in NCBI Blast database (https://blast.ncbi.nlm.nih.gov). Results in this work suggested it is a new β-galactosidase and it has potential to be used in dairy and food processing.     

Purification, gene cloning and characterization of an acidic β-1,4-glucanase from Phialophora sp. G5 with potential applications in the brewing and feed industries

An extracellular β-1,4-glucanase (CelG5, ～55.0kDa) was isolated from the culture filtrate of Phialophora sp. G5, and its encoding gene was cloned. The deduced amino acid sequence of CelG5 was at most 73.6% and 44.0%, respectively, identical with a hypothetical protein from Sordaria macrospora and an experimentally verified GH 7 endo-β-1,4-glucanase of Neurospora tetrasperma FGSC 2508. Native CelG5 had pH and temperature optima of pH 4.5-5.0 and 55-60°C. The enzyme showed some properties superior than most fungal β-1,4-glucanases, such as high activity over a wide pH range (exhibiting >50% of the maximum activity at pH 2.0-7.0), excellent stability in extreme acidic to alkaline conditions (pH 2.0-9.0), and strong resistance against pepsin and trypsin (retaining 89% and 94% activity, respectively). Recombinant CelG5 produced in Pichia pastoris had a molecular mass and a pH optimum similar to native CelG5, but with maximal activity at 65°C. Application tests showed that native CelG5 was stable under simulated gastric conditions (retaining >70% activity), and had capacity to decrease the viscosity of barley-bean feed (8.9% by 200U CelG5) and mash (6.1% by 50U CelG5) and increase the filtration rate of mash (18.4% by 50U CelG5). These properties make CelG5 a good candidate for utilization in the animal feed and brewing industries.     

Cloning, over-expression and characterization of an alkali-tolerant endo-β-1,4-mannanase from Penicillium freii F63

A glycosyl hydrolase family 5 endo-β-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 gl(-1) in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60 °C and exhibited good stability over a broad pH range from acidic to alkaline (>85.0% activity at pH 4.0-9.0, >70.0% activity at pH 10.0 and 43.7% even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co(2+), Cu(2+), Mn(2+) and β-mercaptoethanol and was strongly inhibited by Hg(2+) and SDS. The specific activity, K(m) and V(max) values were 47.5 U mg(-1), 7.8 mg ml(-1) and 70.4 μmol min(-1)mg(-1), respectively, for locust bean gum, and 40.3 U mg(-1), 2.3 mg ml(-1) and 61.7 μmol min(-1)mg(-1), respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries.     

Purification and partial characterization of β-glucanase produced by <Emphasis Type="Italic">Trichoderma viride</Emphasis> TP09 isolated from sewage of beer-making

As an initial investigation to improve the insoluble yeast β-1, 3-glucan solubility, a novel β-glucanase from Trichoderma viride TP09 was purified in the culture supernatant and partially characterized. By 70% saturation ammonium sulfate and chromatography on DEAE-Sepharose CL-6B column, β-glucanase was purified 28.7-fold, with recovery of 45.2% of the initial activity. The molecular weight of this enzyme was estimated to be 54.6 KD by SDS-PAGE. The optimum pH and the optimum temperature for the enzyme were 5.0 and 50 °C, respectively. The enzyme showed high stability within the range of pH 3.0–5.0 and thermostability between 30 and 70 °C. The enzyme activity was inhibited by Fe³⁺, Mg²⁺, Mn²⁺, Cu²⁺, and stimulated by Zn²⁺, Ca²⁺, Fe²⁺. Substrate specificity studies revealed the enzyme to be a β-1, 3–1, 4-glucanase. The β-glucanase showed preference for β-1, 3 linkage and β-1, 4 linkage, but had no activity on α-1, 4 and α-1, 6 linkage. The above results indicated that the enzyme extracted from T. viride TP09 of the beer-making sewage could be used as a potential predominant tool to enhance solubility of the insoluble yeast β-1, 3-glucan. These findings may lead to an enhanced solubility and expedite the progress of application in immunotherapy.     

Preparation and characterisation of arabinoxylan and (1,3)(1,4)-β-glucan alternating multilayer edible films simulated those of wheat grain aleurone cell wall

Various observations show a lamellar organisation of wheat aleurone cell walls, that likely acted as barriers to regulate the water diffusion rate in wheat grains. In this study, arabinoxylan (AX) and (1,3)(1,4)-β-glucan (BG) alternating multilayer films which mimic cell wall of aleurone structure were prepared. Twenty layers of alternating multilayer films of AX/BG were prepared with individual thicknesses of 20 µm. The water diffusion and water mobility of the films were determined using dynamic vapour sorption and time-domain nuclear magnetic resonance spectroscopy. The AX/BG alternating multilayer films exhibited unique mechanical and hydration properties, which could be explained by their unique nanostructure and microporosity. The edible films with this structure were found to exhibit high ultimate stress and ultimate strain, and low rates of water diffusion.