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.     

微泡菌昆布多糖酶Lam128A的酶学性质及酶解产物抗氧化活性

该研究探讨微泡菌ALW1来源的昆布多糖酶Lam128A的酶学性质及其酶解产物的抗氧化活性。利用毕赤酵母异源表达重组蛋白rLam128A,该昆布多糖酶的分子量为70 ku,最适反应温度和最适反应pH值分别为45 ℃和pH值5.5;温度低于45 ℃时稳定,分别在pH值3.0、5.0和11.0条件下处理96 h后,残余酶活力不低于60%。还原试剂二硫苏糖醇（DTT）对昆布多糖酶活力有促进作用,rLam128A对螯合剂乙二胺四乙酸（EDTA）表现出良好的稳定性,对去垢剂Tween 20和Tween 80、变性剂尿素表现出一定的稳定性。昆布多糖经rLam128A水解后,酶解产物对DPPH、ABTS和OH·自由基的半数抑制剂量IC50分别为7.12、1.01和 2.40 mg/mL,相比未经酶解处理的昆布多糖表现出更显著的抗氧化活性。微泡菌昆布多糖酶Lam128A的酶学性质为该酶开发利用昆布多糖生物资源提供了理论基础。     

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.     

皱纹盘鲍内脏β-1,3-葡聚糖酶的提取及其酶学性质研究

从鲍鱼内脏提取β-1,3-葡聚糖酶粗酶并研究其酶学性质。以昆布多糖为底物监控酶的活性。鲍鱼内脏经匀浆后,采用3倍体积0.1mol/L的磷酸氢二钠-柠檬酸缓冲液(pH6.0)浸提12h。浸提液经离心后收集上清液,并采用60%饱和度的硫酸铵对上清液中的蛋白进行盐析沉淀得β-1,3-葡聚糖酶粗酶。研究表明,粗酶中β-1,3-葡聚糖酶的最适反应温度为40℃,酶活力在40℃以下稳定;酶的最适pH为6.0,酶活力在pH4.0～7.0范围内稳定。Mn2+能明显激活酶活力,而Cu2+、Fe3+、Hg+对酶活力有抑制作用,其中Fe3+的抑制作用最强;亮肽酶素和1,10-菲啰啉对酶活力有明显的抑制作用,而E-64对酶活力有一定的激活作用;此酶对海藻酸钠也有一定水解能力。     

工业酿酒酵母重组β-1,3-1,4-葡聚糖酶部分酶学性质的研究

用刚果红法测定β-1,3-1,4-葡聚糖酶的酶活力,研究重组酿酒酵母(S.cerevisiae)菌株SC-βG分泌表达的重组β-1,3-1,4-葡聚糖酶的部分酶学性质,并与出发菌株枯草芽孢杆菌(B.subtilis)表达的原始酶的性质进行比较。结果表明,重组酶保持了与原始酶相同的底物专一性。 重组酶的最适反应温度为35℃,而原始酶为55℃。重组酶的热稳定性也发生了改变,40℃热处理20min只保留63.4%的最初酶活力,但温度再升高时对热处理敏感度降低,70℃的热处理20min仍保留45.9%的最初酶活力;而原始酶50℃时稳定,60℃以上的热处理酶活力损失很大。与原始酶相比,重组酶的最适pH值下降为pH5.0,而原始酶为pH6.5;相比原始酶在pH7.0有最大稳定性,重组酶在pH5.5时有最大稳定性。重组β-1,3-1,4-葡聚糖酶的最适反应条件与原始酶相比更接近啤酒的实际生产条件。     

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.     

Induction of TNF-alpha production from human peripheral blood monocytes with beta-1,3-glucan oligomer prepared from laminarin with beta-1,3-glucanase from Bacillus clausii NM-1

We prepared a beta-1,3-glucan oligomer (DP> or = 4) from laminarin (DP: 25-30) derived from Laminaria digitata with beta-1,3-glucanase, and examined its effect on human peripheral blood monocytes. Conditioned medium prepared by incubating monocytes (MC-CM) with the beta-1,3-glucan oligomer showed strong inhibitory activity against the proliferation of human leukemic U937 cells. Since the beta-1,3-glucan oligomer had no direct cytotoxic effect on U937 cells up to 1000 microg/ml, the cytotoxicity of the MC-CM may be due to cytotoxic cytokines produced from monocytes stimulated by the beta-1,3-glucan oligomer. On the other hand, the MC-CM prepared with original laminarin had little effect on the growth of U937 cells. The cytotoxicity of the MC-CM prepared with the beta-1,3-glucan oligomer was significantly reduced by an anti-TNF-alpha antibody, but the anti-TNF-beta antibody had no effect. Our results suggest that the enzymatically depolymerized beta-1,3-glucan oligomer induces TNF-alpha production from human monocytes.     

β-1,3- 葡聚糖酶基因克隆、表达及抗菌活性的研究

通过RT-PCR的方法分别从小麦和水稻的cDNA中克隆获得β-1,3-葡聚糖酶基因(Glu基因),分别命名为TaGlu9507和OsGlu30。它们的序列分析表明这两个克隆均含一个1002bp的开放阅读框(ORF),编码334个氨基酸,各自的N-端含有一个长20和29个氨基酸残基的信号肽序列。将不含信号肽序列的TaGlu9507和OsGlu30编码区DNA片段分别克隆进pET28-a(+)表达载体,并转入大肠杆菌BL21(DE3),经0.5mmol/L IPTG诱导3h后获得了高量表达,表达量分别占大肠杆菌可溶性蛋白的49.7%和26.7%,表达产物对黑曲霉、酵母等真菌生长均有较为明显的抑制作用。本结果更进一步表明β-1,3-葡聚糖酶基因是植物真菌病防治的潜在目的基因群之一。     

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-β1,3-GLUCANASE FROM GREEN MALT

An endo-β1,3-glucanase from a green malt extract was purified by DEAE- and CM-cellulose ion exchange chromatography followed by molecular sieve chromatography on BioGel P-100. A final enzyme preparation had two protein components on disc electrophoresis, one of which was in-active. The enzyme had a pH optimum of 5·0 for activity on laminarin and 5·8 on carboxymethyl pachyman. The activity was stable up to 60°C and was stimulated by NaCl. The isoelectric point of the enzyme was 9·8.     

裂褶菌产内切β-1,3-葡聚糖酶的特性研究

对裂褶茵所产内切β-1,3-葡聚糖酶进行有效分离纯化并用电泳法对其纯度进行鉴定,进而研究其酶学特性.结果表明:经过DEAE-Sephadex A-50离子交换层析和Sephadex G-75凝胶过滤分离纯化得到电泳纯分子量约为45 kD的内切β-1,3-葡聚糖酶,其最适pH为5.0,最适温度为45℃;Fe<'2+>、B...     

Structural feature and antioxidant activity of low molecular weight laminarin degraded by gamma irradiation

Recently, it has been reported that low molecular weight laminarin had enhanced biological activities. In this study, laminarin was degraded by gamma irradiation, and the changes in its structure and antioxidant property were investigated. Gel permeation chromatography data showed that the average molecular weight of the irradiated laminarin decreased significantly as the irradiation dose increased. The absorbance at 290 nm, from UV spectra, was increased, depending on the irradiation dose and caused by the formation of carbonyl groups. From the NMR analysis, the ratio of β-1,3- to β-1,6-linkages was not changed, indicating the random scission of chains by gamma irradiation. The antioxidative activities, measured by Rancimat test, were all increased in the gamma-irradiated laminarin, depending on the absorbed dose, presumably due to the carbonyl groups formed by gamma irradiation. Therefore, gamma irradiation could be a promising method for preparing low molecular weight laminarin with enhanced biological activities.     

Purification and characterization of a new endo-β-1,3-glucanase exhibiting a high specificity for curdlan for production of β-1,3-glucan oligosaccharides

Endo-β-1,3-glucanase (Endo23) was purified from a Trichoderma reesei GIMCC 3.498 fermentation broth using anion exchange and 2-stage size exclusion chromatography. Purification of 44.5× and a 12% recovery yield of enzyme activity were achieved. The Mw and isoelectric point were estimated to be 24 kDa and 3.85 using SDS-PAGE and IEF, respectively. The highest substrate specificity was observed for water-insoluble curdlan. The optimal conditions for hydrolyzing curdlan were pH 5.0 and 50°C. The main hydrolytic products were glucobiose and glucotriose. Minor amounts of glucose and glucotetraose were detected. Hg²⁺, Fe²⁺, Fe³⁺, and Sn²⁺ inhibited the hydrolysis activity of Endo23 at 5 and 50 mM. K⁺ slightly promoted Endo23 activity. Endo23 belongs to the category EC3.2.1.39. The peptide sequences of Endo23 showed identity with conserved sequences that typically exist in β-1,3-glucanases of the glycoside hydrolase family. The Endo23 sequence was partially similar to a hypothetical lignocellulase from Penicillium oxalicum 114-2.     

木霉LE02 β-1，3-葡聚糖酶酶学特性的研究

对木霉菌株LE02所产β-1,3-葡聚糖酶的酶学特性进行了研究。结果表明,该酶最适反应温度为55℃、最适反应pH值为5.0。Co~(2+)、K+、Zn~(2+)、Li~+、Ba~(2+)、Cu~(2+)以及1.0mmol/L的Fe~(2+)对该酶没有影响,Cd~(2+)和10.0mmol/L的Mg~(2+)对酶具有部分抑制作用,而低浓度的Hg~(2+)、5.0mmol/L以上的Mn~(2+)和10.0 mmol/L的Fe~(3+)能强烈抑制该酶的活性。该酶只能作用于β-1,3-糖苷键,以Larinami为底物时其米氏常数K_m值为128.34μg/mL,最大反应速度V_m为23.01μg/(min·mL)。经过SDS-PAGE测定的分子质量近似为80.137ku。     

Effect of barley β-glucan on murine RAW264.7 macrophages against virulent Salmonella enterica serovar Typhimurium

Heavily evidenced in antimicrobial effects of microbial β-(1,3)(1,6)-glucan, the botanical β-(1,3)(1,4)-glucan is mostly shown to effectively control blood cholesterol level. S. Typhimurium belongs to food-borne zoonoses often causing worldwide epidemic outbreaks in animals and human with severe diarrhea and gastrointestinalitis. Cereal soups are commonly prescribed as neutraceutical for rehydration purpose and a sustaining therapy. Botanical β-(1,3)(1,4)-glucan is easily released in boiled cereal soup. β-(1,3)(1,4)-Glucans on modulating host defense to enteric infectious agents is seldom reported. Our results demonstrated that barley β-(1,3)(1,4)-glucans effectively increased the murine macrophage cell line RAW264.7 against S. Typhimurium infection through antibacterial lysozyme activity (P < 0.001), not the production of intracellular reactive oxygen species. Furthermore, barley β-(1,3)(1,4)-glucans upregulated the gene expressions of its receptor dectin-1. In conclusion, barley β-(1,3)(1,4)-glucan induces a mild immune response with increasing antibacterial lysozymes through up-regulating its receptors dectin-1 and lysozyme M gene expressions.     

Purification and characterization of a novel extracellular beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from ezo abalone Haliotis discus hannai

A novel extracellular alkaline stable beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from the ezo abalone Haliotis discus hannai was purified by ammonium sulfate precipitation, DEAE-Sepharose FF ion exchange chromatography and Sephacryl S-200HR gel filtration. The molecular weight of the purified enzyme was estimated to be 71 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was very stable at pH 5.3 to 11.5 but unstable at pH 4.0 to 4.5. The optimum temperature and thermostability of the enzyme increased in the presence of CaC1, The enzyme hydrolyzed R-1,3-glucan from marine organisms, but did not show activity against any other beta-1,3-glucans. The major hydrolysis products of beta-1,3-glucan from Laminaria digitata and Eisenia bicyclis were laminaritriose and laminaritetraose, respectively. The N-terminal amino acid sequence of the purified enzyme was similar to that of several beta-1,3-glucanases in the glycoside hydrolase family 16.     

Enzymatic Properties of β‐1,3‐Glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo‐β‐1,3‐glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N‐terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β‐1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β‐1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β‐1,6‐linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo‐β‐1,3‐glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β‐1,3 linkage between the 3rd and 4th glucosyl residue.     

Chitosan and postharvest decay of fresh fruit: Meta-analysis of disease control and antimicrobial and eliciting activities

Consumers are increasingly aware of the importance of regular consumption of fresh fruit in their diet. Since fresh fruit are highly sensitive to postharvest decay, several investigations focused on the study natural compounds alternative to synthetic fungicides, to extend their shelf life. A long list of studies reported the effectiveness of the natural biopolymer chitosan in control of postharvest diseases of fresh fruit. However, these findings remain controversial, with many mixed claims in the literature. In this work, we used random-effects meta-analysis to investigate the effects of 1% chitosan on (a) postharvest decay incidence; (b) mycelium growth of fungal pathogens Botrytis cinerea, Penicillium spp., Colletotrichum spp. and Alternaria spp.; and (c) phenylalanine ammonia-lyase, chitinase and β-1,3-glucanase activities. Chitosan significantly reduced postharvest disease incidence (mean difference [MD], −30.22; p < 0.00001) and in vitro mycelium growth (MD, −54.32; p  < 0.00001). For host defense responses, there were significantly increased activities of β-1,3-glucanase (MD, 115.06; p = 0.003) and chitinase (MD, 75.95; p < 0.0002). This systematic review contributes to confirm the multiple mechanisms of mechanisms of action of chitosan, which has unique properties in the natural compound panorama. Chitosan thus represents a model plant protection biopolymer for sustainable control of postharvest decay of fresh fruit.     

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.     

β-Glucoside metabolism in Oenococcus oeni: Cloning and characterisation of the phospho-β-glucosidase bglD

The structural gene for a phospho-β-glucosidase from the oenologically important lactic acid bacterium (LAB) Oenococcus oeni has been cloned and its protein product characterised. This gene is found in a putative β-glucosidase operon of 2178 base pairs encoding 4 genes designated bglA to bglD. The bglA, B and C genes were not cloned and characterised, however, are thought to be phosphoenolpyruvate dependent phospho transferase system (PEP-PTS) components IIC, IIA and IIB which regulate the uptake, phosphorylation and translocation of β-glucosides across the cytoplasmic membrane. The cloned bglD was sequenced and expressed in Escherichia coli followed by purification. The purified bglD protein has 480 residues, a molecular mass of 55.5 kDa and shows high homology to known phospho-β-glucosidases. bglD exhibited high activity towards the phosphorylated β-glucoside para-nitrophenol-β-d-glucopyranoside-6-phosphate with a pH optimum of 5.5 and maintained similar levels of activity between temperatures of 4 °C and 40 °C. The enzyme was not active against non-phosphorylated β-glucosides.     

酸酶水解-HPLC法检测香菇多糖中β-D-葡聚糖含量

为了更有效地监控食品和医药行业中香菇多糖制品的质量,建立了一种香菇多糖中主要活性成分β-D-葡聚糖含量测定的新方法:香菇多糖样品经酸部分水解后用β-1,3-葡聚糖外切酶和β-葡糖苷酶进一步完全水解以测定总葡聚糖含量;另用淀粉葡萄糖苷酶水解样品中α-葡聚糖;根据HPLC测定的总葡聚糖与α-葡聚糖含量之差计得β-D-葡聚糖含量。分别用该方法和苯酚硫酸法及刚果红法测定已知含量的β-D-葡聚糖对照样品的结果表明:该方法最接近于对照品标示值,且重复性好,可用作为香菇多糖产品中β-D-葡聚糖含量测定的专属方法。