THE ACTION OF ENDO-β-1,3-GLUCANASES ON BARLEY AND MALT β-GLUCANS

The β-glucan extracted from ungerminated barley with water at 40 °C has a much lower specific viscosity than the corresponding material isolated from a wort prepared at 65 °C from a two-day germinated barley malt. Both glucans are similar in that they are polymers of β-D-glucose, with approximately 74% of the linkages in the β-1,4 configuration and 26% in the β-1,3 configuration. However, the two glucans are not hydrolysed to the same extent either by a partially purified bacterial endo-β-1,3-glucanase or by a homogeneous endo-β-1,3-glucanase from malted barley. The malt glucan is readily hydrolysed, causing a rapid decrease in specific viscosity but with no measurable increase in reducing power, whereas barley glucan undergoes only limited hydrolysis under similar conditions. Thus, different β-glucan preparations from barley or malt may be identical in the proportion of β-1,3 to β-1,4-linkages but the overall arrangement of linkages, and hence susceptibility to enzyme attack, differs according to the source and the method of extraction of the glucan. The molecular weights of both β-glucan preparations and the products of their enzyme hydrolysis have been determined by agarose gel permeation chromatography. A simple model which illustrates the underlying structural relationships of the β-glucans from barley and malt is suggested.     

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...     

THE DEVELOPMENT OF β-D-GLUCANASES DURING THE GERMINATION OF BARLEY AND THE EFFECT OF KILNING ON INDIVIDUAL ISOENZYMES†

Two endo-1,3;1,4-β-D-glucanase isoenzymes developed in response to gibberellic acid, during the germination of barley. Two endo-1, 3-β-D-glucanases, one present in ungerminated, steeped grain, also developed but did not appear to be markedly stimulated by the hormone. A comparison of crude and partially purified malt extracts highlighted the errors that are involved in the specific determination of endo-1, 3;1, 4-β-glucanase activity in crude extracts. The development and effect of kilning on individual malt isoenzymes was demonstrated by carboxymethylcellulose (CM-cellulose) chromatography profiles. Kilning and dry-milling of germinated barley caused losses of 80–90% in the specific endo-1,3;1,4-glucanase activity. The effect was less pronounced if wet-milling was substituted for dry-milling. Extraction studies and CM-cellulose chromatography profiles indicated that both endo-1,3;1, 4-β-glucanase isoenzymes were heat labile and were particularly susceptible to oxidation. In contrast, endo-1,3-β-glucanase activity and cellobiase activity in malt extracts were less affected by the kilning process or extraction procedures. Preliminary results suggested that one of the endo-1,3-β-glucanase isoenzymes was more sensitive to kilning.     

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.     

裂褶菌产内切β-1,3-葡聚糖酶的分离纯化

采用不同饱和度的硫酸铵溶液对内切β-1,3-葡聚糖酶粗酶液进行分段盐析,以确定最佳盐析条件,将盐析处理后的酶液经透析浓缩、DEAE-Sephadex A-50离子交换层析、Sephadex G-75凝胶过滤层析等进一步分离纯化,浓缩纯化后的含酶组分,经过SDS-PAGE电泳分析其纯度并初步确定其分子量。结果显示:经过纯化后的内切β-1,3-葡聚糖酶的比活力由20.90U/mg提高到933.37U/mg,纯化倍数为44.7倍,酶活回收率为11.6%,电泳分析呈单一条带,分子量近似为45ku。     

β-葡聚糖酶[EC. 3. 2. 1. 73]的研究 Ⅰ.产酶菌种的筛选和培养

β-葡聚糖酶主要用于啤酒工业,它能专一地分解粘度很高的各种大麦β-葡聚糖中β-1.3和β-1.4糖苷键,使麦汁粘度降低,从而缩短麦汁和啤酒的过滤时间,增加产量,并改善啤酒的质量。 试验所用菌种Bacillus Subtilis是从34株细菌中筛选并经诱变获得,它具有较高的产β-葡聚糖酶的能力。 在以淀粉、蔗糖和硫酸铵为主的培养基、pH7.2～7.5、温度为37℃条件下,进行搅拌通风培养两天。酶活力约为30U/ml。 发酵液采用絮凝剂处理,经硅藻土过滤,再将滤液经超滤或真空低温浓缩,即获得了深棕色的液体酶制剂。若进一步用酒精沉淀或进行喷雾干燥,即可制得粉末状酶制剂产品。 β-葡聚糖酶应用于啤酒糖化,对加速麦汁过滤、提高麦汁收得率,均有明显效果。     

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。     

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.     

裂褶菌发酵产β-1,3-葡聚糖酶条件的优化

本文通过裂褶菌发酵培养获得β-1,3-葡聚糖酶,并对其发酵条件进行了研究.分别探讨了培养基中的碳源、氮源及无机盐对产酶的影响,并采用正交试验找到最佳的产酶培养基配方为葡萄糖2%,牛肉膏0.3%,NH4Cl0.1%,KH2PO40.05%,MgSO40.05%;对培养温度、起始pH值、接种量和培养时间等条件进行了优化,得...     

β-1,3-1,4-葡聚糖酶基因的克隆与序列分析

依据GenBank中收录的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)β-1,3-1,4葡-聚糖酶基因序列,设计特异性引物,以提取的解淀粉芽孢杆菌基因组DNA为模板,利用聚合酶链式反应(PCR)扩增获得758 bp的β-1,3-1,4-葡聚糖酶基因(bgl),将此目的基因克隆至pTG19-T Easy载体中,经PCR、限制性内切酶鉴定和克隆片段的序列测定、比较,结果表明该克隆片段扩增准确、可靠。序列比较发现,此片段与解淀粉芽孢杆菌(B.amyloliquefacines,M15674)、枯草芽孢杆菌(B.subtilis,D00518)和地衣芽孢杆菌(B.licheniformis,AY365256)分别有99%、95%和94%的同源性。     

THE DEVELOPMENT OF β-GLUCAN SOLUBILASE DURING BARLEY GERMINATION

β-Glucan solubilase in either germinating barley or in endosperm slices treated with gibberellic acid is synthesized before endo-β-glucanase, α-amylase and protease. In common with these enzymes, β-glucan solubilase is synthesized much sooner in endosperm slices than in whole grain. Gibberellic acid stimulates β-glucan solubilase synthesis in endosperm slices and most of the activity is rapidly released into the surounding medium, irrespective of whether the hormone is present. Inhibitors of RNA and protein synthesis block the formation of β-glucan solubilase. Unlike β-glucanase, α-amylase and protease, β-glucan solubilase is present in significant quantity in untreated barley where it is concentrated in the embryo-containing half of the grain. The only β-glucan solubilase activity in barley is due to an acidic carboxypeptidase. Malt contains a small amount of a second solubilizing enzyme which appears to be an endo-β1, 3-glucanase.     

RELATIONSHIP BETWEEN LEVELS OF GIBBERELLIC ACID AND THE PRODUCTION AND ACTION OF CARBOHYDRASES OF BARLEY

Different hydrolytic enzymes require different levels of gibberellic acid to induce their maximal production and release into the endosperm of barley. Barley-endo-β-glucanase requires a higher level of gibberellic acid to induce maximal production than does α-amylase. Although gibberellic acid also increases the level of barley endo-β-1,3 glucanase, this enzyme, unlike the barley-endo-β-glucanase, develops to significant levels when gibberellic acid is absent. In gibberellic acid-treated aleurone layers β-glucanases degrade the cell wall mainly to glucose. Xylose and cellobiose appear when the aleurone wall has undergone extensive enzymic hydrolysis. Laminaribiose and arabinose are found whether or not gibberellic acid is present in the medium. In addition to the degradation of the endosperm cell walls, β-glucanases may also play an important role in the release of enzymes from the aleurone into the endosperm during malting.     

微生物木聚糖酶及其应用

木聚糖(Xylan)是植物半纤维素的主要成分,是一种复杂的多聚五碳糖。木聚糖酶(Xylanase,EC 3.2.1.8)以内切方式作用于木聚糖主链,产生不同链长的寡糖和少量的木糖,是木聚糖降解酶系中最为关键的酶。本文综述了微生物木聚糖酶系统、微生物木聚糖酶的分类及其来源分布、微生物木聚糖酶的特性、产生及在食品、造纸、饲料行业的应用。     

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。     

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.     

使用分离自啤酒废水的木霉菌TP09固态发酵提纯β-葡聚糖酶

使用木霉TP09固态发酵,提纯并鉴定了β-1,3-葡聚糖酶的部分性质。采用饱和度为70%的（NH4）2SO4及DEAE-琼脂糖凝胶CL-6B柱层析纯化,β-葡聚糖酶相对粗酶溶液纯化了28.7倍,酶回收率为45.2%。经SDS-PAGE分析,该酶分子量近似54.6KD。酶最适反应pH为5.0,最适温度为50℃。在pH3.0～5.0、温度30～70℃酶活相对稳定。Fe^3＋、Mg^2＋、Mn^2＋以及Cu^2＋对该酶有抑制作用,Zn^2＋、Ca^2＋和Fe^2＋则有激活作用。底物选择性研究表明该酶为β-1,3-葡聚糖酶、β-1,4-葡聚糖酶。该酶可作用于含β-1,3、β-1,4糖苷键的底物,对含α-1,4和α-1,6糖苷键的底物无作用。表明分离自啤酒废水的木霉TP09生产的β-1,3-葡聚糖酶可增强不溶性β-1,3-葡聚糖的可溶性,促进了其在免疫疗法中的应用。     

Fungal β-1,3-1,4-glucanases: production,proprieties and biotechnological applications

β-1,3-1,4-glucanases (or lichenases; EC 3.2.1.73) comprise one of the main enzymes used in industry during recent decades. These enzymes hydrolyze β-glucans containing β-1,3 and β-1,4 linkages, such as cereal β-glucans and lichenan. The β-1,3-1,4-glucanases are produced by a variety of bacteria, fungi, plants and animals. A large number of microbial β-1,3-1,4-glucanases have potential application in industrial processes, such as feed, food and detergent industries. The present review summarizes the available studies with respect to β-1,3-1,4-glucanases production conditions, enzyme biochemical properties and potential industrial application. © 2018 Society of Chemical Industry     

A Novel α-Glucosidase of the Glycoside Hydrolase Family 31 from Aspergillus sojae

We characterized an α-glucosidase belonging to the glycoside hydrolase family 31 from Aspergillus sojae. The α-glucosidase gene was cloned using the whole genome sequence of A. sojae, and the recombinant enzyme was expressed in Aspergillus nidulans. The enzyme was purified using affinity chromatography. The enzyme showed an optimum pH of 5.5 and was stable between pH 6.0 and 10.0. The optimum temperature was approximately 55 °C. The enzyme was stable up to 50 °C, but lost its activity at 70 °C. The enzyme acted on a broad range of maltooligosaccharides and isomaltooligosaccharides, soluble starch, and dextran, and released glucose from these substrates. When maltose was used as substrate, the enzyme catalyzed transglucosylation to produce oligosaccharides consisting of α-1,6-glucosidic linkages as the major products. The transglucosylation pattern with maltopentaose was also analyzed, indicating that the enzyme mainly produced oligosaccharides with molecular weights higher than that of maltopentaose and containing continuous α-1,6-glucosidic linkages. These results demonstrate that the enzyme is a novel α-glucosidase that acts on both maltooligosaccharides and isomaltooligosaccharides, and efficiently produces oligosaccharides containing continuous α-1,6-glucosidic linkages.     

β-GLUCANASE: THE SUCCESSFUL APPLICATION OF GENETIC ENGINEERING

The now well established principles of genetic engineering are described in relation to the solution of problems associated with β-glucans in beer. The application of these techniques has enabled the isolation of a Bacillus subtilis endo-1, 3–1, 4-β-D-glucanase gene which expresses a biologically active enzyme in yeast.^15,16 Although this enzyme is capable of hydrolysing beer β-glucans during fermentation, thereby enhancing beer filtration, insufficient β-glucanase is produced in yeast to enable successful commercial implementation. The requirements for the efficient production of β-glucanase in genetically manipulated brewing yeast are described.     

特基拉芽孢杆菌来源β-1,3-1,4-葡聚糖酶重组菌发酵培养基的优化

为了提高重组大肠杆菌(Escherichia coli BL21DE3)(pET-28a(+)-bgl)发酵产β-1,3-1,4-葡聚糖酶的能力,研究了发酵培养基中各类碳源及氮源的影响,并通过响应面分析法优化培养基各组分的含量。结果表明,甘油为最适碳源,酵母粉及胰蛋白胨为氮源。优化的培养基组成是:yeast extract终浓度为20 g/L,胰蛋白胨12.5 g/L,甘油14.1 mL/L,KH2PO42.17 g/L,K2HPO42.74 g/L。三角瓶发酵产β-葡聚糖酶酶活(2 978.2 U/mL),与初始培养基(1 671.9 U/mL)相比,提高了1.78倍。研究结果表明,发酵培养基的优化对重组大肠杆菌发酵生产工业酶具有重要作用。