多重破壁技术提取葡萄酒泥废酵母β-葡聚糖研究

以葡萄酒泥废酵母为试材,采用高压均质法和冻融法协同破碎酵母细胞壁,并辅以复合蛋白酶和脂肪酶酶解技术,研究多重破壁技术对β-葡聚糖纯度的影响。在单因素实验基础上,利用Box-Behnken实验设计原理,以酵母浓度、均质时间和冻融加水量为实验因素,以β-葡聚糖纯度为响应值,优化葡萄酒泥酵母β-葡聚糖提取工艺。结果表明:葡萄酒泥酵母β-葡聚糖最优提取工艺为均质压力70 MPa,酵母浓度13%,均质时间34 min,冻融加水量25%,在此条件下提取所得酵母β-葡聚糖纯度为91.69%,得率为13.23%,该方法为酵母葡聚糖的开发利用提供了参考依据。     

灰树花菌丝体β-葡聚糖提取工艺优化

以β-葡聚糖得率为考察指标,考察了热水浸提法、热水-复合酶法、超声波法、超声波-复合酶法对灰树花菌丝体β-葡聚糖得率的影响。影响提取的关键因素为超声功率、超声时间、复合酶添加量、酶解温度,采用正交试验对提取工艺进行优化。结果表明,采用超声波-复合酶法所得β-葡聚糖得率最大,灰树花菌丝体β-葡聚糖最佳提取条件为超声功率300 W,超声时间15 min,复合酶添加量1.5%,酶解温度40℃。在此条件下,灰树花菌丝体β-葡聚糖得率可达2.80 mg/g。     

不同提取方法对酵母葡聚糖性质的影响

以啤酒废酵母为原料,分别就酵母β-葡聚糖的提取方法及不同提取方法对所得β-葡聚糖相关性质的影响进行了研究.结果表明,与碱法、碱酸法相比,酶碱法提取的多糖含量最高,且β-葡聚糖分子量也最大;但三种提取方法所得β-葡聚糖的持油性、冻融稳定性等差异不明显.β-葡聚糖能产生较大的粘度且具有较好的热稳定性,可作为食品的增稠剂、稳定剂.     

β-1,3-1,4-葡聚糖酶在毕赤酵母中的克隆与表达

β-1,3-1,4-D-葡聚糖酶是一类专一性降解β-1,3-1,4-葡糖苷键中的β-1,4-糖苷键,产生小分子还原糖的水解酶,广泛应用于啤酒工业和饲料工业中.本研究根据毕赤酵母密码子偏好性优化β-1,3-1,4-葡聚糖酶基因序列,采用PCR法将其插入毕赤酵母表达载体pPICZαA,经Sac I线性化后电击整合入毕赤酵母X-33基因组,构建重组酵母;经菌落PCR验证和摇瓶筛选,获得一株X-33/pPICZαA-bgl,甲醇诱导96h后,酶活力达308.5U/mL,经SDS-PAGE电泳,实际蛋白分子量约为33ku.β-1,3-1,4-D-葡聚糖酶最适反应pH为5.0,最适反应温度为50℃.     

酵母β-葡聚糖提取方法及免疫活性研究

β-葡聚糖是酵母细胞壁的成分之一,具有抗肿瘤、抗感染以及免疫调节等一系列生物学活性。基于其增强机体免疫功能的功效,β-葡聚糖作为一种功能性食品添加剂引起关注。探寻一种高效、快捷、环保且满足纯度需求的β-葡聚糖提取方法成为研究的热点。该文综述高压微射流均质法、超声波法等具有应用潜力的酵母β-葡聚糖提取方式,并且阐述酵母β-葡聚糖免疫活性机制以及在动物模型和细胞模型的研究进展。     

从酵母细胞壁提取β-1,3-D-葡聚糖的研究

分别采用酸法、碱法来提取酵母细胞壁中的β-1，3-D-葡聚糖。用紫外光谱法、纸层析法法和红外光谱法分析多糖成分。结果发现：经酸法（醋酸溶液浓度为0．5mol／L）提取的β-1，3-D-葡聚糖产品中除含有葡聚糖外，还含有一定量的甘露聚糖和蛋白质成分；而用碱法（氢氧化钠溶液浓度为1．0mol/L）提取时，产品为高纯度的β-1，3-D-葡聚糖。本文从其水解机理上探讨了产生上述两种不同结果的原因，指出碱法提取是从酵母中提取β-1，3-D-葡聚糖的高效方法。     

从酵母细胞壁提取β-1,3-D-葡聚糖的研究

采用酸法、碱法提取酵母细胞壁中的β-1，3-D-葡聚糖。用纸层析法和紫外光谱法进行多糖成分分析。结果表明，经酸法提取的β-1，3-D-葡聚糖产品中除含有葡聚糖外，还含有甘露聚糖和蛋白质；而用碱法提取时，产品为高纯度的β-1，3-D-葡聚糖。     

海藻糖合酶基因在毕赤酵母中的表达

首次构建来源于恶臭假单胞菌海藻糖合酶基因（AE015451.1）的真核表达载体,探索在毕赤酵母中的表达。PCR扩增目的基因,经EcoRⅠ/XbaⅠ双酶切后连接至同样双酶切的pPICZaA中,经PCR检测和序列测定准确后,通过电击法将重组质粒转入毕赤酵母GS115中,利用含Zeocin的YPD平板筛选到阳性转化子,提取阳性转化子基因组并利用特异性引物PCR得到一条与目的基因大小相同的条带,说明目的基因转入成功,诱导重组蛋白表达并用SDS-PAGE检测可见一条约76 ku与目的蛋白大小预测相符合的蛋白条带,最后HPLC检测重组蛋白可将麦芽糖特异转化为海藻糖,说明外源表达的海藻糖合酶具有一定酶活。通过PCR、SDS-PAGE和HPLC结果说明成功构建重组pPICZaA-TreS质粒并整合到巴斯德毕赤酵母的基因组上,且海藻糖合酶基因得到预期的表达并具有活性,为下一步研究奠定基础。     

微波辅助碱—酶法提取酵母β-1,3-葡聚糖工艺优化

为建立酵母β-1,3-葡聚糖高效、快速的提取方法,以细胞壁为原料,在传统碱—酶法基础上,辅助微波加热,采用响应面法优化提取工艺,通过二次回归模型分析得出最佳工艺为:微波功率420 W,加热时间5min,酶添加量2 550U/g。该条件下β-1,3-葡聚糖总糖含量预测值为87.44%,验证值为87.94%。     

人溶菌酶基因在乳酸克鲁维酵母中的表达

人溶菌酶是一种天然广谱抑菌物质,在食品和医药工业有潜在应用前景。为获得高活性的人溶菌酶制剂,采用乳酸克鲁维酵母表达系统,对经密码子优化的人溶菌酶基因(h LYZ)进行分泌表达。将人工合成h LYZ插入到乳酸克鲁维酵母表达载体p KLAC1,构建重组载体p KLAC1-h LYZ,并用电脉冲法将SacⅡ线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过全细胞PCR鉴定,最后获得了一株多拷贝整合的基因工程菌h LYZ1。工程菌可以分泌表达分子量约14 ku的目的蛋白质,与预期大小相符。摇瓶发酵培养128 h,酶活最高达到1430 U/mL。抗菌活性检测结果显示,重组人溶菌酶对溶壁微球菌、大肠杆菌和枯草芽孢杆菌有较好的溶菌活性。本研究成功地在乳酸克鲁维酵母中表达了重组人溶菌酶,表达的蛋白具有较高的酶活性,试验结果为利用乳酸克鲁维酵母表达系统规模化生产重组人溶菌酶奠了基础。     

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.     

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。     

面包酵母FX-2酶联辅助自溶工艺研究

该文主要研究了外源酶添加对面包酵母（Saccharomyces cerevisiae）FX-2自溶过程的影响。 首先通过单一对照实验,发现甘露 聚糖酶、木瓜蛋白酶和β-葡聚糖酶能显著提高酵母自溶效率。 其次,以上述三种酶为对象,考察了酶添加量、pH和温度对面包酵母酶 联辅助自溶过程的影响,在单因素试验的基础上,采用正交试验设计分别确定了各酶的最佳助溶条件。 最后,进一步比较验证了单 酶、双酶和多酶联用的助溶能力,并获得最优的酶法辅助自溶工艺：13%酵母乳调pH至5.5,添加0.6‰木瓜蛋白酶,于57.5 ℃条件下助 溶30 min后,调节温度为55 ℃、pH为6.0、添加0.2‰ β-葡聚糖酶,助溶至10 h。 该条件下,细胞死亡率、酵母乳离心上清液干物质含量及 细胞壁水解度分别达100%、9.40%和54.41%,辅助自溶效果好。     

An improved method for whole protein extraction from yeast Saccharomyces cerevisiae

A new method for protein extraction from yeast Saccharomyces cerevisiae cells is described. This method involves the use of LiAc and NaOH to enhance the permeability of yeast cell wall prior to protein extraction with SDS-PAGE sample buffer. It was safe and efficient compared to other methods reported so far in the literature. The proteins extracted with this new method retained their immunoreactive properties and are suitable for most applications in molecular biology studies.     

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.     

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.     

Characterization of a disulphide-bound Pir-cell wall protein (Pir-CWP) of Yarrowia lipolytica

In this work we have studied the disulphide-bound group of cell wall mannoproteins of Yarrowia lipolytica and Candida albicans. In the case of Y. lipolytica, SDS-PAGE analysis of the beta-mercaptoethanol-extracted material from the purified cell walls of the yeast form, showed the presence of a main polypeptide of 45 kDa and some minor bands in the 100-200 kDa range. This pattern of bands is similar to that obtained in identical extracts in Saccharomyces cerevisiae (Moukadiri et al., 1999), and besides, all these bands cross-react with an antibody raised against beta-mercaptoethanol-extracted material from the purified cell walls of S. cerevisiae, suggesting that the 45 kDa band could be the homologue of Pir4 of S. cerevisiae in Y. lipolytica. To confirm this possibility, the amino-terminal sequences of two internal regions of the 45 kDa protein were determined, and degenerate oligonucleotides were used to clone the gene. The gene isolated in this way codes a 286 amino acid polypeptide that shows homology with the Pir family of proteins of S. cerevisiae (Russo et al., 1992; Toh-e et al., 1993), accordingly we have named this gene YlPIR1. Disruption of YlPIR1 led to a slight increase in the resistance of the cells to calcofluor white, Congo red and zymolyase, but did not cause changes in cell morphology, growth rate or morphological transition.     

Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9.

Mannoproteins were isolated from Saccharomyces cerevisiae mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion-exchange chromatography. The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa. These compounds were absent in an SDS extract of cell walls or in a hot-citrate extract of mnn9 cells. The carbohydrate part of the purified mannoproteins consisted of (N-acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4. O-Glycosidically linked chains, containing 70% of the mannose, were released by mild beta-elimination. N-Glycosidically linked chains, representing 80% of the (N-acetyl)glucosamine and 20% of the mannose, were released by peptide N-glycosidase F (PNGase F) digestion. Complete degradation of protein by alkaline hydrolysis released besides the N- and O-glycosidically linked chains, another type of carbohydrate chain containing the residual (N-acetyl)glucosamine, mannose and most of the glucose in a molar ratio of 1:17:18. Glucose was beta-glycosidically linked. The results indicate that beta-glucose is linked to PNGase F-resistant N-linked chains present on cell wall mannoproteins. We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall.     

An Enzymatic Method for the Determination of Yeast Cell Wall Glucan in Foods

Processed cell wall β-glucans prepared from strains of baker's or brewer's yeast can be used as a thickening agent in aqueous food systems and they provide a fat-like mouthfeel. This article describes an analytical procedure for the determination of the glucan content added to a variety of food products. The method is based on measurement of reducing sugars produced when the food sample, after appropriate pretreatment, is treated with a commercial microbial β-glucanase (Zymolyase) that is specific for the β-linkages present in the glucan polysaccharide of the added yeast cell walls.     

酶法制备葛根纤维低聚糖

以葛根膳食纤维为原料,采用酶法制备功能性纤维低聚糖,并采用高效阴离子交换色谱法测定所制备样品的糖分组成。研究酶制剂、酶解时间和酶用量对纤维低聚糖组分及含量的影响。结果表明：选择β-葡聚糖酶制备纤维低聚糖优于纤维素酶,酶解产物主要成分为葡萄糖、纤维二糖和纤维三糖。β-葡聚糖酶法制备纤维低聚糖,酶解时间2h、酶用量3U/g时所得酶解产物中纤维二糖和纤维三糖所占比例均较高,纤维低聚糖得率为11.8g/100g。