β-葡萄糖苷酶在工农医领域的应用

β-葡萄糖苷酶能够水解多种β-葡萄糖苷.它与其他纤维素酶共同作用,可以将自然界中含量丰富的纤维素水解成人类可以直接利用的能源物质--葡萄糖;β-葡萄糖苷酶在医学上也有重要的应用,它可用于一些肿瘤疾病的诊断和治疗.人缺乏β-葡萄糖苷酶,可以引起葡萄糖苷-N-脂酰鞘氨醇在巨噬细胞溶酶体内积累,引发戈谢病(Gaucher disease).     

利用黑曲霉β-葡萄糖苷酶催化香兰素葡萄糖苷水解

本文通过在香兰素葡萄糖苷溶液中加入黑曲霉菌种发酵制取的β-葡萄糖苷酶,进行酶促反应实验,定时取样进行高效液相分析检测,结果表明在反应过程中,溶液中香兰素葡萄糖苷的含量呈逐步下降趋势,香兰素的量呈逐步增加趋势;而在没有加β-葡萄糖苷酶的对照实验中,整个反应过程中香兰素葡萄糖苷的含量基本没有出现什么变化,在反应液中也没有检测到香兰素,这说明黑曲霉β-葡萄糖苷酶能够催化香兰素葡萄糖苷分解为香兰素的反应.     

Talaromyces piceae来源β-葡萄糖苷酶在毕赤酵母中的重组表达及发酵优化

β-葡萄糖苷酶在利用高浓度葡萄糖生产低聚龙胆糖的工业应用中有很大潜力,但巨额的加酶量和菌株的蛋白表达能力一直是其工业化的瓶颈.实验室前期研究发现,蓝状菌Talaromyces piceae来源的β-葡萄糖苷酶TpBgl3A能以较低加酶量高转化率制备低聚龙胆糖,但由于其在毕赤酵母中发酵水平尚不理想,使得加酶成本仍不能满足...     

用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因

目的:为提高β-葡萄糖苷酶的产量,用毕赤酵母取代理氏木霉用于生产,以弥补理氏木霉在大规模生产中的缺陷。方法:用套叠PCR法从理氏木霉基因组中扩增β-葡萄糖苷酶基因(bglⅠ)。用T4DNA连接酶和限制性DNA内酶将bglⅠ重组于P.pastoris表达载体pPIC9K的多克隆位点,获得含bglⅠ的重组表达载体pPIC9K-bglⅠ。通过电转法将其pPIC9K-bglⅠ载体转化于P.pastoris基因组,筛选高G418抗性以及高表达bglⅠ酶的重组子作为工程菌。结果:用BMGY-BMMY培养基体系,在摇瓶中发酵48 h,表达BglⅠ30 mg/L,在P.pastoris中表达的BglⅠ能水解对硝基苯-β-D-葡萄糖苷具有β-葡萄糖苷酶活性。其酶活力为56 U/L发酵液。结论:通过这种方法,可以成功地用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因。     

黑曲霉β-葡萄糖苷酶的酶学特性研究

研究黑曲霉β-葡萄糖苷酶的酶学特性,采用酶学研究方法,通过硫酸铵沉淀、Sephadex G-25脱盐和Sephadex G-100纯化了β-葡萄糖苷酶,并进行了黑曲霉β-葡萄糖苷酶的最适反应温度、最适pH、热稳定性、pH稳定性及米氏常数等特性研究,采用SDS-PAGE凝胶电泳测定了分子量。研究表明,β-葡萄糖苷酶的最适反应温度为70℃、最适反应pH为4.5;在40、50和60℃下较稳定,80℃以上稳定性差;β-葡萄糖苷酶在pH为3、7、8、9的缓冲液中的稳定性很差,在pH为4、5、6的缓冲液中稳定性较好,其中在pH为5时,稳定性最好;酶的Km=41.67 mmol/L,Vmax=23.81 U/L;其分子量为65.2 ku。β-葡萄糖苷酶在饲料工业具有良好的应用前景。     

重组毕赤酵母生产β-葡萄糖苷酶发酵条件初步研究

为提高重组毕赤酵母(P.pastoris KM71/pPIC9K-bgl)生产β-葡萄糖苷酶的产量,在摇瓶条件下对重组P.pastoris产β-葡萄糖苷酶的发酵过程进行了优化,得到最佳的条件:生长阶段甘油浓度为30 g/L,接种量为10%,诱导阶段甲醇的初浓度为4%,过程补加甲醇0.5%,诱导温度30℃,pH7.5,诱导周期120 h,酶活可达到245 U/mL。在此基础上,在3 L发酵罐上进行初步放大,流加甘油提高细胞密度至OD_(600)为170,开始流加甲醇诱导,最终BGL酶活达到1 175 U/mL。比摇瓶提高了4.8倍,为β-葡萄糖苷酶工业化生产打下了坚实的基础。     

扣囊复膜孢酵母b-葡萄糖苷酶基因在工业酿酒酵母中的表达

本文以工业酿酒酵母菌株( Saccharomyces cerevisiae Y )为研究对象,针对其复杂的生理生化遗传特性,建立了相对应的转化体系。以pRS41H质粒为基础载体,构建了含有工业酿酒酵母自身的gpd2启动子、终止子和扣囊复膜孢酵母的b-葡萄糖苷酶基因bgl的重组质粒pRS-gb。电击转化进入工业酿酒酵母细胞,潮霉素抗性筛选,获得重组菌。该重组菌可以在以纤维二糖为唯一碳源的培养基中生长,培养36 h,b-葡萄糖苷酶酶活达到0.967 u/ml。以纤维二糖为唯一碳源的酒精发酵中,酒精度可以达到0.92 g/l。这对工业生产中利用纤维素为原料发酵生产酒精具有重要意义。     

一种来源于蜗牛酶的β-葡萄糖苷酶的纯化

通过DEAE-Sepharose离子交换分段层析、DEAE-Sepharose离子交换梯度层析和Sephadex G-100凝胶过滤层析三种方法的联用,从中华白玉蜗牛消化酶中提纯出一种β-葡萄糖苷酶。该酶在SDS-PAGE上呈单一蛋白质条带。应用SDS-PAGE和凝胶过滤层析测定其分子量,提示该酶是由4个分子量为110~115 kD的相同亚基组成的同源四聚体。pNPG为底物的动力学参数Km和Vmax分别为0.182 mmol/L和0.189μmol/(min.mg)。     

长梗木霉β-葡萄糖苷酶基因的克隆及表达

【目的】以实验室筛选获得的一株长梗木霉GM2(Trichoderma longibrachiatum)为材料,克隆出其β-葡萄糖苷酶(β-Glucosidase)基因bgl并在大肠杆菌和酵母中进行表达。【方法】利用同源克隆扩增出其β-葡萄糖苷酶基因bgl全长序列,分别亚克隆到质粒pET-32a(+)和pPICZα-B中,构建其原核表达载体pET32a(+)-bglI和真核表达载体pPICZα-B-bgl。【结果】bgl基因序列全长2 369 bp,含两个内含子,编码744个氨基酸。在大肠杆菌BL21(DE3)中表达bgl,重组蛋白以包涵体形式存在,上清液中没有β-葡萄糖苷酶的酶活。将载体pPICZα-B-bgl电转化入毕赤酵母GS115,得到78 kD左右重组蛋白,与预测大小相符。按9%接种量接入50 mL YP培养基(初始pH 5.5),30°C振荡培养96 h,添加终浓度1%的甲醇诱导后β-葡萄糖苷酶酶活达60 U/mL。重组酶bgl催化水杨苷水解反应的最适pH为5.0,最适温度为70°C;另外,此bgl在pH 3.0 10.0和40°C 60°C范围内具有比较好的稳定性。【结论】长梗木霉GM2的β-葡萄糖苷酶在P.pastoris中获得可溶性表达,并证明有一定的活性。     

烟曲霉β-葡萄糖苷酶的基因克隆、表达及酶学性质分析

探索获得优良的β-葡萄糖苷酶基因,对实现其工业化生产具有重要意义。烟曲霉Aspergillus fumigatus基因组中含有一个bgl基因(1 752 bp),编码的蛋白约65 kDa,推测为属于糖苷水解酶家族的β-葡萄糖苷酶。将bgl基因克隆并构建了重组表达载体pGEX-bgl,转化大肠杆菌Escherichia coli BL21(DE3),经IPTG诱导获得表达。重组蛋白经亲和层析纯化后,以七叶苷为底物进行了酶学分析,结果表明该酶的最适温度是45℃,最适pH在5.5~6.0之间,对七叶苷的Km值为17.7 mmol/L。该酶在pH 4~7范围内稳定;70℃保温2 h后仍能保持60%的活性。金属离子和化学试剂对酶活性有不同程度的影响,Ca2+对重组酶有轻微的激活作用,而SDS可强烈抑制其活性。由于其相对于真菌来源的其他葡萄糖苷酶稳定性较高,为进一步的研究与应用奠定了基础。     

拟康氏木霉eg1基因的克隆及在酿酒酵母中的分泌表达

从拟康氏木霉3.3002基因组中克隆了内切葡聚糖酶EGI基因,该基因全长1566 bp,由3个外显子2个内含子组成,编码461个氨基酸.编码蛋白EGI的N端为22aa组成的信号肽,其后依次为催化结构域、连接肽和结合结构域.采用重叠PCR法获得无内含子的内切葡聚糖酶基因eg1,并将其成熟肽编码序列插入酿酒酵母分泌型表达载...     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

PCR differentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using specific primers

The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5'-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3'-SSU end) and the 3'-specific primers were located in the LSU gene (positions 560-590 from the 5'-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.     

Oenococcusoeni苹果酸-乳酸酶基因克隆及其在酿酒酵母中的表达

苹果酸-乳酸酶是苹果酸-乳酸发酵过程中负责苹果酸转化为乳酸的功能酶。在进行酒酒球菌SD2a的苹果酸-乳酸酶基因(mleA)克隆测序基础上,以PGK1强启动子和ADH1终止子为调控元件,以大肠杆菌-酵母菌穿梭质粒YEp352为载体,构建了重组表达质粒并转化酿酒酵母YS58。酵母转化子用SD/Ura平板筛选鉴定。斑点杂交检测表明目的基因mleA转化到受体菌中,SDSPAGE检测表明获得的转化子表达了约60kDa的目标蛋白。获得的转化子在添加了L苹果酸的培养基中培养4d;取培养液上清用HPLC检测L苹果酸及L乳酸含量,采用t检验进行差异显著性分析,结果表明mleA基因进行了功能性的表达,将L苹果酸转化成L乳酸,L苹果酸和L乳酸含量分别与对照差异极显著和显著,苹果酸的相对降低率平均为20.95%。在有选择压力条件下,重组质粒相对稳定,而在无选择压力条件下,传代培养10d后大约有65%的重组质粒丢失。     

Detection of inactive precursors of beta-glucanases in Saccharomyces cerevisiae

Accumulation and secretion of beta-glucanases have been studied in vivo by using a thermosensitive secretory mutant of Saccharomyces cerevisiae blocked at the endoplasmic reticulum level (sec 18-1). When incubated at the restrictive temperature no accumulation of active glucanases was observed. Following a shift to permissive conditions in the presence of cycloheximide a rise in the internal activity took place. The increase in total glucanase activity was partially due to the activation of an exo-glucanase that hydrolyzes PNPG. It is concluded that glucanases are synthesized in inactive precursor forms and are converted to the active forms in their secretory pathway.     

瑞氏木霉木糖醇脱氢酶基因的分离与鉴定

将在木聚糖上生长的瑞氏木霉(Trichoderma reesei)RutC-30的cDNA文库全部质粒转化已携带有毕赤氏酵(Pithia stipitis)木糖还原酶基因的重组酿酒酵母(Saccharomycescerevisiae)菌株H475,在H475中构建了瑞氏木霉的cDNA表达亚文库。在以木糖为唯一碳源的选择性酵母合成培养基上,从该亚文库中筛选到瑞氏木霉木糖醇脱氢酶cDNA基因．该基因片段长为1．3kb。Southern、Norhern印迹杂交分析和蛋白质凝胶电泳结果表明该基因确实来源于瑞氏木霉,所编码蛋白质分子量约为40kDa。携带有毕赤氏酵母木糖还原酶和瑞氏木霉木糖醇脱氢酶基因的重组酵母能够在以木糖为唯一碳源的培养基上生长,并能将90％以上的木糖转化为木糖醇、乙醇和其它副产品。     

Studies on azole-induced cell death in Saccharomyces cerevisiae

Abstract The Saccharomyces cerevisiae strain XL16-5B exhibited a fungicidal response to treatment with ketoconazole. Cell death became apparent during prolonged treatment over 72 h following an initial period over 24 h where viable cells were found and limited cell division occurred. Sterol analysis showed some differences between XL16-5B and the strain XY729-5a, which had a fungistatic response to ketoconazole. In particular, the level of ergosterol was higher in XL16-5B and remained high during treatment.