甲基对硫磷水解酶的酿酒酵母表面展示及在甲基对硫磷降解中的应用

PCR扩增假单胞菌WBC-3的甲基对硫磷水解酶基因,插入表面展示质粒pYD1的多克隆位点,构建pYD1-MPH重组质粒。重组质粒转化酿酒酵母EBY100,2%半乳糖诱导甲基对硫磷水解酶表达,并利用免疫荧光检测甲基对硫磷水解酶在酿酒酵母细胞表面的表达展示。研究了表面展示甲基对硫磷水解酶的酶学性质和酵母工程菌对水体中甲基对硫磷的降解效果。结果表明成功构建具有全细胞甲基对硫磷水解酶催化活性的酵母工程菌,经2%半乳糖诱导48 h,表面展示甲基对硫磷水解酶比酶活力为18.2 U/mg细胞干重。表面展示甲基对硫磷水解酶的最适作用pH为9.5,最适作用温度为30℃,在p H4.0-10.5之间和45℃以下稳定性较好,Mn2+、Co2+、Zn2+、Ca2+、Hg2+、K+、Ni2+对表面展示甲基对硫磷水解酶活性有激活作用,Na+、Fe3+、Ag+对展示酶活力有抑制作用。工程菌在1 h内对淡水中20 mg/L的甲基对硫磷的降解率在80%以上。     

表面展示表达植酸酶的重组酿酒酵母构建及酒精发酵

以淀粉为原料的同步糖化发酵是目前乙醇生产的主要途径之一。然而原料中含有的植酸不仅影响酒精发酵效率,而且也会导致环境中难以被植物吸收的磷含量的增加,加剧环境污染。将来源于大肠杆菌的植酸酶基因与酵母编码α-凝集素C端编码序列连接并置于α-因子分泌信号肽下游,构建植酸酶表面展示表达重组载体pMGK-AG-phy并转化工业酿酒酵母,成功获得了在细胞表面锚定表达植酸酶的重组菌PHY。重组酵母的植酸酶表达水平达到6.4 U/g(菌体湿重),其最适温度为55℃,最适pH 4.0,在pH 3.5–4.5范围内具有较高的活性。以玉米粉为原料的同步糖化发酵实验表明,重组酵母PHY的生长速度高于出发菌株,同时酒精产量相较于出发菌株提高了3.7%。更为重要的是发酵后酒糟中植酸磷含量与对照相比降低了91%。构建的表面展示表达植酸酶的重组工业酿酒酵母能够有效降低植酸含量,提高了酒糟的利用价值,减少磷排放,对燃料酒精的环境友好生产具有重要的借鉴意义。     

表面展示表达果胶酯酶的重组酿酒酵母构建及乙醇发酵

【目的】以淀粉为原料的乙醇发酵工艺仍然是当前燃料乙醇的主要生产方式。然而,一些原料中含有的果胶物质不仅降低了乙醇产率,而且会导致醪液粘度增大,从而会进一步影响传质和传热、增加设备负担等。构建能够自主降解果胶质的重组酿酒酵母并应用于燃料乙醇生产是值得探索的领域。【方法】论文将来源于黑曲霉的果胶酯酶基因克隆于α因子信号肽下游并通过酵母α-凝集素C-端蛋白的介导构建了在细胞表面锚定表达果胶酯酶的重组酿酒酵母PE。【结果】重组酵母的果胶酯酶表达水平达到2.6 U/g(菌体湿重),并进一步鉴定了重组果胶酯酶性质。以甘薯粉为原料的同步糖化发酵实验中,重组酵母PE的乙醇浓度和乙醇转化率分别达到95 g/L和88.1%,与出发菌株相比提高了2.2%。更重要的是,表面展示果胶酯酶能够显著降低发酵过程中的发酵液粘度。【结论】通过在工业酿酒酵母表面展示表达果胶酯酶不仅能够提高糖化酶等的作用效果和酿酒酵母的代谢能力,而且能够显著降低乙醇生产过程中发酵液的粘度,将对工业规模乙醇生产在降低设备负担、节约能耗方面具有一定的潜在价值。     

基于壳聚糖结合模块构建新型酿酒酵母孢子表面展示系统

【目的】基于当前细胞表面展示体系存在的问题,旨在构建一个新型的普适性强、抗逆性好、高效稳定的酿酒酵母孢子表面展示系统。【方法】首先,根据酵母孢子固定化酶的特性,通过查阅文献寻找潜在的与孢子壁壳聚糖层高度亲和的壳聚糖结合模块;其次,利用绿色荧光蛋白(green fluorescent protein,GFP)与结合模块融合表达,在体外和孢子内分别验证结合模块与孢子壁的亲和能力;之后,选择Saccharomyces cerevisiae AH109来源的α-半乳糖苷酶(α-galactosidase,MEL1)评估新型展示体系的效能。【结果】首先,选择Paenibacillussp. IK-5来源壳聚糖酶的碳水化合物结合模块32 (carbohydrate binding module 32,CBM32)作为壳聚糖结合模块。其次,将大肠杆菌表达纯化后的融合蛋白CBM32-GFP与dit1Δ孢子共孵育,通过GFP荧光定位以及荧光强度验证CBM32在体外与孢子壁具有较好的亲和能力;CBM32-GFP在dit1Δ孢子内的荧光定位与结合能力证明了其在孢子内能够与孢子壁紧密结合;最后,以MEL1替换GFP应用到新型展示体系中,与只表达MEL1的孢子相比,CBM32-MEL1孢子酶活不仅提高了68.65%,最高酶比活力达到460.59 U/g DCW (dry cell weight, 菌体干重),重复使用能力也得到了显著提高;此外,该体系提高了MEL1的稳定性和可操作性。【结论】本研究首次提出利用结合模块来构建一个新型酿酒酵母孢子表面展示体系,为真核来源的多糖基化位点修饰以及多亚基结构蛋白提供了可靠的细胞表面展示平台,为实现工业化应用孢子固定化酶提供了理论依据。     

利用a凝集素在酿酒酵母表面展示解脂耶氏酵母脂肪酶Lip2

[目的]将解脂耶氏酵母胞外脂肪酶Lip2展示在酿酒酵母表面,构建全细胞催化剂.[方法]采用PCR方法扩增得到解脂耶氏酵母胞外脂肪酶Lip2成熟肽编码基因LIP2,将其连接到AGA2基因的下游构建表面展示载体pCTLIP2.分别以橄榄油、三丁酸甘油酯和对硝基苯酚棕榈酸酯(pNPP)为底物检测展示的脂肪酶酶活.在此基础上,对野生菌及工程菌的酶学性质进行比较.[结果]展示Lip2的酿酒酵母重组菌株在半乳糖的诱导下,表现出水解橄榄油、三丁酸甘油脂以及pNPP的活性,20℃诱导72h时酶活达到最高,为182 U/g干细胞.对展示的Lip2的酶学性质研究表明,其最适温度为40℃,最适pH为8.0,温度稳定性比自由酶有所提高,50℃温浴4 h后残余酶活为其最大酶活的23.2%.以不同碳链长度的对硝基苯酚酯为底物检测其底物特异性,结果显示其水解C8,C12,C16对硝基苯酚酯活性相近,均远高于对硝基苯酚丁酸酯(C4)的水解酶活.[结论]对于Lip2,a凝集素系统是一个有效的展示系统,利用该系统成功将Lip2展示在酿酒酵母表面,从而构建了酿酒酵母全细胞催化剂,该全细胞催化剂具有良好的潜在应用前景.     

酵母表面展示系统研究进展

酵母表面展示系统是继噬菌体展示技术创立后发展起来的真核展示系统,酵母的蛋白质折叠和分泌机制与哺乳动物细胞非常相似,对人的蛋白质表达和展示更具优越性.酵母细胞颗粒大,可用流式细胞仪进行筛选和分离.目前报道的两种酵母展示系统分别以α或a凝集素作为融合骨架.在蛋白质的定向进化、口服疫苗的研制等多方面均有报道.     

酿酒酵母表面展示表达系统及应用

酵母细胞表面展示表达系统是一种固定化表达异源蛋白质的真核展示系统,即把异源靶蛋白基因序列与特定的载体基因序列融合后导入酵母细胞,利用酿酒酵母细胞内蛋白转运到膜表面的机制（GPI锚定）使靶蛋白定位于酵母细胞表面并进行表达。它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白,可应用于生物催化剂、细胞吸附剂、活疫苗、环境治理、蛋白质文库筛选、高亲和抗体、生物传感器、抗原／抗体库构建、免疫检测及亲和纯化、癌症诊断等领域。国内对这一方面研究较少,本文主要介绍了该技术的基本原理、研究现状、应用及其发展前景。     

木糖异构酶在酿酒酵母表面表达及对木糖代谢影响的初步研究

利用α-型酿酒酵母(Saccharomyces cerevisiae)表面展示系统的载体,将来源于嗜热细菌Thermus thermophilus的木糖异构酶基因xylA,插入到酿酒酵母蔗糖酶信号肽序列与α-凝集素的C端编码序列之间,形成融合表达框,构建重组质粒pSY-xy222,转化酿酒酵母H158。含重组质粒的菌株H158-SXI木糖异构酶活性测定表明,细胞壁上酶活测定值为1.53 U,木糖异构酶在酿酒酵母细胞壁上得到活性表达。木糖葡萄糖共发酵结果显示,重组菌株木糖利用率较出发菌株提高了17.8%。     

酵母细胞表面展示系统的研究进展及其应用

酵母表达体系不但有原核表达体系的特点,同时具有真核细胞翻译后蛋白加工修饰的过程,因此,以酵母为基础的表面展示体系在诸多展示系统中有独特的优势。将酶,抗原,抗体和六聚组氨酸等不同蛋白和多肽展示在酵母细胞表面,可实现各种各样的用途。本文主要概述了酵母细胞展示的理论基础、展示体系类型及应用研究的进展。     

重组鲑鱼降钙素前体多肽的制备及其性质研究

硫氧还原蛋白的第 38位 Met突变为 Ala的 Trx- s CT- Gly基因在 E.coli BL2 1 ( DE3)中得到高效表达 .用 Thio Bind亲和树脂纯化表达的融合蛋白 .结果说明 38位的 Met突变为 Ala不影响融合蛋白与树脂的特异性结合 ,融合蛋白的纯度达 90 %以上 .在含有 3mol/L尿素的 70 %甲酸中 ,室温 48h,至少 80 %融合蛋白被溴化氰裂解开 .采用 CM-纤维素吸附 ,用稀盐酸解吸附 ,得到纯度为92 %的降钙素前体多肽 s CT- Gly.氨基酸的序列分析结构表明 ,重组 s CT- Gly的 N端 1 0个氨基酸与预期一致 .在强酸性条件下 ,没有发生氨基酸的脱酰胺反应 ,氨基酸组成分析与预期基本一致 .质谱法测定的分子量为 3492 ,毛细管电泳测定的等电点 p I为 6.46,大鼠降钙素比活性为 1 90 IU/mg左右 ,与天然的人降钙素相当 .     

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.     

酿酒酵母菌培养条件的研究

目的：优化酿酒酵母液体发酵得到菌体的最佳条件。方法：通过单因素试验,以吸光度为指标,研究碳源、氮源、接种量、pH值及无机离子对酿酒酵母菌生长的影响。结果：酿酒酵母生长的最佳碳源是葡萄糖,最佳氮源是蛋白胨,最佳接种量2％,最佳初始pH为4．5,添加无机盐硫酸亚铁能够促进其生长。结论：得到了酿酒酵母液体生长的最佳培养基配方。     

Explanation for Unusual Potency of Salmon Calcitonin

THE calcitonins are polypeptide hormones of thirty-two amino-acids which lower serum calcium in mammals by inhibiting bone resorption¹. During evaluation of these hormones as a means of treating skeletal disorders in man, particularly Paget's disease of bone^{2, 3}, the surprising observation was made that calcitonin from the salmon (SCT) is 20–200 times more potent than porcine calcitonin (PCT) and at least ten times more potent than human calcitonin^{3, 4}. SCT is far more potent than any mammalian calcitonin yet tested in a wide variety of animal species^{5, 6}. This unusual potency of salmon calcitonin could reflect either a greater hormone affinity for receptor sites or a greater resistance to metabolic destruction. We now report evidence which supports the latter possibility, infused SCT disappears from the circulation of the dog much more slowly than does PCT.     

酿酒酵母菌培养条件的研究

目的:优化酿酒酵母液体发酵得到菌体的最佳条件。方法:通过单因素试验,以吸光度为指标,研究碳源、氮源、接种量、pH值及无机离子对酿酒酵母菌生长的影响。结果:酿酒酵母生长的最佳碳源是葡萄糖,最佳氮源是蛋白胨,最佳接种量2%,最佳初始pH为4.5,添加无机盐硫酸亚铁能够促进其生长。结论:得到了酿酒酵母液体生长的最佳培养基配方。     

鲑鱼降钙素及其类似物的合成

鲑鱼降钙素及其类似物的合成潘和平王良友陈正英王芳王会信（军事医学科学院基础医学研究所,北京１００８５０）ＳｙｎｔｈｅｓｉｓｏｆＳａｌｍｏｎＣａｌｃｉｔｏｎｉｎａｎｄＩｔｓＡｎａｌｏｇｓＰａｎＨｅ－ＰｉｎｇＷａｎｇＬｉａｎｇ－ＹｏｕＣｈｅｎＺｈｅｎｇ－...     

鲑鱼降钙素在链霉菌中的分泌表达

利用聚合酶链式反应(PCR)技术分别扩增鲑鱼降钙素的编码序列(sCT-Gly)和抗生链霉菌melCl的信号肽编码序列及其上游的调控序列,将鲑鱼降钙素编码序列融合在melCl信号肽编码序列后,定向克隆到链霉菌质粒载体pIJ680中的新霉素磷酸转移酶基因的启动子(Paph)下游,得到的表达质粒pMS680转化变铅青链霉菌(Streptomyces lividans TK54)进行分泌表达。酶联免疫测定法(EIA)显示重组菌株在YEME培养基中的分泌表达水平于96h达到最高,表达量大于100μ/L培养液。实验表明表达产物能降低大鼠血清钙的浓度。高压液相色谱(HPLC)结果表明,表达产物中主峰的保留时间与鲑鱼降钙素标准品的保留时间基本一致。以上结果提示,鲑鱼降钙素在链霉菌中获得了成功的分泌表达。     

Surface display of active lipases Lip7 and Lip8 from Yarrowia Lipolytica on Saccharomyces Cerevisiae

For unsuspecting bacteria, the difference between life and death depends upon efficient and specific responses to various stressors. Facing a much larger world, microbes are invariably challenged with ever-changing environments where temperature, pH, chemicals, and nutrients are in a constant state of flux. Only those that are able to rapidly reprogram themselves and express subsets of genes needed to overcome the stress will survive and outcompete neighboring microbes. Recently, low shear stress, emulating microgravity (MG) experienced in space, has been characterized in a number of microorganisms including fungi and prokaryotes ranging from harmless surrogate organisms to bona fide pathogens. Interestingly, MG appears to induce a plethora of effects ranging from enhanced pathogenicity in several Gram-negative enterics to enhanced biofilm formation. Furthermore, MG-exposed bacteria appeared better able to handle subsequent stressors including: osmolarity, pH, temperature, and antimicrobial challenge while yeast exhibited aberrant budding post-MG-exposure. This review will focus on MG-induced alterations of virulence in various microbes with the emphasis placed on bacteria.     

Genetic Characterization of Pathogenic Saccharomyces Cerevisiae Isolates

Saccharomyces cerevisiae isolates from human patients have been genetically analyzed. Some of the characteristics of these isolates are very different from laboratory and industrial strains of S. cerevisiae and, for this reason, stringent genetic tests have been used to confirm their identity as S. cerevisiae. Most of these clinical isolates are able to grow at 42°, a temperature that completely inhibits the growth of most other S. cerevisiae strains. This property can be considered a virulence trait and may help explain the presence of these isolates in human hosts. The ability to grow at 42° is shown to be polygenic with primarily additive effects between loci. S. cerevisiae will be a useful model for the evolution and genetic analysis of fungal virulence and the study of polygenic traits.     

Mutations Affecting the Trna-Splicing Endonuclease Activity of Saccharomyces Cerevisiae

Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.     

Preparation and Characterization of Salmon Calcitonin–biotin Conjugates

This study was performed to prepare and characterize the biotinylated Salmon calcitonin (sCT) for oral delivery and evaluate the hypocalcemic effect of biotinylated-sCTs in rats. Biotinylated sCTs was characterized by using high performance liquid chromatography (HPLC) and MALDITOF-MS. The effect of biotinylation on permeability across Caco-2 cell monolayers was examined. Their hypocalcemic effect was determined in rats. Mono- and di-bio-sCTs were separated by reverse phase HPLC. The molecular weights of mono-bio-sCT and di-bio-sCT were determined to be 3,660.5 and 3,900.2 Da, respectively. The permeability of biotinylated-sCTs across Caco-2 cell monolayers was observed with a significant enhancement compared with sCT. Intrajejunal (ij) administration of mono-bio-sCT and di-bio-sCT resulted in sustained reduction in serum calcium levels, with a maximum reduction (% max(d)) of 21.6% and 30% after 4 h and 6 h of application, respectively. The biotin conjugation of sCT may be a promising strategy for increasing the oral bioavailability of sCT and achieving sustained calcium-lowering effects.