产甘油假丝酵母胞浆3磷酸甘油脱氢酶在甘油形成中的作用

为了研究胞浆３磷酸甘油脱氢酶（ｃｔＧＰＤ, ＥＣ １．１．１．８）在产甘油假丝酵母（ ＣａｎｄｉｄａｇｌｙｃｅｒｏｌｇｅｎｅｓｉｓＺｈｕｇｅ ）甘油合成机制中的作用,考察了发酵实验过程中ｃｔＧＰＤ的活力水平以及高渗环境对该酶酶活水平和甘油形成的影响．结果表明,该菌种中,ｃｔＧＰＤ 的表达在相对低渗的发酵过程中处于一个较高的水平,并在３６ ｈ 和６０ ｈ 处分别出现两次峰值;高渗环境可使ｃｔＧＰＤ酶活增加,但幅度不大．发酵试验表明,随着高渗溶质浓度增大,可在一定程度上恢复细胞产甘油能力,但总水平下降．     

产甘油假丝酵母稳定期酸胁迫耐受性研究

研究了产甘油假丝酵母稳定期的酸胁迫耐受性。结果表明,在一定的酸性pH值范围内,pH值降低而酸胁迫压力增加,可以促进产甘油假丝酵母胞内聚磷酸盐积累,从而保证细胞免受酸胁迫的过度影响,维持较高甘油生产水平所需的活细胞数;但过高的酸性环境会严重损伤细胞,降低细胞合成聚磷酸盐的能力,从而使细胞存活率和甘油产量下降。产甘油假丝酵母胞内积累聚磷酸盐,可以增强其对营养饥饿和强酸胁迫等的抵抗力,从而提高其在生长稳定期的存活率。     

产甘油假丝酵母酸性磷酸酯酶突变株的选育及其性质研究

运用化学诱变法从高产甘油的工业生产菌株产甘油假丝酵母（Ｃａｎｄｉｄａｇｌｙｃｅｒｏｌｇｅｎｅｓｉｓ）ＷＬ２００２－５（Ｈ－）筛选获得两类酸性磷酸酯酶突变株．一类为阻遏型酸性磷酸酯酶缺失突变株;另一类为对磷调节不敏感的部分去阻遏调节突变株,并研究了它们的发酵性能     

高强度产甘油假丝酵母突变株的选育及其发酵性状

以一株产甘油假丝酵母为出发菌株 ,采用化学诱变 ,通过玉米浆和外加无机磷组成的磷质量浓度为 3 40mg/L的磷源选择培养基 ,得到了一株发酵时间较原菌株缩短了 8h左右 ,产甘油能力 (甘油产量约为 1 40 g/L ,甘油转化率为 5 6% )和原菌株相似的突变株 ,并研究了突变株的发酵性状 .     

产甘油假丝酵母在丙三醇发酵过程中的有机酸种类及其变化

HPLC分析研究了产甘油假丝酵母 (Candidaglycerolgenesis)发酵过程中有机酸的种类和含量变化 .C .glycerolgenesis主要在菌体生长期产生有机酸 ,使发酵液总酸增加 .确定了发酵过程形成的有机酸含有乙酸、乳酸、丙酮酸、酮戊二酸、苹果酸等 .建立了HPLC测定有机酸的方法 .分析发现 :有机酸中乙酸质量浓度最高约为 1.6 g/L .随着发酵时间的延长 ,发酵液总酸含量逐渐下降 ,而乙酸和α -酮戊二酸在发酵后期有所回升 .     

产甘油假丝酵母甘油合成关键酶基因CgGPD1多拷贝表达载体的构建及其在大肠杆菌中的表达

采用PCR的方法从产甘油假丝酵母(Candida glycerinogenes) 中扩增出3-磷酸甘油脱氢酶的编码基因及侧翼序列 CgGPD1,分别构建了含不同 CgGPD1 拷贝数的根癌农杆菌双元载体pCAM3300-zeocin-CgGPD1(Ⅰ)、pCAM3300-zeocin-CgGPD1-CgGPD1(Ⅱ)和pCAM3300-zeocin-CgGPD1 -CgGPD1-CgGPD1(Ⅲ).在大肠杆菌JM109中,研究了其在不同质量浓度NaCl、葡萄糖胁迫下表达情况.结果表明,在大肠杆菌中GPDH的活性随着NaCl、葡萄糖质量浓度的升高而增加.当NaCl质量浓度达到2.5 g/dL时,GPDH的酶活比在0.5 g/dL NaCl下平均提高31.2%; 当葡萄糖质量浓度提高至10 g/dL时,GPDH的酶活比2 g/dL葡萄糖下平均提高31.8%;在相同的NaCl、葡萄糖质量浓度下,GPDH的活性随着 CgGPD1 拷贝数的增加而升高,JM109(Ⅱ)比JM109(Ⅰ)的GPDH酶活平均提高8.2%,JM109(Ⅲ)比JM109(Ⅱ)的GPDH酶活平均提高9.9%.以上结果表明,在大肠杆菌中 CgGPD1 基因的表达同样受渗透压胁迫调节.     

产朊假丝酵母生长条件的优化

通过安瓿管复壮的产朊假丝酵母,用YPD培养基进行生长曲线的测定,得出12h是此种酵母菌的生长最优条件。对产朊假丝酵母进行单因素条件筛选,包括培养温度、摇床转速、碳氮比,实验得出温度在30℃、转速180r/min、碳氮比(葡萄糖和蛋白胨的质量比)为3∶1时菌体密度最高。通过采用L9(34)正交实验设计确定各因素对酵母菌生长的影响,优化条件为培养时间13h、温度30℃、转速160r/min、碳氮比约3∶1,此时菌体密度最高。     

产朊假丝酵母利用有机酸的研究

在有机酸为唯一碳源的培养中培养产朊假丝酵母(Candida utilis)时,以L-苹果酸、乳酸、琥珀酸或柠檬酸为碳源的培养基经过48h后,有机酸浓度均由初始浓度5.0g/L下降到0.0g/L。以乙酸、草酸和富马酸为碳源的培养基有机酸浓度始终没有明显变化,说明产朊假丝酵母能够利用细胞外的L-苹果酸、乳酸、琥珀酸和柠檬酸,不能利用乙酸、草酸和富马酸。当葡萄糖和L-苹果酸、乳酸、琥珀酸和柠檬酸中的某种有机酸共同做碳源时,葡萄糖浓度均可以在32h内从20.0g/L降到0.0g/L,而各有机酸在0~24h内浓度变化不大,24~48h浓度均有不同程度的下降,说明当培养基中有葡萄糖时,有机酸的利用受到抑制。当浓度均为2g/L的L-苹果酸、乳酸、琥珀酸和柠檬酸同时做碳源培养产朊假丝酵母时,乳酸大约经过40h浓度首先降到0.0g/L,L-苹果酸、柠檬酸、琥珀酸浓度在0~16h过程中没有明显变化,16~48h下降趋势明显,最后也都被菌体完全利用,说明乳酸比较容易被菌体利用,而L-苹果酸、琥珀酸和柠檬酸在被菌体利用先后顺序上没有明显区别。     

产朊假丝酵母发酵培养基的优化研究

对产朊假丝酵母高产菌体量的液体发酵培养基进行了研究,结果表明其优化后的培养基为:葡萄糖40g/L,蛋白胨10g/L,KH2PO42g/L,MgSO4·7H2O0.3g/L。摇瓶发酵结果菌体干重达到6.37g/L,比基础培养基发酵结果增加了47%。     

高渗环境假丝酵母胞内甘油和海藻糖代谢研究

研究高渗环境中假丝酵母(T酵母)细胞内海藻糖和甘油的代谢情况。利用微波破碎法这一传统的物理破壁法来提取T酵母细胞内甘油和海藻糖,并通过高效液相色谱仪测定细胞内甘油和海藻糖的含量。经过高效液相色谱测定,可以得到不同盐浓度下T酵母胞内甘油和海藻糖含量。T酵母在高渗环境中通过对甘油和海藻糖的积累,同时阻止甘油的外排作用来抵抗外界环境对其生长的影响。     

高渗工业菌株Candida glycerinogenes整合型表达载体的构建及验证

构建应用于工业用产甘油假丝酵母(Candida glycerinogenes)CGMCC NO.6830的整合型表达载体,建立一种可利用底物形成的渗透压调控表达外源基因的体系。以p UC19质粒为基本骨架,C.glycerinogenes CGMCC NO.6830的18S r DNA为整合位点,运用其3-磷酸甘油脱氢酶基因启动子PCggpd启动外源基因表达,腐草霉素抗性基因ble作为重组酵母转化子筛选标记,获得应用于产甘油假丝酵母的稳定的表达型整合载体;以绿色荧光蛋白基因gfp作为报告基因考察该表达质粒的性能,实现了外源基因gfp的渗透压调控表达。     

Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer

The URA3 gene of Candida glycerinogenes WL2002-5, an industrial glycerol producer encoding orotidine-5'-phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein.     

Cloning and characterization of a novel NAD+‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter

A 3950 bp genomic fragment from Candida glycerinogenes, WL2002‐5, containing the CgGAP gene encoding a glyceraldehyde‐3‐phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers, was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids, with a molecular mass of 36 kDa. The CgGAP protein consisted of an N‐terminal NAD⁺‐binding domain and a central catalytic domain. Six stress‐response elements were found in the upstream region of the CgGAP gene. The influence of CgGAP on glycolysis was investigated. Functional analysis revealed that Saccharomyces cerevisiae transformed with CgGAP was restored to the wild‐type phenotype when cultured in high‐osmolarity medium, suggesting that it is a functional GAP protein. Promoter studies in S. cerevisiae using the green fluorescent protein (gfp) gene as a reporter showed that the GAP promoter (P_CgGAP) is constitutively expressed in S. cerevisiae cells grown on glucose. Copyright © 2013 John Wiley & Sons, Ltd.     

A simple and efficient procedure for transformation of Schizosaccharomyces pombe

We describe a simple and efficient procedure for transformation of Schizosaccharomyces pombe. Sz. pombe colonies grown on minimal (SD) plates were directly removed and suspended in a 100 microl reaction mixture containing 70 microl PLATE solution (50% polyethylene glycol-4000, 100 mM lithium acetate, 10 mM Tris-HCl, pH 4.9, and 1 mM EDTA), 10 microl plasmid DNA (1 microg), 10 microl carrier DNA (100 microg) and 10 microl sterile distilled water. After incubation at 30 degrees C for 1 h followed by heat shock treatment at 42 degrees C for 15 min, the reaction mixture was spread on a selection plate. The transformation efficiency obtained using the procedure was approximately 8000 transformants/microg DNA. The method is simple and time-saving, making it especially useful for a large number of samples and when a high transformation efficiency is not required.     

Transformation of Candida albicans with a synthetic hygromycin B resistance gene

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2‐containing plasmids or single‐copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT‐1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild‐type C. albicans. We used PCR to fuse CaHygB or SAT‐1 to approximately 1 kb of 5′ and 3′ noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild‐type C. albicans strains. Homologous targeting frequencies were approximately 50–70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates. Copyright © 2010 John Wiley & Sons, Ltd.     

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.     

皱褶假丝酵母脂肪酶CRL1在毕赤酵母中的高效表达及其催化合成维生素E醋酸酯

本研究将经密码子优化的皱褶假丝酵母脂肪酶(Candida rugosa lipase) CRL1基因克隆到毕赤酵母分泌型表达载体pHKA,构建得到重组菌株GS115/pHKA-CRL1;经摇瓶发酵并结合甲醇诱导120 h处理,该菌株对底物对硝基苯酚丁酯(p NPB)的水解活力达到39.73 U/mL。通过对AOX1启动子和α分泌信号肽同时进行改造,进一步获得重组菌株GS115/pHKA-AOX1m/αm-CRL1,其脂肪酶活力提高达到63.63 U/mL。发酵液上清液经超滤浓缩、硫酸铵沉淀后,再经脱盐和阴离子交换层析处理,获得纯化的重组CRL1。该纯化工艺的纯化倍数为5.41倍,回收率为33.81%。而纯化重组CRL1的比酶活为984.5U/mg。重组CRL1的最适温度为40℃,最适pH为7.5;经40℃保温6 h,仍保留52.99%的相对活力;在偏酸性环境中较为稳定。以摇瓶发酵、真空冷冻干燥后的重组CRL1为催化剂,在无溶剂体系中催化合成维生素E醋酸酯,在最适反应条件下(200 mg D-α-生育酚、1 mL乙酸酐、100 mg CRL1、反应温度60℃、转速200 r/min、反应时间9 h),D-α-生育酚的转化率在97.00%以上。     

检测霜霉威的两种方法的比较

目的比较胶体金法与液相色谱-质谱质谱联用法(LC-MS/MS)在霜霉威检测中的应用。证实胶体金试纸检测蔬菜中霜霉威残留的可靠性,并以此为基础建立一个新的检测手段。方法通过胶体金试纸条半定量方法检测的试样,再用液/质谱法联用定量检测试样中的霜霉威。结果胶体金试纸检测为阴性的100个试样,经液/质谱法联用检测结果为未检出,符合率为100%。添加霜霉威的胶体金试纸检测阳性的试样经液/质谱法联用检测,符合率为100%,添加的Cutoff值样品回收率达到93%。结论与液/质联用检测法相比较,胶体金法检测霜霉威具有操作简便、观察直观、快速、省时的特点,特异性强、敏感性较高,从而为检测霜霉威提供了一种快速的检测方法。