中温α-淀粉酶基因在枯草芽孢杆菌中的整合及高效表达

从中温α-淀粉酶生产菌株枯草芽孢杆菌(Bacillus subtilis)264中克隆得到中温α-淀粉酶基因(amy L)并构建了重组表达质粒p AX01-amy L。该质粒转化枯草芽孢杆菌264后利用同源重组机制将由木糖操纵子引导的amy L表达盒整合入枯草芽孢杆菌染色体的lac A位点,以增加中温α-淀粉酶基因的拷贝数。经淀粉平板产酶初筛及Southern blot鉴定,成功分离获得了可高效分泌表达中温α-淀粉酶的基因工程菌ZHWY。该菌株在摇瓶发酵75 h后α-淀粉酶酶活可达到730 U/m L,比酶活为156 U/mg总蛋白,与原始菌株264相比提高了70%,且继代过程中表达水平稳定。在5 L发酵罐中,枯草芽孢杆菌ZHWY发酵所产中温α-淀粉酶酶活最高达到1450 U/m L,具有良好的工业应用前景。     

α-淀粉酶和糖化酶的表达及酿酒酵母工程菌的构建

将切除了５′端非编码区５０碱基对片段的黑曲霉糖化酶ＧＡＩｃＤＮＡ与大麦α淀粉酶基因重组进大肠杆菌酵母穿梭载体,构建重组表达质粒ｐＭＡＧ１１,转化酿酒酵母ＧＲＦ１８,获得含α淀粉酶和糖化酶双基因的酵母工程菌ＧＲＦ１８（ｐＭＡＧ１１）．在酵母ＰＧＫ基因启动子和终止信号的调控下,α淀粉酶和糖化酶基因获得高效表达,９９％的表达产物分泌至胞外．在含ｗ＝１５％的可溶性淀粉的ＹＰＳ培养基中培养４４ｈ,淀粉水解率达到９９％,并能发酵产生酒精     

耐酸耐温α-淀粉酶基因在大肠杆菌中的功能表达

兼顾大肠杆菌与枯草芽孢杆菌密码子偏好性,优化获得了1个耐酸耐温淀粉酶的基因amyCN1,并将其在大肠杆菌中实现了功能表达。纯化后的重组α-淀粉酶(AMY1)表征结果表明:在最适pH 5.5和最适温度75℃条件下表观米氏常数(Km)值和催化效率(kcat/Km)值分别为20.93 g/L和98.20 L/(g.s);低浓度的Co2+(1 mmol/L)可以提高30%的淀粉酶活力,Mn2+抑制了大部分活力,包括Ca2+在内的其他金属离子影响不显著;高浓度的EDTA(≥50 mmol/L)抑制其活力;生物信息学分析表明,AMY1具有α-淀粉酶家族典型的3-结构域分布,具有1个Ca2+结合位点和5个Zn2+结合位点,活性中心催化氨基酸残基D198和E222位于(β/α)8桶中β折叠片C端一侧。AMY1优良的低pH耐受性和耐温性,有助于木薯淀粉生产燃料乙醇工业液化与糖化同步发酵工艺的实现。     

中温α-淀粉酶基因的克隆及在枯草芽孢杆菌中的高效表达

利用高效表达栽体pWB980,实现了Bacillus subtilis BF7658中温α-淀粉酶基因amy在B.subtilis DB403中的高效表达,活力达到770 U/mL.经多步纯化,重组酶AMY的比活达到35.8 U/mg,纯化倍数为1.7,获得凝胶电泳条带单一的蛋白样品,经SDS-PAGE检测,重组酶AM...     

β—淀粉酶基因在黄单胞菌中的克隆及表达

广泛寄主范围载体ｐＫＴ２１０可在辅助质粒ｐＲＫ２０１３的协助下转移进入黄单胞菌（革兰氏阴性）中并能稳定存在；来源于枯草杆菌的β－淀粉酶基因与载体ｐＫＴ２１０形成重组质粒ｐＹＬ１，并以其转化大肠杆菌；通过三亲本接合将ｐＹＬ１引入带有利福平抗性的黄单胞菌ＮＫ－０１－Ｒ中，通过抗性选择接合子，并测定接合子的淀粉酶水解能力及产胶能力。本文获得一株黄原胶产量比出发菌株提高２０％，且淀粉酶水解能力显著提高的工     

α-淀粉酶的超滤研究

对用超滤法浓缩分离α-淀粉酶过程进行了研究,讨论了料液浓度、膜面液体流速、操作压力等因素对超滤过程的影响。实验结果表明,用醋酸纤维素超滤浓缩α-淀粉酶的过程可用扩散模型来描述,超滤时宜控制压力在0.3～0.4MPa范围内。用超滤法能有效地浓缩α-淀粉酶,酶的总回收率大于90％。此外,通过实验建立了分离过程的传质模型,为超滤法浓缩α-淀粉酶的工业设计提供了依据。     

枯草杆菌α-淀粉酶的活性研究

研究了酸度、温度、钙离子对枯草杆菌α－淀粉酶活性的影响。研究表明，ｐＨ值对酶活影响较大，最适作用温度随底物种类及其浓度而异。研究确立了酶一步失活模型，指出在较高温度和长时间作用的条件下，应有钙离子的保护，以发挥α－淀粉酶的效能     

α-淀粉酶的研究与应用

介绍了α-淀粉酶的分离纯化和活力测定方法,耐高温α-淀粉酶和耐酸性α-淀粉酶的研究状况及α-淀粉酶在食品和医药工业中的应用,并指出α-淀粉酶具有很大的经济、社会和环境效益。     

乙腈对α-淀粉酶催化活性的影响

乙腈对α-淀粉酶活性具有抑制作用，这种抑制作用随着乙腈溶液浓度的增大而增强。动力学分析表明乙腈的抑制类型为线性混合型抑制作用。紫外吸收光谱和荧光发射光谱分析显示酶的二级结构在乙腈作用下发生了变化，导致了酶活性的丧失。     

α-淀粉酶固态发酵的研究

研究了固态法发酵生产α－淀粉酶的工艺,用麸皮作原料,其优化条件是:在接种量0.08%,拌水比1：0.6,pH6.0,培养温度37℃,葡萄糖浓度15%,发酵72小时,其酶活力可达1742u/g。与液态发酵相比,固态发酵能有效地克服分解代谢产物的阻遏作用。     

Unequal crossing over in the ribosomal DNA of Saccharomyces cerevisiae

Unequal sister chromatid exchanges occur at the ribosomal DNA locus of yeast during mitotic growth. The frequency of unequal crossing over, as measured by the deletion or duplication of an inserted genetic marker (LEU2), is sufficient to maintain the sequence homogeneity of the rDNA repeat units.     

十字花科黑腐病菌一个致病相关基因的鉴定

十字花科黑腐病菌(Xanthomonas campestris pv.campestris,简称Xcc)8004菌株的一个转座子插入突变体186807,其Tn5gusA5插入位点位于一个推测的双组分调控系统的感受蛋白基因XC2229中.该突变体对寄主植物满身红萝卜的致病力显著降低.为了进一步证实XC2229基因与该菌致病力之间的相关性,本工作构建了该基因的缺失突变体DM2229.DM2229与186B07在寄主上的致病力基本一致,均显著低于野生型Xcc 8004.生化及表型检测结果表明,DM2229的胞外多糖(exopolysaccharides,EPS)产量降低,细胞运动能力减弱.用带有完整XC2229基因的pLALR6互补DM2229,互补菌株CDM2229在EPS合成、细胞运动能力以及致病力方面与野生型Xcc 8004基本一致.这些实验结果表明,XC2229是一个与EPS合成、细菌运动相关的基因.推测该基因通过调控EPS的生成和运动能力而影响Xcc的致病力.     

rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用

根据酵母整合质粒的设计要求,PCR扩增特定的2．2kb rDNA片段,并以此替换酿酒酵母(Saccharomyces cereristae)整合载体YIp5的URA3片段;在此基础上,引入G418抗性基因KanMX和酵母磷酸甘油激酶(phosphoglycerale kinase,PGK)组成型强启动子和终止子序列(PGKp-t),构建适合酿酒酵母工业菌株高拷贝整合表达载体pYMIKP,以细菌木糖异构酶(xylose isomerase,XI)基因xy/A为目标基因,通过载体pYMIKP引入到酵母工业菌株NAN-27中,酵母转化子在非选择培养条件下,连续生长50世代质粒稳定性为99．72％,目标基因高拷贝重组菌的木糖异构酶比酶活是对照菌株的67．2倍,达到0．672U／mg蛋白,实现了外源基因在酿酒酵母工业菌株中的稳定高效表达。     

Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae.

An unusual protein splicing reaction joins the N-terminal segment (A) and the C-terminal segment (C) of the 119K primary translation product (ABC) of the yeast VMA1 gene to yield a 69K vacuolar H(+)-ATPase subunit (AC) and an internal 50K polypeptide (B). This 50K protein is a site-specific DNA endonuclease that shares 34% identity with the homothallic switching endonuclease. The site cleaved by the VMA1-derived endonuclease exists in a VMA1 allele that lacks the derived endonuclease segment of the open reading frame. Cleavage at this site only occurs during meiosis and initiates 'homing', a genetic event that converts a VMA1 allele lacking the endonuclease coding sequence into one that contains it.     

Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae

The migration of genetic information from ancestral prokaryotic endosymbionts into eukaryotic nuclei is thought to have had an important role in the evolution of mitochondria and chloroplasts. Here we describe an assay for the detection of movement of DNA between mitochondria and the nucleus in yeast. Because recombinant plasmid DNA replicates after transformation into mitochondria of yeast strains lacking endogenous mitochondrial DNA we were able to propagate the nuclear genetic marker URA3 in mitochondria. As expected, the wild-type URA3 gene in mitochondria failed to complement the uracil auxotrophy (Ura-) caused by a nuclear ura3 mutation. But selection of Ura+ prototrophs from a Ura- strain carrying URA3 on a plasmid in its mitochondria enabled us to detect plasmid movement to the nucleus. Conversely, as the plasmid used also contained the mitochondrial gene COX2 required for respiratory growth, we were able to set up corresponding selections to detect migration of DNA from the nucleus to the mitochondria. Our results show that, in yeast, DNA escapes from mitochondria and appears in the nucleus at a surprisingly high frequency (approximately 2 x 10(-5) per cell per generation). But the rate at which DNA makes the journey in the opposite direction--nucleus to mitochondria--is apparently at least 100,000 times less.     

Expression of the SOD gene from Trichoderma harzianum in Saccharomyces cerevisiae

Superoxide dismutases are metalloproteins which play a major role in defense against oxygen radicalmediated toxicity in aerobic organisms. Such proteins are important endogeneity cytoprotection factor involving defence. A 751-bp full-length cDNA sequence of an SOD gene was isolated from the Trichoderma harzianum. The full-length cDNA of the SOD gene consists of one 465-bp open reading frame nucleotide, which encodes a 15.7-kDa polypeptide consisting of 154 amino acid residues. Sequence analysis revealed that SOD gene has more than 72%-86% amino acid sequence homology with those of other fungi. The SOD gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2-SOD. SOD expressed by pYES2-SOD was induced by galactose. We test whether SOD could offer abiotic stress resistance when it was introduced into yeast ceils. A transgenic yeast harboring T. harzianum SOD was generated under the control of a constitutively expressed GAL promoter. The results indicated that SOD yeast transformants had significantly higher resistance to salt and drought stress.     

短小芽孢杆菌C-9葡聚糖内切酶基因的克隆及其在酿酒酵母中的表达

从短小芽孢杆菌(Bacillus pumilus)C-9中克隆得到葡聚糖内切酶基因,该基因包含1980 bp核苷酸,编码559个氨基酸,其N端有一个预测的29个氨基酸的信号肽序列;以YIp5质粒为骨架,构建rDNA介导的多拷贝整合载体,实现葡聚糖内切酶在酿酒酵母中的高效、稳定表达。与对照菌株相比,含有葡聚糖内切酶基因的重组酿酒酵母可以在以羧甲基纤维素为唯一碳源的培养基上生长。     

ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae

The oncogenes of Harvey and Kirsten murine sarcoma viruses (v-rasH and v-rasK) and their cellular homologues (c-rasH and c-rasK) constitute two members of the ras gene family. Each functional member of the ras gene family encodes a 21,000 molecular weight protein (p21ras). ras genes have been detected in a wide variety of vertebrate species, including Xenopus laevis (R. E. Steele, personal communication), and in Drosophila melanogaster. We report here the detection of ras-related genes in the yeast Saccharomyces cerevisiae, and the isolation of two ras-related molecular clones, c-rassc-1 and c-rassc-2, from the DNA of Saccharomyces. Both c-rassc-1 and c-rassc-2 hybridize specifically to probes prepared from mammalian ras DNA. Sequencing of c-rassc-1 reveals extensive amino acid homology between the protein encoded by c-rassc-1 and the p21 encoded by c-rasH. Our studies suggest that these clones can be used to elucidate the normal cellular functions of ras-related genes in this relatively simple eukaryotic organism.     

转座子诱变甘蓝黑腐病菌所获胞外多糖突变体的类型

根据对转座子Ｔｎ５ｇｕｓＡ５诱变甘蓝黑腐病菌所获得３９株胞外多糖突变体的３种胞外酶活性和在含２％葡萄糖的ＮＹＧＡ培养基上的菌落形态的检测结果，将突变株分成５种类型。Ｓｏｕｔｈｅｒｎ杂交结果表明它们的突变位于基因组中的８个不同的位置，其中１个位于已鉴定的ｒｐｆ调控基因簇