黑曲霉糖化酶基因在酿酒酵母基因组中的整合及其在整合子中的稳…

把黑曲霉糖化酶ｃＤＮＡ连同酵母ａ因子启动子及其分泌序列,通过转化整合到酿酒酵母染色体ＤＮＡ上,获得了整合型的分解淀粉酵母转化子。Ｓｏｕｔｈｅｒｎ印迹分析证明了糖化酶ｃＤＮＡ对酵母染色体ＤＮＡ的整入。整合型转化子在以可溶性淀粉为碳源的培养基中分泌糖化酶活力达２．５ｕ／ｍｌ,在非选择性培养基中连续转移１０次,糖化酶分泌活力稳定不变。     

水解淀粉的酿酒酵母菌的构建

把黑曲霉糖化酶cDNA,酵母磷酸甘油激酶基因启动子区和终止区以及酵母Ty因子的δ序列构建成整合型的糖化酶表达分泌质粒pKG 1。该质粒转化酿酒酵母Y33得到整合型转化子。转化子分泌糖化酶活力在3.0μ/ml以上,在以5％可溶性淀粉为碳源的培养基中静止培养7d,淀粉利用率达86％,生成酒精的浓度与以5％葡萄糖为碳源时相等。     

黑曲霉糖化酶在酿酒酵母中的表达和分泌

从黑曲霉糖化酶高产株T2l合成的糖化酶cDNA,经5’端和3’端改造后克隆到酵母质粒YFDl8上,转化酿酒酵母。转化子的淀粉培养基平板检测,培养滤液蛋白电泳和糖化酶活力分析都表明,含有糖化酶基因表达质粒的酵母转化子能有效地分泌有功能的糖化酶到细胞外。实验证明酵母a园子启动子和分泌信号序列能促使黑曲霉糖化酶cDNA在酵母中表达和分泌．实验还表明．黑曲霉糖化酶原的翻译后加工序列很可能亦能被酵母识别,加工生成有功能的成熟的糖化酶。以上成功为构建有实用意义的淀粉水解酵母工程菌迈出了重要的一步。     

黑曲霉糖化酶cDNA的改造及其在酿酒酵母中的表达

应用PCR技术扩增黑曲霉糖化酶cDNA不含非编码区50bp的5’端740bp的序列与该cDNA3’端1400bp的序列连接,获得切除了5’端非编码的糖化酶cDNA。将改造后的cDNA插到质粒pMA91的酵母PGK基因的启动子和转录终止信号之间,构建了含黑曲霉糖化酶基因的表达载体pMAG17。用原生质体转化法将重组质粒pMAG17引入酿酒酵母GRF18。酿酒酵母GRF18转化子在淀粉平板上产生水解透明圈,表明糖化酶已在酵母中表达并分泌至培养基中。测定转化子的胞外酶活力及淀粉水解率。结果表明：改造后的糖化酶基     

α－淀粉酶和糖化酶在酿酒酵母中的表达和分泌

将地衣芽孢杆菌α－淀粉酶基因及黑曲霉糖化酶cDNA重组进大肠杆菌．酵母穿梭质粒,转化酿酒酵母,构建能分解淀粉的酵母工程菌。酶活力测定和酶学性质分析的结果显示：在酵母MF—αl因子及磷酸甘油酸激酶基因的启动子和终止信号的调控下,α－淀粉酶和糖化酶基因在酵母中获得高表达并向胞外分泌这两种酶。构建的酵母工程菌在含10％淀粉的培养基中6天内能水解97%的淀粉。重组质粒能在酵母中较稳定地存在。     

大麦α-淀粉酶和黑曲霉糖化酶在酿酒酵母中的表达和分泌

将大麦α 淀粉酶和黑曲霉糖化酶cDNA重组进同一大肠杆菌 酵母穿梭质粒构建含双基因的表达分泌载体 pMAG1 5 .用原生质体转化法将 pMAG1 5引入酿酒酵母 (S .cerevisiae  GRF1 8) ,在酵母PGK基因的启动子和转录终止信号及本身的信号序列的调控下 ,实现大麦α 淀粉酶和糖化酶的高效表达 ,99%以上的酶活力分泌至培养基中 .构建的酿酒酵母菌株GRF1 8( pMAG1 5 )在含 1 5 %可溶性淀粉的培养基中 ,培养 47h能水解 99%的淀粉 ,并能发酵产生酒精     

α淀粉酶和糖化酶在酿酒酵母中的表达和分泌

将地衣芽孢杆菌α-淀粉酶基因及黑曲霉糖化酶cDNA重组进大肠杆菌-酵母穿梭质粒,转化酿酒酵母,构建能分解淀粉的酵母工程菌。酶活力测定和酶学性质分析的结果显示:在酵母MF-α1因子及磷酸甘油酸激酶基因的启动子和终止信号的调控下,α-淀粉酶和糖化酶基因在酵母中获得高表达并向胞外分泌这两种酶。构建的酵母工程菌在含10％淀粉的培养基中6天内能水解97％的淀粉,重组质粒能在酵母中较稳定地存在。     

枯草芽孢杆菌bgls基因在酵母染色体上整合

将枯草杆菌β－1,3—1,4－葡聚糖酶基因(bgls)2．7kbEcoRl片段和酵母染色体Rdna片段克隆到整合型不含酵母自主复制序列(Autonomusly replicating sequence)的大肠杆菌／酵母菌穿梭质粒YIP5上,构建成YIP5-bgls—Rdna的杂种质粒PCZH,转化S．Cerevisiae并得到表达。稳定性测定表明．Y 33(PCZH101)和Y 33(PCZH104)在无选择压力的YEPD培养基中繁殖70代以上,两个转化子90％以上的酵母细胞仍含有质粒并且遗传特征与染色体行为相似。以bgls基因作探针;与酵母染色体DNA的Southern bloot分子杂交证实bgls基因已整合到酵母菌染色体上。     

黑曲霉糖化酶和木聚糖酶基因在工业用酿酒酵母中的共表达

构建了黑曲霉糖化酶、木聚糖酶基因双表达的酵母YIp型载体pNEW4,通过与G418抗性质粒共转化,将糖化酶和木聚糖酶基因表达元件整合到多倍体酒精生产用酵母S. cerevisiae2.346染色体上,获得了整合型分泌表达这两种酶的工业酿酒酵母工程菌株GX11,研究了重组糖化酶和木聚糖酶在酵母工程菌中的表达及性质。     

黑曲霉木聚糖酶在工业酒精酵母中的分泌表达

用重叠延伸PCR方法从黑曲霉 (Aspergillusniger)UV 11的基因组DNA中克隆出木聚糖酶的cDNA基因 ,构建了由酵母乙醇脱氢酶 (ADH1)启动子和终止子引导表达、木聚糖酶自身信号肽引导分泌、rDNA序列介导的酵母整合型分泌表达质粒pAX2。用pAX2与酵母YEp型G4 18抗性质粒共转化野生型工业酒精酵母S .cerevisiae 2 346 ,获得了整合型分泌表达木聚糖酶的酵母重组菌株XY2。发酵分析表明该工程菌能够明显提高酒精生产率     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

AIMS: Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. METHODS AND RESULTS: First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4.5-5.0 and 65 degrees C. The enzyme was stable at 60 degrees C, the enzyme activity kept 80% after 60 min incubation at 70 degrees C. The half-life was 40 and 10 min under incubation at 80 and 90 degrees C respectively. CONCLUSIONS: A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.     

Cloning of the alpha-amylase cDNA of Aspergillus shirousamii and its expression in Saccharomyces cerevisiae.

alpha-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined. The alpha-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active alpha-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.     

Secretion of alpha-amylase and multiple forms of glucoamylase by the yeast Trichosporon pullulans

Trichosporon pullulans IGC 3488 produced extracellular alpha-amylase and glucoamylase activities when grown in batches in a medium containing corn steep liquor and soluble starch or corn starch. alpha-Amylase, unlike glucoamylase activity, was secreted biphasically. For both amylases the maximum concentration was found in stationary phase cultures. The amylolytic enzymes, previously concentrated by ammonium sulfate precipitation, were separated into a glucoamylase fraction and an alpha-amylase fraction by Ultrogel AcA 54 gel filtration. Pullulanase activity was located in the glucoamylase fraction, whereas cyclodextrinase activity was restricted to the alpha-amylase fraction. Isoamylase and alpha-glucosidase were not detected. Electrophoretic analysis showed that alpha-amylase activity was due to a single protein. Glucoamylase, however, occurred in multiple forms. The four glucoamylases and the alpha-amylase were glycoproteins.     

Purification and characterization of an extracellular glucoamylase from the yeastCandida tsukubaensis CBS 6389

The starch-degrading yeastCandida tsukubaensis CBS 6389 secreted amylase at high activity when grown in a medium containing soluble starch. The extracellular α-amylase activity was very low. The major amylase component was purified by DEAE-Sephadex A-50 chromatography and Ultrogel AcA 44 gel filtration and characterized as a glucoamylase. The enzyme proved to be a glycoprotein with a molecular weight of 56000. The glucoamylase had a temperature optimum at 55°C and displayed highest activity in a pH range of 2.4–4.8. Acarbose strongly inhibited the purified glucoamylase. Debranching activity was present as demonstrated by the release of glucose from pullulan.     

A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger

Abstract The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10–20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase ( gla A), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any gla A coding sequence. The portion of gla A used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.     

Cloning of the α-Amylase cDNA of Aspergillus shirousamii and Its Expression in Saccharomyces cerevisiae

α-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3? non-coding regions, respectively, so far determined. The α-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active α-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.