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体pYEα中,构建成pYEα-Peg1重组质粒,转化酿酒酵母.重组转化子经β-半乳糖诱导,检测表达产物的分子大小以及酶活,结果表明,转化子在刚果红平板上可产生明显的水解圈;酶活检测显示该基因能在酿酒酵母中表达有生物活性的EG I并分泌到胞外;SDS-PAGE电泳显示EGI蛋白分子量比预期目的蛋白稍偏大.     

古菌Pyrococcus furiosus嗜热α-淀粉酶基因在酿酒酵母中的表达

用PCR方法从嗜热古菌Pyrocccus furiosus的基因组DNA中扩增出胞外α-淀粉酶成熟肽结构基因,插入pUC19中构建成质粒pUC19-amy,将pUC19-amy外源片段接入酿酒酵母表达载体pXY212多克隆位点,构建成载体pYX212-amy,电转化酿酒酵母W303-1A,转化子成功表达出有活性的高嗜热α-淀粉酶,重组酶具有P.furiosus产生的胞外α-淀粉酶相似的酶学性质,最适pH为5.0,最适温度约为90℃,在121℃下热处理30min酶活仍能保持50%以上。     

玉米△12脂肪酸脱氢酶基因（FAD2）在酿酒酵母中的表达

玉米△12脂肪酸脱氢酶是催化油酸形成亚油酸的关键酶。将其编码基因FAD2（GenBank登陆号：DQ496227）克隆到酿酒酵母表达载体pYES2．0中,构建成重组质粒pYE／FAD2,转化到酿酒酵母进行诱导表达,同时以pYES2．0转化子为对照。气相色谱（Gc）分析表明,重组转化子亚油酸的含量占酵母总脂肪酸的1．54％,而对照未检测到亚油酸。表明FAD2基因具有编码△12脂肪酸脱氢酶的功能。为探索转译起始密码子周边序列的改变对FAD2基因表达产生的影响,将该基因的起始密码子上游序列进行修改,构建重组表达载体pYE／FAD2—1,转化酿酒酵母进行表达。GC分析表明,pYE／FAD2—1转化子的亚油酸含量占总脂肪酸含量的8．81％,是对照pYE／FAD2转化子的近5倍。     

细菌的β—1,4—内切葡聚糖酶基因在酿酒酵母（Saccharomyces cerevi…

将大肠杆菌质粒ｐＡ２含气单胞菌的β－１,４－内切葡聚糖酶基因直接连于大肠杆菌／酵母菌穿梭载体ｐＶＣ７２７组建成重组质粒,质粒ｐＶＣ含强启动子。首先,通过菌落染色方法和Ｃｏｎｇｏ－Ｒｅｄ染色方法筛选到大肠杆菌ＤＨ５α转化子,然后以重组质粒转化酿酒酵母ＢＪ１９９１并得到表达。最后,对酶反应的最适ｐＨ和适宜温度进行了测定。     

细菌的β-1,4一内切葡聚糖酶基因在酿酒酵母(Saccharomyces cerevisiae)中的表达

将大肠杆菌质粒pA2含气单孢菌（Aromonds.sp.212）的β-1,4-内切葡聚糖酶基因直接连干大肠杆菌／酵母菌穿梭载体pVC727组建成重组质粒,质粒PVC含强启动子。首先,通过菌落染色方法和Congo－Red染色方法筛选到大肠杆菌DH5α转化子,然后以重组质粒转化酿酒酵母BJ1991并得到表达。最后,对酶反应的最适pH和适宜温度进行了测定。     

古菌Pyrococcus furiosus嗜热α-淀粉酶基因在酿酒酵母中的表达*

用PCR方法从嗜热古菌Pyrococcusfuriosus的基因组DNA中扩增出胞外α 淀粉酶成熟肽结构基因 ,插入 pUC19中构建成质粒 pUC19 amy。将 pUC19 amy外源片段接入酿酒酵母表达载体 pYX2 12多克隆位点 ,构建成载体 pYX2 12 amy ,电转化酿酒酵母W 30 3 1A。转化子成功表达出有活性的高嗜热α 淀粉酶。重组酶具有与P. furiosus产生的胞外α 淀粉酶相似的酶学性质 :最适 pH为 5 0 ,最适温度约为 90℃ ,在 12 1℃下热处     

一种新型酿酒酵母附加型分泌表达载体的构建

用化学法合成克鲁维酵母的菊粉酶基因的信号肽序列(INU),将其嵌入酵母附加型表达质粒pYES2,得到一套新型的分泌表达载体pYES2I,pYES2Ⅱ,pYES2Ⅲ。然后用PCR方法分别扩增大肠杆菌的天冬酰胺酶基因(ASN)和短芽孢杆菌α乙酰乳酸脱羧酶(ALDC)基因,连接到INU下游,得到重组质粒pASN和pALDC。分别将这两个重组质粒转化酿酒酵母菌株INVScⅠ中表达,胞内和胞外的酶活分析表明ASN和ALDC基因都能在酿酒酵母中分泌表达,表明菊粉酶信号肽序列能很好地将酿酒酵母中的重组蛋白分泌到胞外。稳定性分析表明,重组酵母菌株在没有选择压力的条件下连续接种培养100h,未发现重组质粒的不稳定性。     

Secreted expression of the pullulanase in Bacillus amyloliquefaciens BF7658

根据文献报道的核苷酸序列合成Bacillus deramificans普鲁兰酶成熟肽编码基因BdP.将BdP基因插入芽孢杆菌分泌表达载体pUC980信号肽编码区下游,获得重组质粒pUC980-BdP,重组质粒转化中温α-淀粉酶生产菌解淀粉芽孢杆菌BF7658菌株.摇瓶发酵实验表明,重组转化子发酵液有明显普鲁兰酶酶活,约48 h酶活达到最高水平,为2.8 ASPU/mL.酶学性质分析表明,重组酶最适作用温度约为60℃,最适反应pH为5.0,60℃保温3h仍保存50％的活性.重组酶性质适合淀粉糖化工艺的要求.     

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...     

普鲁兰酶基因在解淀粉芽孢杆菌BF7658菌株中的分泌表达

根据文献报道的核苷酸序列合成Bacillus deramificans普鲁兰酶成熟肽编码基因BdP。将BdP基因插入芽孢杆菌分泌表达载体pUC980信号肽编码区下游,获得重组质粒pUC980-BdP,重组质粒转化中温α-淀粉酶生产菌解淀粉芽孢杆菌BF7658菌株。摇瓶发酵实验表明,重组转化子发酵液有明显普鲁兰酶酶活,约48h酶活达到最高水平,为2．8ASPU／mL。酶学性质分析表明,重组酶最适作用温度约为60℃,最适反应pH为5．0,60℃保温3h仍保存50％的活性。重组酶性质适合淀粉糖化工艺的要求。     

Enhanced production of Trichoderma reesei endoglucanases and use of the new cellulase preparations in producing the stonewashed effect on denim fabric

Trichoderma reesei strains were constructed for production of elevated amounts of endoglucanase II (EGII) with or without cellobiohydrolase I (CBHI). The endoglucanase activity produced by the EGII transformants correlated with the copy number of the egl2 expression cassette. One copy of the egl2 expression cassette in which the egl2 was under the cbh1 promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A T. reesei strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the cbh2 locus with the coding region of the egl2 gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.     

Enhanced production of cellobiohydrolases in Trichoderma reesei and evaluation of the new preparations in biofinishing of cotton

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).     

Enhanced Production of Trichoderma reesei Endoglucanases and Use of the New Cellulase Preparations in Producing the Stonewashed Effect on Denim Fabric

Trichoderma reesei strains were constructed for production of elevated amounts of endoglucanase II (EGII) with or without cellobiohydrolase I (CBHI). The endoglucanase activity produced by the EGII transformants correlated with the copy number of the egl2 expression cassette. One copy of the egl2 expression cassette in which the egl2 was under the cbh1 promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A T. reesei strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the cbh2 locus with the coding region of the egl2 gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.     

Heterologous expression and characterization of endoglucanase I (EGI) from Trichoderma viride HK-75

Endoglucanase I (EGI) secreted from Trichoderma viride HK-75 has a unique transglycosylation activity. The genomic and cDNA clones encoding EGI (egl1) of T. viride HK-75 were isolated and characterized. The coding region of egl1, composed of 1392 bp, was found to encode a polypeptide of 464 amino acids that has extensive similarity (93.8%) with EGI of T. reesei. Expression of the egl1 gene in E. coli as a fusion protein (with N-terminal thioredoxin and C-terminal histidine tag) led to a large production of a nonglycosylated protein of 62.5 kDa. However, it formed an insoluble inclusion body. Upon denaturation with 8 M urea followed by dialysis and successive purification, the enzymatically active recombinant EGI (rEGI) was obtained at a level as high as 18.3 mg/l of 1,000 ml of culture. The rEGI had 67.8% activity for carboxymethyl cellulose (CMC), compared to native EGI (nEGI). The optimum pH and optimum temperature of rEGI were lower than those of nEGI by 0.5 and 5 degrees C, respectively. The rEGI also had narrower CMCase ranges than nEGI in pH and temperature stabilities. However, the catalytic and transglycosylation abilities against cellotriose of rEGI were comparable to those of nEGI. These results suggest that the glycosylation is important for the stabilities of EGI but not critical for the essential enzymatic capacity.     

The gene encoding squalene epoxidase from Saccharomyces cerevisiae: cloning and characterization.

The gene (ERG1) encoding squalene epoxidase (ERG) from Saccharomyces cerevisiae was cloned. It was isolated from a gene library, prepared from an allylamine-resistant (AlR) S. cerevisiae mutant, by screening transformants in a sensitive strain for AlR colonies. The ERG tested in a cell-free extract from one of these transformants proved to be resistant to the Al derivative, terbinafine. From this result, we concluded that the recombinant plasmid in the transformant carried an allelic form of the ERG1 gene. The nucleotide sequence showed the presence of one open reading frame coding for a 55,190-Da peptide of 496 amino acids. Southern hybridization experiments allowed us to localize the ERG1 gene on yeast chromosome 15.     

Cloning of a DNA sequence that complements glutamine auxotrophy in Saccharomyces cerevisiae

Glutamine (gln) requiring mutants of Saccharomyces cerevisiae have been isolated. They synthesize small amounts of glutamine synthetase (GS), which is more thermolabile than the enzyme from the parental strain. The gln auxotrophy was complemented in transformation experiments using an S. cerevisiae gene library constructed in the plasmid vector YEp13. The transformants were mitotically unstable and synthesized almost tenfold higher amounts of GS than wild-type cells. This activity was as thermoresistant as that from the wild-type strain. A recombinant plasmid was isolated from one of the transformants and partially mapped. Upon reintroduction into the auxotrophic strain, the transformation frequency to gln prototrophy was the same as that for the marker LEU2 gene. The evidence presented suggests that we have cloned the structural gene for GS from S. cerevisiae.     

Expression of fungal thermotolerant endo-1,4-β-glucanase in transgenic barley seeds during germination

The malting quality of two barley cultivars, Kymppi and Golden Promise, was modified to better meet the requirements of the brewing process. The egl1 gene, coding for fungal thermotolerant endo-1,4--glucanase (EGI, cellulase), was transferred to the cultivars using particle bombardment, and transgenic plants were regenerated on bialaphos selection. Integration of the egl1 gene was confirmed by Southern blot hybridization. The transgenic seeds were screened for the expression of the heterologous EGI. Under the high-pI -amylase promoter, the egl1 gene was expressed during germination. The heterologous enzyme was thermotolerant at 65 °C for 2 h, thus being suitable for mashing conditions. The amount of heterologous EGI produced by the seeds (ca. 0.025% of soluble seed protein), has been shown to be sufficient to reduce wort viscosity by decreasing the soluble -glucan content. A decrease in the soluble -glucan content in the wort improves the filtration rate of beer.