稻瘟病菌一假定几丁质酶在毕赤酵母中的表达

本研究从稻瘟病菌菌株Guy11中提取总RNA,利用RT-PCR方法扩增到一假定几丁质酶基因MGG_08054.6的cDNA序列,将其亚克隆至带有His-tag标签的酵母表达载体pPIC3.5K中,构成重组质粒pPIC3.5K-ws,电击转化至毕赤酵母菌菌株GS115,通过MD-MM平板筛选及PCR验证获得重组酵母转化子GS115-PICH-ws;阳性转化子在甲醇诱导下,成功分泌出重组蛋白.SDS-PAGE分析表明,重组蛋白分子质量为48 ku,与其理论计算值基本相符;Ni-NTA法分离纯化后的重组蛋白经DNS法测定具有几丁质酶活性.     

甜味蛋白Brazzein基因合成及其在酵母中表达的初步研究

采用pGAPZαA为表达载体,SMD1168毕赤酵母为受体系统,构建分泌型的Brazzein毕赤酵母重组菌。按照毕赤酵母偏爱密码子设计Brazzein基因,共设计4对引物,采用SOE-PCR法合成Brazzein基因,构建克隆载体pUC57-Bra与酵母表达载体pGAPZαA-Bra。将重组质粒线性化,电转导入Pichia.pastoris SMD1168中,用高浓度的Zeocin筛选高拷贝转化子。将重组菌株接种于YPD培养基诱导表达,进行SDS-PAGE分析。成功构建了分泌型的Brazzein毕赤酵母重组菌,SDS-PAGE表明,目标蛋白的相对分子量与理论值一致,诱导72h后的目的蛋白表达量达0.12g/L。     

利用透明颤菌血红蛋白在低氧条件下提高毕赤酵母中植酸酶的表达

为了提高毕赤酵母(Pichia pastoris)中重组植酸酶的表达,在来源于Pichia stipitis的低氧诱导启动子PsADH2控制下,进行了透明颤菌血红蛋     

三角褐指藻Δ6脂肪酸脱饱和酶基因克隆及其表达分析

γ-亚麻酸在人类的健康与营养中具有重要的功能,利用基因工程手段提高其产量是满足其需求日益增大的途径之一。Δ6脂肪酸脱饱和酶是γ-亚麻酸合成途径中的关键酶,催化亚油酸（C18∶2 Δ9,12）到γ-亚麻酸（C18∶2 Δ6,9,12）的反应。通过克隆三角褐指藻的Δ6脂肪酸脱饱和酶基因,并连入酵母表达载体pPIC3.5K中,在毕赤酵母中进行了表达,结果表明：重组酵母经甲醇诱导后表达转入的外源基因,气-质联用分析显示重组毕赤酵母中含有γ-亚麻酸和SDA（18∶4 Δ6,9,12,15）,其含量分别为2.5%和0.8%。     

毕赤酵母分泌表达SAMS及表达产物活性分析

[目的]利用毕赤酵母分泌表达S-腺苷甲硫氨酸合成酶（SAMS）。[方法]以酿酒酵母（Saccharomyce cerevisiae）基因组DNA为模板,通过PCR扩增出S-腺苷甲硫氨酸合成酶基因2（sam2）,构建重组毕赤酵母（Pichia pastoris）表达载体pPIC9Ks-am2,转化毕赤酵母GS115,以甲醇诱导表达分泌蛋白SAM合成酶,并用HPLC测定重组蛋白的酶活力。[结果]经SDS-PAGE鉴定重组蛋白分子量约50kD,比理论值42.5 kD稍大,可能是分泌过程中糖基化等加工造成。体外酶促反应结果显示,伴随甲醇诱导及初步纯化过程的进行,蛋白活力逐步提高,纯化后比活力为61.48 U/mg。[结论]该研究首次实现了SAM合成酶的胞外表达,为开发和建立体外酶促法生产SAM工艺奠定了基础。     

鸡白细胞介素-2在毕赤酵母中的表达

将鸡白细胞介素-2（ChIL-2）基因插入酵母分泌型表达载体pPIC9K构建重组表达载体pPIC9K／ChIL-2,通过电转化构建毕赤酵母（Pichia methanolica）GS115甲醇利用型重组表达菌株．经SDS—PAGE与Western blot分析,证实ChIL-2基因在重组GS115中成功表达出约16ku与14ku两条特异性条带,这与天然ChIL-2相对分子质量大小一致,提示ChIL-2可能为糖基化蛋白质．     

RETRACTED ARTICLE: Effect of calcium chloride on the biocontrol efficacy of two antagonistic yeasts against <Emphasis Type="Italic">Penicillium expansum</Emphasis> on apple fruit

Two antagonistic yeasts, Candida membranaefaciens and Pichia guilliermondii, were evaluated for the control of the blue mold of apple caused by Penicillium expansum. Dual culture, cell-free metabolite and volatile tests were used for in vitro assay. Yeast strains of two genera inhibited growth of P. expansum; inhibition varied from 30.27% to 44.19% in dual culture, from 79.40% to 90.57% in the volatile metabolite test, and from 72.99% to 88.77% in the cell-free metabolite test. Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen P. expansum, but did not affect the colony-forming units (CFU) of the yeasts C. membranaefaciens and P. guilliermondii in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of P. expansum in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro, as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 10⁷ CFU ml^-1, growth of the pathogen was completely limited in vitro, and no infection was found in apple fruits treated with or without calcium. This article has been retracted because part of the data shown has already been published before, by different authors. An erratum to this article can be found at     

黑木耳漆酶基因在毕赤酵母中的表达及其酶学性质研究

应用SignalP 3.0 Server软件对黑木耳漆酶基因lac1(GenBank No.AY450405)编码的蛋白质序列进行分析,发现在其N端存在18个氨基酸的信号肽序列。通过RT-PCR克隆了lac1基因,分别构建带有自身信号肽的lac1基因毕赤酵母表达载体pPICN-lac1和以α因子信号肽替换自身信号肽的lac1基因毕赤酵母表达载体pPIC9-lac。将这两个载体转化巴斯德毕赤酵母(Pichia pastoris)GS115细胞,获得基因工程菌GS115-pPICN-lac1与GS115-pPIC9-lac。SDS-PAGE分析和酶活性测定结果表明,在转化后者中漆酶基因得到了有效分泌表达,而在转化前者中漆酶基因未能得到有效分泌表达。进一步研究确定GS115-pPIC9-lac菌株液体发酵的最适pH为4.0,在此发酵条件下,其分泌表达的漆酶最高酶活为0.149U·mL-1。酶学性质研究表明,该酶的最适反应温度为40℃,最适反应pH为5.0,在最适反应条件下其pH稳定性和热稳定性均较好。     

在毕赤酵母中表达人溶菌酶蛋白的研究

将化学合成的人溶菌酶基因htYZ（444bp）构建到毕赤酵母分泌型表达载体pPICZ上;载体质粒用SacⅠ线性化后电击法转化毕赤酵母菌株GS115,在含Zeocin的抗性培养基筛选后PCR鉴定获得了9株重组菌株。重组菌株经甲醇诱导后取表达上清液进行SDS-PAGE电泳和Western blot分析。结果显示,重组hLYZ在毕赤酵母中成功表达,在诱导60h时表达量最高。用镍亲和层析柱纯化重组hLYZ,用Bradford法测得其含量约为324mg·L^-1。比浊法活性测定结果显示所表达的重组人溶菌酶活性达到4473U·mL^-1。本研究利用毕赤酵母分泌型表达载体成功表达了重组人溶菌酶,井探索了简单有效的纯化和活性测定方法,为利用毕赤酵母系统规模化生产重组人溶菌酶奠定了技术基础。     

基于响应曲面法优化蜂胶的乙醇提取工艺

蜂胶具有优良的保健效果,一直被作为功能食品而加以应用。为了满足实际生产中提高蜂胶产品质量和生产效率的要求,在充分模拟实际生产条件的基础上,利用响应曲面法对蜂胶提取率和总黄酮含量的影响因素（提取时间、乙醇浓度、固液比、提取次数、提取温度）进行了分析。结果表明,针对本实验中采用的蜂胶毛胶样品,回归模型预测的蜂胶提取率达到38.324 6%,总黄酮含量达到199.776 0 mg/g;最佳提取条件为提取时间24 h,乙醇浓度95%,固液比1∶10,提取次数2次,提取温度为室温。为了达到指导实际生产的目的,试验结论须得到生产车间的实际检验。