青虾泛素特异性蛋白酶9基因Usp9全长cDNA序列的克隆及表达分析

本研究首次克隆了青虾的Usp9 (ubiquitin-specific protease 9)基因全长 cDNA序列,并在青虾中使用荧光定量 PCR 技术首次检测了 Usp9 基因在不同组织和发育阶段中的表达变化。青虾 Usp9 基因cDNA全长 1259 bp,包括 162 bp的 5’ UTR, 978 bp的开放阅读框以及 116 bp的 3’ UTR。氨基酸序列比对分析显示,青虾 Usp9蛋白与太平洋牡蛎(Crassostrea gigas)、凤仙花熊蜂(Bombus impatiens)及灰短尾负鼠(Monodelphis domestica)的蛋白相似度分别为 99%、 97%和 95%。应用 MEGA5.1软件对青虾与其他物种的 Usp9氨基酸进行系统进化树分析,结果表明,青虾 Usp9与同为甲壳纲的钩额型蚤状溞聚为一支,具有最近的亲缘关系。采用荧光定量 PCR技术检测了 Usp9基因的时空表达,水温 28℃时, Usp9基因在青虾发育的后幼体发育期第 5天 PL5出现表达高峰。Usp9基因在 6种成体组织中均有表达,表达水平依次为：脑＞腹神经＞精巢＞卵巢＞肌肉＞肠。而在雌雄组织差异表达分析表明,精巢中Usp9基因表达量比卵巢的比值为 2.86,脑组织中雄虾的表达量比雌虾为 2.27,差异均为极其显著(P＜0.01)。这表明Usp9基因的表达和雄性偏向基因趋势是一致的（雄性表达：雌性表达≥1.5）。本研究为进一步开展青虾Usp9基因功能等相关研究奠定了基础,并可为青虾性别的遗传调控机制研究提供参考。     

利用抑菌率对羊骨酶解产物的制备中最佳用酶的筛选

利用碱性蛋白酶、中性蛋白酶、胃蛋白酶、胰蛋白酶、动物蛋白酶、酸性蛋白酶和木瓜蛋白酶分别在各自最适条件下对羊骨进行酶解,以大肠杆菌、枯草杆菌和藤黄球菌为供试菌种,以抑菌性为指标,对7种酶作用下所得酶解产物的抑菌性进行评定,以选出水解最佳用酶。结果表明,在各自最适条件下,胃蛋白酶酶解产物(pH值为3.0,37℃,底物质量浓度0.2kg/L,酶添加浓度2000U/g,作用时间6h)的抑菌效果最佳。     

大丽轮枝菌菌体对链霉菌胞外蛋白酶活性及抑菌效果的影响

 为研究拮抗链霉菌对棉花黄萎病原大丽轮枝菌（Verticillium dahliae Kleb.）的抑菌机理,以大丽轮枝菌菌体为唯一碳氮能源,采用液体培养法及生长速率法研究供试病原菌菌体对4株拮抗链霉菌胞外蛋白酶合成的诱导作用及抑菌活性物质产生的影响。结果表明：大丽轮枝菌菌体可以诱导供试链霉菌合成蛋白酶;诱导粗酶液对病原真菌菌丝有溶解作用;当菌体添加量为10 g·L^-1,28℃培养7 d时菌株Z13蛋白酶活性高达4.24 U;以大丽轮枝菌菌体为碳氮能源时供试链霉菌能产生活性较强的抑菌物质;当菌体添加量为20 g·L^-1,发酵液稀释5倍时菌株B49所产抑菌活性物质的最大抑菌率达95.7%。     

酶法水解牦牛血制备蛋白多肽粉的技术研究

摘 要：[目的][方法]利用中性蛋白酶将牦牛血红蛋白水解,探讨各因素对中性蛋白酶水解牦牛血红蛋白的影响以及水解度的关系,并对酶解液进行了活性炭脱色效果的研究。通过单因素和正交试验（L16(45)）,[结果]确定了中性蛋白酶水解血红蛋白的适宜条件为 pH 7.0,温度 45℃,酶底物浓度比 4000 U/g 蛋白质液,底物浓度 5%,酶解时间 7 h。通过正交试验,确定酶解液的最佳脱色工艺条件为活性炭用量 4%,脱色温度 75℃,pH 5.0,脱色时间 60 min。     

Detection of protease genes in field soil applied with liquid livestock feces and speculation on their function and origin

The origin of soil protease in field soil was estimated using culture-dependent and independent approaches. Overall soil protease activity was much higher in field soils with an annual application of liquid livestock feces (120 t ha⁻¹ year⁻¹ and 600 t ha⁻¹ year⁻¹) compared with the activity recorded in other field soils, and the character of the soil proteases became highly homogeneous (approximately 70% metalloprotease in a 600 t field). Selective incubation studies suggested that bacteria were the most important source of soil protease. There were significantly higher correlations between serratial metalloprotease and the overall soil protease in both feces-applied fields in terms of the effect of inhibitors, and the bacteria, which produced serratial metalloprotease, were suggested to proliferate in both the 120 t and 600 t fields. The gene homologous to serratial metalloprotease gene was amplified in directly extracted DNA from field soils using selective DNA primer and proteolytic Serratia marcescens was certified to be one source of soil protease in these field soils. Proteolytic S. marcescens and its metalloprotease gene have occasionally been isolated and detected in field soils applied with raw feces, and have rarely been isolated or detected from other field soils. Proteolytic S. marcescens is believed to be introduced in the raw feces and subsequently colonizes the field soil and replaces the indigenous bacteria in the soil.     

油茶籽油的蛋白酶水解法提取工艺研究

采用物理破碎油茶籽细胞和酶降解相结合的方法提取油茶籽油,对油茶籽原材料的预处理时间和温度,酶解方法进行了研究,探索蛋白酶在水性条件下酶解油茶籽的提油工艺及其影响因素,结果表明:油茶籽原材料预处理的适宜加热温度为90℃、加热时间为2 h,适宜的酶解温度为38℃、pH值为8、水解酶用量为0.25%(占油料的质量比重)、酶解时间为4 h。     

Source of soil protease based on the splitting sites of a polypeptide

Abstract

Soil protease is an important enzyme in the nitrogen cycle which plays an essential role in the growth of various crops. We have attempted to identify a microbial source of soil protease. Selective soil incubation using antibiotics was suitable for a rough estimation of the groups of microorganisms responsible for the production of soil protease (Hayano and Watanabe 1990; Hayano 1993). Enumeration of proteolytic microorganisms in the field and analysis of their protease-producing ability enabled to evaluate the potential of various soil microorganisms for soil protease production (Watanabe and Hayano 1993a; Watanabe et al. 1994). For the accurate estimation of the soil protease source, the characteristics of the soil protease and microbial protease must be compared directly based on enzymatic properties as indices.     

甘蓝黑腐病菌rPf基因簇对胞外蛋白酶Ⅰ基因表达的调控

甘蓝黑腐病黄单胞菌（ＸａｎｔｈｏｍｏｎａｓｃａｍｐｅｓｔｒｉｓＰｖ．ｃａｍｐｅｓｔｒｉｓ）产生的胞外蛋白酶Ⅰ在致病的早期阶段起重要作用，该酶以及其它胞外酶和胞外多糖的合成受一致病因子调控基因簇（ｒＰｆ基因簇）的正向调控。本研究利用带有β－半乳糖苷酶报道基因（ｌａｃＺ）的转座子Ｔｎ５－Ｂ２０诱变蛋白酶Ⅰ基因克隆，获得了ｌａｃＺ在蛋白酶Ⅰ基因启动子控制下表达的Ｔｎ５－Ｂ２０插入突变质粒。通过将这种突变质粒导入野生型和各ｒｐｆ基因突变体菌株后，测定ｌａｃＺ基因在细胞生长周期中的表达水平，不仅进一步证实了这些ｒｐｆ基因对蛋白酶Ⅰ基因的正向调控作用，而且明确了它们的调控水平．发现ｒｐｆＡ、ｒｐｆＣ、ｒｐｆＥ、ｒｐｆＧ或ｒｐｆＨ突变后，蛋白酶Ⅰ基因的转录会降低９０％左右，而ｒｐｆＢ突变后，蛋白酶Ⅰ基因的转录只降低４８％。     

农杆菌介导pepA基因对黑曲霉菌丝体的遗传转化

通过重叠延伸PCR将宇佐米曲霉中1 347 bp的酸性蛋白酶基因pepA与黑曲霉耱化酶基因5′同源臂和3′同源臂进行拼接,构建pepA基因表达框.将此表达框插入载体pSZH中,构建黑曲霉同源重组表达载体pSZH-pepA.将裁体pSZH-pepA通过冻融法转化农杆菌AGL1,进而通过农杆菌介导法转化高产糖化酶的黑曲霉菌丝体.以PDA作为共培养培养基,乙酰丁香酮(简称AS)浓度200μmol·mL-1的条件下,28℃恒温静置培养48 h,经潮霉素筛选和PCR鉴定获得3株重组菌株.以出发菌株作为对照,对3株重组菌株静置培养7d后的发酵液上清进行酶活测定,结果发现酸性蛋白酶酶活最高达45.56 U· mL-1,而出发菌株的酸性蛋白酶酶活仅为3.72 U· mL-1,表明酸性蛋白酶基因pepA在高产糖化酶的黑曲霉中得到表达.研究建立了农杆菌介导的pepA基因导入黑曲霉菌丝体的转化体系,为实现酸性蛋白酶在黑由霉菌丝体中高效表达奠定基础.