绵羊瘤胃微生物胞外蛋白酶性质的研究

本试验测定了pH、温度和EDTA对纯化的绵羊的瘤胃微生物胞外蛋白酶活性的影响,并用一已知氨基酸排列顺序的肽段(由15个氨基酸组成)作为底物,进一步测定了蛋白酶的酶切位点。结果表明,(1)纯化蛋白酶的最适pH在6.0～6.5之间,最适温度为40℃左右。(2)不同浓度的EDTA(浓度分别为1,10,25,50,75和100mM)对蛋白酶活性没有影响,因而该酶属于丝氨酸蛋白酶类。(3)纯化蛋白酶是一种内肽酶,其酶切位点在组氨酸—酪氨酸(?)、天冬氨酸—丙氨酸(?)、亮氨酸—赖氨酸(?)和(或)缬氨酸—赖氨酸(?)相连的肽键。(4)胰蛋白酶和纯化的蛋白酶虽同属丝氨酸蛋白酶类,但前者对底物具有高度的专一性,后者对底物的专一性不强,这是瘤胃内饲料蛋白质强烈降解的主要原因之     

温度和食物种类对红褐斑腿蝗取食、生长及肠道消化酶活性的影响

在实验室不同温度(10、25、35℃)下,比较茶叶和小麦苗对红褐斑腿蝗Catantops pinguis取食利用能力的影响及其与蝗虫肠道消化酶活性之间的关系。结果表明,温度对蝗虫的日均取食量、相对生长率影响极显著,均表现为35℃>25℃>10℃,对日均绝对增重(干重)的影响趋势与此相同,但各处理温度间差异不显著,对消化酶活性影响显著,总体表现为高温时活性大于低温,但不同的水解酶表现各不相同。食物种类对近似消化力影响不大,但对小麦苗的取食量极显著高于对茶叶的取食量;三个处理温度下日均绝对增重均以小麦苗为食时高于以茶叶为食,但差异不显著,以小麦苗为食时相对生长率极显著高于以茶叶为食,显示食物对相对生长率影响显著;取食小麦苗时肠道消化酶活力极显著高于以茶叶为食,只有类胰凝乳蛋白酶活力差异不显著。     

Screening and Identification of Protease-producing Bacillus amyloliquefaciens from Pig's Intestines and the Preliminary Study of Its Enzymatic Properties

A Bacillus strains,MY-6 strain which secreted high amount of protease was isolated from healthy pig's intestines,and it was identified as Bacillus amyloliquefaciens by the methods of morphological observation, biochemical tests,16S rDNA sequence amplification and comparison, biolog identification. In order to get an initial understanding of the enzymatic properties of its protease, the dynamic relationship between protease activity and bacterial life cycle, effects of pH,temperature,metal ions and inhibitors on protease activity,and the protease hydrolysis of different substrates were investigated. The results showed that,the protease synthesis of MY-6 strain was synchronized with its spore formation which began in the early stage of the exponential growth phase. The optimum conditions for the protease was 40℃ and pH 7.0,Ca²⁺ and Mn²⁺ had a intensive effects on the protease activity, PMSF could completely inhibit the activity of protease, indicating that the protease was a serine protease.The content of TCA soluble peptides for protein hydrolyzate fish protein, soybean protein, corn protein and casein was 0.83,0.67,0.58 and 0.38 μmol/g,respectively. In conclusion,Bacillus amyloliquefaciens MY-6 strain could be used as an initial selection of probiotic Bacillus to improve protein digestion.     

中华蜜蜂工蜂碱性磷酸酶功能基团的研究

在一定条件下，分别采用二硫代二硝基苯甲酸（DTNB）、N-溴代琥珀酰亚胺（NBS）、苯甲基磺酰氟（PMSF）、溴乙酸（BrAc）、乙酰丙酮、巯基乙醇（ME）等化学修饰剂选择修饰中华蜜蜂碱性磷酸酶多种氨基酸残基，并测定其酶活力变化。结果表明，DTNB、PMSF的修饰不表现对酶的抑制作用，而NBS、BrAc、ME能显著抑制酶的活力，乙酰丙酮也能抑制酶的活力，但是不太明显。因此认为Trp、His是中华蜜蜂工蜂碱性磷酸酶的必需功能基团，部分二硫键对保护酶的催化功能也是必需的。     

Increased abundance of patatins,lipoxygenase and miraculins in a thaxtomin A-habituated potato Russet Burbank somaclone with enhanced resistance to common scab

Potato common scab caused by the actinobacterium Streptomyces scabiei is characterized by the formation of corky lesions on tubers that reduce their marketability. Management of common scab is very complex and often ineffective under various environmental conditions. Using potato varieties that are more resistant to common scab remains one of the most efficient strategies to control this disease. However, very little is known about the factors associated with resistance to common scab. Somaclone RB9 regenerated from thaxtomin A-habituated potato Russet Burbank calli produced tubers more resistant to common scab than the original variety. Comparison of the RB9 tuber proteome with that of Russet Burbank using label-free quantitative proteomic analysis revealed changes in the accumulation of defence-related proteins from the patatin and lipoxygenase (LOX) families, which are involved in the metabolism of lipids, and of two miraculins of the Kunitz-type protease inhibitors family. The implication of LOX during common scab infection was studied using synchronized minitubers developed from leaf-bud cuttings. S. scabiei infection stimulated the accumulation of LOX in both Russet Burbank and RB9 minitubers, but this accumulation was intensified in RB9 minitubers. Infection also increased LOX activity in Russet Burbank and RB9 minitubers. However, LOX activity measured in noninfected RB9 minitubers was similar to that of infected Russet Burbank minitubers, indicating endogenous activation of LOX activity in RB9 minitubers. We discuss how increased LOX abundance and activity in the somaclone RB9 may contribute to improving tuber defence against common scab.     

碱性硅肥对香蕉枯萎病的影响

本研究以香蕉枯萎病菌尖镰孢古巴专化型4号小种为目标菌,采用PDA平板培养、土壤平板培养和盆栽试验三种方法来测定碱性硅肥对香蕉枯萎病病菌的影响。结果表明,使用第一种方法,硅肥在小于1000倍液的范围内对菌丝生长均显示抑制作用,其中原液的抑制率达到最高并与其他浓度处理,以及对照普通肥料有显著的差异（p<0.05）;第二种方法显示与第一种方法的抑菌效果一致;第三种方法显示,硅肥处理的香蕉苗病情指数为43.85%,对照肥料处理的为68.57%,空白对照的为80.00%。本研究认为,碱性硅肥对香蕉枯萎病菌有一定的抑制作用,在生产上应用潜力。     

一株盐芽孢杆菌的筛选及其粗酶的酶学特性研究

从浙江舟山市双峰盐场中筛选出一株分泌蛋白酶的嗜盐菌,结合16S rDNA基因序列鉴定、菌株形貌特征确定其为特氏盐芽孢杆菌(Halobacillustrueperi)命名为HalobacillustrueperiB1。经研究,该菌株最适生长NaCl浓度为5%,表明其为中度嗜盐菌。该蛋白酶在50℃和pH 7.5条件下具有最高的蛋白酶活力。对发酵培养时间的优化实验表明发酵8 h后B1分泌的蛋白酶活力最大。对发酵培养基的优化结果表明,脱脂奶粉为最适氮源和碳源;75 g·L~(-1)为最佳发酵盐浓度,对钙镁离子的依赖不明显。确定优化后的发酵培养基:Na HCO_30.06 g,KCl_2.0g,CaCl_2·2H_2O0.48 g,Mg SO_4·7H_2O 1.0 g,FeCl_3 0.001 g,NaCl 75.0 g,脱脂奶粉10.0 g。在初始pH 7.5,温度为40℃条件下发酵8 h。     

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker <Emphasis Type="Italic">Miichthys miiuy</Emphasis> larvae

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.     

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。