酶解提高谷朊粉乳化性能的蛋白酶筛选

国内外对谷朊粉的酶法改性研究主要集中于单一蛋白酶水解谷朊粉,而多种蛋白酶水解谷朊粉的比较研究较少。为拓宽谷朊粉的应用范围,针对谷朊粉的乳化性能进行了研究。选择中性蛋白酶、碱性蛋白酶、风味蛋白酶、木瓜蛋白酶、复合蛋白酶分别对谷朊粉进行提高乳化性能的比较研究。在蛋白酶最适水解条件下,比较谷朊粉的水解度与水解时间、乳化性能的关系,筛选出风味酶为谷朊粉最佳水解蛋白酶。     

Introduction of new traits into cotton through genetic engineering: insect resistance as example

The main goal of gene transfer into cotton is the development of insect-resistant varieties. The stakes are important since cotton protection against insects uses almost 24% of the world's chemical insecticides market, which is not without consequences on the environment. The first approach was to introduce and express in the cotton genome, genes from the bacterium Bacillus thuringiensis (B.t.) which produces entomopathogenic toxins. The development of an efficient Agrobacterium tumefaciens mediated transformation system was the first step. The expression of B.t. genes was studied and synthetic genes more adapted to a plant genome have been constructed. Studies on their expression in cotton is underway. The second focus was to develop strategies that would minimize the risks of inducing insect resistance. The main approach is to associate several genes coding for entomopathogenic proteins with different modes of action. Genes encoding protease inhibitors were chosen. One possibility is to associate a B.t. gene and a gene encoding a protease inhibitor. Several protease inhibitors were tested in artificial diets on major pests of cotton. The corresponding genes have been introduced into the cotton genome. These various orientations of the research program will be presented. This revised version was published online in July 2006 with corrections to the Cover Date.     

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。     

鸡肉蛋白酶水解工艺条件的研究

为探索鸡肉酶水解工艺条件及其动力学特性，分别采用木瓜蛋白酶、胃蛋白酶、中性蛋白酶、菠萝蛋白酶对鸡肉进行了水解试验，得出最优酶为木瓜蛋白酶；由此选用木瓜蛋白酶做正交试验，考察水解温度、时间、加酶量、pH值和固液比5个因素对酶水解的影响，并确定出最优水解条件为温度50℃，时间7 h，加酶量2.4%(以鸡肉的质量百分数计)，pH 7.0，固液比1∶4；在此条件下，水解度可达26.07%。在此基础上由试验数据推导出描述木瓜蛋白酶水解鸡肉的动力学方程，可为鸡肉酶解生化反应器的设计和鸡肉水解蛋白液或蛋白粉的开发提供一定的理论依据。     

酶法提取芦笋皮中高活性膳食纤维的研究

以芦笋皮为原料，采用酶法水解制备高活性膳食纤维。试验结果表明：用0.54%的脂肪酶(100 U/mg)、0.80%的淀粉酶(25 U/mg)和0.80%的糖化酶(20 U/mg)、1.20%的蛋白酶(100 U/mg)可提取出高活性的膳食纤维。所得膳食纤维纯度高，总膳食纤维含量达83.08%，其中水溶性膳食纤维17.02%，不溶性膳食纤维66.06%。而且生理活性好，持水性和溶胀性分别为886%和4.5 mL/g。     

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％。     

Evidence of peptides in low-molecular-weight fractions of soil organic matter

Summary Organic matter was extracted from three soils, a Berwick sandy loam, a Franklin loamy sand, and a Cumberland silty loam. The extracts were separated into high (>8000 daltons) and low-molecular-weight (<8000 daltons) fractions using gel filtration. Reverse-phase high performance liquid chromatography at 214 nm was used to separate the peptides into low-molecular-weight fractions. Peptide samples were collected with an integrated fraction collector and hydolyzed with an immobilized protease column reactor. High performance liquid chromatography with fluorescence detection was used to determine the amino-acid contents of the collected samples. The results indicated that peptide intermediates are present in soil size fractions. Greater quantities of several amino acids were released from the peptide hydrolyzates of the Berwick sandy loam and Franklin loamy sand, compared with the Cumberland silty loam, an uncultivated soil. These findings indicate that organic intermediates (e.g., peptides) are more prevalent in biologically active soils than in relatively inert soils.     

苜蓿草粉对禾花鲤蛋白酶和淀粉酶活性影响的研究

选取来源一致、规格整齐的2龄禾花鲤1 200尾,采用单因子完全随机设计,分为5个处理,每处理3个重复,每重复80尾鱼,分置于15个试验池中。各处理苜蓿Medicago sativa草粉的添加量分别为0（对照组）、40（试验Ⅰ组）、80（试验Ⅱ组）、120（试验Ⅲ组）和160 g/kg（试验Ⅳ组）,正试期50 d,观测苜蓿草粉对禾花鲤前、中、后肠及肝胰脏蛋白酶和淀粉酶活性的影响。结果表明：1）添加苜蓿草粉对前肠蛋白酶活性有显著影响 (P<0.05),其中试验II组显著高于对照组和试验Ⅳ组,但和试验Ⅰ、Ⅲ组差异不显著 (P>0.05)。添加苜蓿草粉对中、后肠及肝胰脏蛋白酶活性均无显著影响 (P>0.05),但它们有相同的变化趋势,即试验Ⅰ、Ⅱ、Ⅲ组鱼的蛋白酶活性均高于对照组。2）鲤鱼的前肠、中肠、后肠蛋白酶活性依次减弱,而肝胰脏的蛋白酶活性远低于肠道。3）添加苜蓿草粉后前肠及中肠淀粉酶活性比较,试验II组显著高于对照组 (P<0.05),其余各组与对照组差异不显著 (P>0.05),后肠淀粉酶活性与对照组无显著差异 (P>0.05)。添加苜蓿草粉能提高鲤鱼肝胰脏淀粉酶活性,其中试验Ⅱ组显著高于对照组 (P<0.05),其余试验组与对照组无显著差异 (P>0.05)。4）禾花鲤前、中、后肠及肝胰脏4部分淀粉酶的活性有较大的不同,肝胰脏>后肠>中肠>前肠。     

重组球孢白僵菌表达目标产物类枯草杆菌蛋白酶的发酵特性研究

采用单因子筛选和正交试验,研究了碳源、氮源、微量元素及无机盐等营养成分对重组球孢白僵菌(Beauveria bassiana)菌株Bb0062-15-4-CDEP-1产生目标产物类枯草杆菌蛋白酶CDEP-1的影响,并通过摇瓶发酵和30 L发酵罐发酵对其发酵特性进行了研究.结果表明,其优化发酵培养基为2.0%玉米粉,1.0%麦麸,0.25%豆粕粉,0.4%NaNO3,0.1%MgSO4·7H2O.摇瓶发酵结果表明,在对数生长期,随着菌体生物量的增加,CDEP-1产量上升,二者呈一定的平行关系;当菌体生长到稳定期,CDEP-1产量继续上升,并达到产酶高峰;此后,随着菌体生长的衰退,CDEP-1产量出现快速下降趋势.扩大培养时,30 L发酵罐中60 h产酶量最高,比摇瓶培养(168 h)缩短了108 h,单位体积的产酶量比摇瓶培养提高80%.结果对研究和利用该球孢白僵菌重组菌株的代谢产物,提高真菌杀虫剂的杀虫效果奠定了基础.     

Ontogenic changes in the digestive enzyme patterns and characterization of proteases in Indian major carp Cirrhinus mrigala

Digestive enzymes of Cirrhinus mrigala (Ham.) were studied during ontogenic development. Specific amylase activity was detected in first feeding fish. The enzyme activity decreased up to day‐18 and then it increased with the age of fish to reach the highest level on day‐34. Protease activity was 28.61 ± 8.90 mU mg protein^?1 min^?1 on day‐4 and increased with the age throughout the study period. Trypsin activity was 31.86 ± 1.12 mU mg protein^?1 min^?1 on day‐4. The activity decreased up to day‐10 and from day‐12 onwards increased up to day‐26. Chymotrypsin activity was 14.56 ± 2.74 mU mg protein^?1 min^?1on day‐4 and constantly increased up to day‐26. A significant increase in lipase activity was observed between days‐24 and 34. SDS‐PAGE and substrate SDS‐PAGE showed the diversity of protein (17.4–127.8 kDa) and protease activity bands (16.6–88.8 kDa) during ontogenesis. Soybean trypsin inhibitor, phenyl methyl sulphonyl fluoride, N‐α‐p‐tosyl‐l ‐lysine chloromethylketone and N‐tosyl‐l ‐phenylalanine chloromethylketone inhibited the protease activity up to 79.72–97.21, 65.55–94.83, 45.41–75.31 and 40.78–64.72%, respectively. Inhibition study in substrate SDS‐PAGE revealed the abundance of serine proteases and the presence of isoforms of trypsin and chymotrypsin. Ethylenediamine‐tetraacetate showed 5.56–22.78% inhibition of metal ion‐specific enzyme activity.