Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

Non-target toxicology of a new mosquito larvicide, trypsin modulating oostatic factor

Trypsin modulating oostatic factor (TMOF), a peptide hormone originally isolated from the ovaries of adult Aedes aegypti, is currently under commercial development as a new pesticide chemistry with a novel mode of action for the control of larval mosquitoes. The objective of the current research is to evaluate potential risks of the use of TMOF as an insecticide on non-target organisms. TMOF (YDPAP₆) was degraded in vitro (as determined by HPLC and LC/MS) to DPAP₆, PAP₆, and then AP₆ by leucine aminopeptidase, a pancreatic enzyme found in the digestive system of vertebrates. The rate of degradation of TMOF and PAP₆ was significantly greater than that of DPAP₆, while no metabolism of AP₆ was found. TMOF technical insecticide was produced on a commercial scale by recombinant yeast (heat-killed before application). The technical TMOF when administered in a single dose by gavage to male and female mice at 2000 mg dry weight/kg body weight produced no negative effects as compared to controls up to 12 days after treatment. When male and female mallard ducks were treated by gavage with 1250 mg dry weight of technical TMOF/kg body weight each day for 5 days, again no toxic effects were noted through 35 days after the last treatment. TMOF technical insecticide was also applied to the shaved skin of male and female rabbits at the rate of 2000 mg/kg for 1-2 days, with no effect. The end point observations in these in vivo experiments were mortality; changes in growth rate, behavior, body structure, and color; and possible lesions observed during necropsy. Finally, Daphnia incubated with technical TMOF in rearing water at the level of 1.0 × 10⁶ yeast cells/ml (10 mg/ml) also demonstrated no negative effects on mortality, growth, molting, time to first brood, and production of viable neonates. It appears from these studies that TMOF can be degraded by vertebrate digestive proteases and technical TMOF is not toxic to the non-target organisms examined.     

壳聚糖固定化胰蛋白酶研究

以自制的壳聚糖为载体,以戊二醛为交联剂,制备了壳聚糖固定化胰蛋白酶。经测定该酶活性回收率达到72.7%,操作半衰期为12 d,表观米氏常数为5.88 mg/ml,最适pH值为8.0,最适温度为40℃;与游离胰蛋白酶相比,酶的耐热性和操作半衰期有明显提高,但表观米氏常数也略有增大。     

胰蛋白酶基因的系统发育分析

下载NCBI数据库中的动物胰蛋白酶基因的氨基酸序列,用CLUSTAL X2程序进行序列比对,用MEGA3.1软件中的Neighbor-joining法构建系统发育树.序列分析表明,胰蛋白酶氨基酸有多处保守基序,且保守性较高,最典型的保守序列有催化三联体结构:His(组氨酸)-Asp(天冬氨酸)-Ser(丝氨酸)以及金有6个保守的半胱氨酸形成3对肽内二硫键.另外,此序列还具有特异性的底物结合位点,这就决定了胰蛋白酶的专一性.系统发育分析结果显示,脊椎动物与无脊椎动物被分成2个大的分支,而脊椎动物中trypsinX3物种被单独分为一支,这表明typsinX3物种与脊椎动物的同源性较低.     

酶法提取猕猴桃皮和渣中果胶的工艺研究

[目的]去除猕猴桃皮和渣中的淀粉和蛋白质,提取并制备膳食食用果胶。[方法]通过单因素和正交试验设计,分别确定0.4%淀粉酶和0.4%胰蛋白酶提取猕猴桃皮和渣中果胶的适宜工艺条件。[结果]0.4%淀粉酶可有效去除猕猴桃皮和渣中的淀粉,其适宜工艺条件为：料液比1∶10.0,50℃下酶解60 min;0.4%胰蛋白酶可有效去除猕猴桃皮和渣中的蛋白质,其适宜工艺条件为：料液比1∶10.0,35℃下酶解60 min。在最佳工艺条件下,经离心、浓缩得到果胶,其中猕猴桃皮和渣中的果胶得率分别为3.10%和1.39%。[结论]通过改良酶法,建立了猕猴桃皮和渣中果胶提取的适宜工艺。     

不同热处理对黑豆干物质消化率,蛋白质消化率的影响

为了研究热处理的温度和时间对黑豆干物质消化率(DMD)、蛋白质消化率(PD)及脲酶活性的影响规律,对经过110℃和121℃下分别处理3,9和15min的饲用黑豆进行了离体干物质消化率和离体蛋白质消化率及脲酶活性的测定。结果表明:在121℃处理15min的样本干物质消化率和蛋白质消化率最高,分别为75.13%,90.91%,脲酶活性最低,为0.04mg/min·g。作者认为:在本研究范围内,湿热处理最适宜的温度是121℃,时间是15min。     

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.     

大豆抗营养因子对生长蛋鸡生产性能、氮代谢和小肠内胰蛋白酶活性的影响

为了观察不同梯度大豆抗营养因子（Ａｎｔｉｎｕｔｒｉｔｉｏｎａｌｆａｃｔｏｒｓ、简称ＡＮＦｓ）对不同生理阶段的生长蛋鸡的影响，本试验采用４８０只海兰褐蛋用雏鸡，以熟豆饼日粮为对照，从３或９周开始给饲３个水平的生大豆日粮（４％，９％和１３％），每期每个日粮４个重复。在３～８周和９～１８周观察雏鸡的采食量，日增重和饲料效率。在第８和１７周分别测定各组的粗蛋白质和干物质的代谢率。并于８、１３和１８周末每个重复随机抽取试鸡两只屠宰，观察小肠内胰蛋白酶活性的变化。结果表明，３个水平的大豆ＡＮＦｓ对育雏期雏鸡的增重没有影响，育成期对照组的增重明显高于采食生大豆组（Ｐ＜０．０５），大豆ＡＮＦｓ使饲料效率降低，但对氮和干物质的代谢影响不显著；８、１３和１８周龄雏鸡小肠内胰蛋白酶活性亦无显著变化。     

缺失SBTi—A2大豆杂交后代品系籽粒化学成分分析

对一个杂交组合后代１０个稳定缺失胰蛋白酶抑制剂（ＳＢＴｉ－Ａ２）品系及其亲本籽粒化学成分组成进行的分析比较表明，大豆籽粒中缺失ＳＢＴｉ－Ａ２对其蛋白质含量及天门冬氨酸、甘氨酸和丙氨酸含量有一定影响，表现为缺失ＳＢＴｉ－Ａ２品系显著低于高亲和双亲平均值；而对其他氨基酸和脂肪含量及脂肪酸组成基本没有显著的影响。     

Proteolytic activity in soil: A review

The aim of this work is to review current knowledge on inputs, sources and regulation of protease activities in soils from different ecosystems, while exploring limitations to proteolysis and N mineralisation. Extracellular proteases enter the soil via microbial production and other sources, including plant root exudates, animal excrements, decomposition processes and leaching from agro-industrial fertilisers. The synthesis and activities of proteases in soil are regulated by many factors, including climate, soil properties and the presence of organic compounds of plant and microbial origin. Two particularly important areas for future research are the regulation of proteolysis by low-molecular-weight organic compounds, including amino acids, sugars, flavonoids, plant hormones and siderophores, as well as the identification and characterisation of proteinaceous protease inhibitors of plant and microbial origin in the soil. Despite all the work that has been performed on soil proteases, our understanding of the roles of extracellular plant root proteases in N nutrition is weak. Furthermore, the regulation of soil proteolytic activities of different ecosystems, especially in terms of pollutant inputs and the impact of climate change, requires investigation. Other areas that pose important questions for the future include assessments of protease inhibitor inputs to the soil, regulation of these inhibitors via naturally occurring soil organic compounds and the interactions between soil organisms.