不同菜豆品种4种抗营养因子水平差异分析

菜豆中植物凝集素、皂苷、胰蛋白酶抑制剂和植酸等抗营养因子是引起菜豆食物中毒的内源物。以56份食荚菜豆品种为材料,测定了鲜荚中4种抗营养因子含量或活性。结果表明,供试菜豆品种群体4种抗营养因子水平均存在极显著差异。植物凝集素含量(均值为1.743 mg·g~(-1))和胰蛋白酶抑制剂活性(均值为1.680 mg·g~(-1))变异系数均在100%以上,品种频率分布曲线主峰明显,但在极高和极低区域均有品种间断分布,出现了品种水平的数量等级差异;皂苷含量(均值为3.730 mg·g~(-1))和植酸含量(均值为3.102 mg·g~(-1))变异系数均低于41%,品种频率分布曲线呈近正态分布,主峰明显,双尾有低频率连续分布。相关分析表明,植物凝集素含量与胰蛋白酶抑制剂活性呈极显著正相关。聚类分析将供试56份菜豆品种划分为3个品种群,近80%品种聚于抗营养因子中等水平品种群,近12%品种居于较低水平。由于菜豆植物凝集素是食用致毒性的主要内源物,同时其和胰蛋白酶抑制剂等均与菜豆田间抗病虫性密切相关,预示着筛选出极低水平植物凝集素的菜豆品种是可行的,但同时可能会降低其田间抗病虫性。     

维生素B6对史氏鲟幼鱼胰蛋白酶活性的影响

试验研究了饲料中不同水平维生素B6对史氏鲟（Acipenser schrencki）幼鱼胰蛋白酶活性的影响。在温度为（20±0.5）℃、pH值为6.8～7.5的条件下,研究了低蛋白含量（28%）饲料中,添加不同水平的维生素B6（0、15、30、45、60、75mg/kg）对史氏鲟幼鱼[（34.07±3.44）g]胰蛋白酶活性的影响。结果表明,饲料中的维生素B6添加量为45～60mg/kg时,对幼鲟胰蛋白酶的激活作用最大,并可相应提高对饲料蛋白质的消化率。     

Marker assisted accelerated introgression of null allele of kunitz trypsin inhibitor in soybean

Development of kunitz trypsin inhibitor (KTI)-free soybean is crucial for soy-food industry as the heat inactivation employed to inactivate the anti-nutritional factor in regular soybean incurs extra cost and affects protein solubility. In the presented work, a null allele of KTI from PI542044 was introgressed into cultivar ‘JS97-52’ (recurrent parent) through marker assisted backcrossing. Foreground selection in BC₁F₂, BC₂F₂ and BC₃F₂ was carried out using the null allele-specific marker in tandem with SSR marker Satt228, tightly linked with a trypsin inhibitor Ti locus. Background selection in null allele-carrying plants through 106 polymorphic SSR markers across the genome led to the identification of 9 KTI-free lines exhibiting 98.6% average recurrent parent genome content (RPGC) after three backcrosses, which otherwise had required 5–6 backcrosses through conventional method. Introgressed lines (ILs) were free from KTI and yielded at par with recurrent parent. Reduction of 68.8–83.5% in trypsin inhibitor content (TIC) in ILs compared to the recurrent parent (‘JS97-52’) was attributed to the elimination of KTI.     

豇豆胰蛋白酶抑制剂和硫氧还蛋白的融合表达和活性测定

采用融合蛋白表达技术，通过1个人工设计合成的柔性接头，将豇豆胰蛋白酶抑制剂(cowpea trypsin inhibitor,CpTI)基因与表达载体上的硫氧还蛋白(thioredoxin )基因连接，构建了融合表达载体。将表达载体转入大肠杆菌(Escherichia coli )GI724，通过色氨酸诱导获得高效表达，产物以可溶状态存在。活性测定显示，融合蛋白具有抑制胰蛋白酶的生物学活性。以大豆胰蛋白酶抑制剂为参照，将融合蛋白热处理后进行活性测定，发现它具有较好的热稳定性。将肠激酶(enterokinase)与融合蛋白在37 ℃作用16 h，可获得切掉thioredoxin的CpTI蛋白。活性测定显示，切除thioredoxin后CpTI的胰蛋白酶抑制活性比同样处理条件下的thioredoxin-CpTI明显下降，比活力仅为原来的80%，说明融合蛋白具有更高的稳定性。研究结果为thioredoxin-CpTI作为一种生物农药的应用奠定了基础。     

胰蛋白酶对壳聚糖的降解研究

用粘度法和吸光度法，研究胰蛋白酶非专一性降解壳聚糖过程中温度、PH值、反应时间、酶浓度、底物浓度。金属离子对胰蛋白酶降解壳聚糖反应的影响，确定了以壳聚糖为底物的胰蛋白酶的一些催化特性：最适温度为30℃，最适PH值为5.0,10-180min内酶反应速度恒定，酶浓度在0.1-0.5g/L的范围内，酶反应速度与酶浓度呈线性关系，米氏常数Km为9.54g/L。     

0～14日龄乳鸽小肠主要消化酶发育规律的研究

以0～14日龄雄乳鸽为研究对象,测定3日龄、8日龄和14日龄乳鸽十二指肠和空肠中淀粉酶、总蛋白水解酶、胰蛋白酶、糜蛋白酶和脂肪酶活性随年龄的变化趋势。结果表明：十二指肠中淀粉酶、总蛋白水解酶、胰蛋白酶和糜蛋白酶活性随日龄增加而呈上升趋势,脂肪酶活性在3～8日龄显著降低,8～14日龄活性升高;空肠中淀粉酶、总蛋白水解酶和脂肪酶活性先降低后升高,而胰蛋白酶和糜蛋白酶活性先升高后降低,并在8日龄时达到峰值。     

豆粕固态限定发酵和强化发酵的研究

本文采用短乳杆菌、酵母菌、枯草芽孢杆菌和地表芽孢杆菌进行豆粕固态限定发酵和强化发酵的研究,对发酵过程中小分子蛋白含量、菌量、pH及胰蛋白酶抑制剂活性等进行了测定.结果表明:纯菌条件下,接种短乳杆菌和地衣芽孢杆菌实验组限定发酵的小分子蛋白含量分别为31.0%和28.3%,高于接种酵母菌试验组的22.4%.且小分子蛋白积累高峰时间为48～72 h;接种地表芽孢杆菌实验组的胰蛋白酶抑制剂降解率高于接种短乳杆菌或酵母菌实验组.豆粕天然发酵试验组的小分子蛋白含量和胰蛋白酶抑制剂降解率分别为22.2%和44.1%.接种短乳杆菌、地衣芽孢杆菌试验组的小分子蛋白含量和胰蛋白酶抑制剂降解率分别为32.4%、99.5%和29.3%、99.7%.表明接种强化发酵有助于小分子蛋白含量的提高和胰蛋白酶抑制剂的降解.     

Purification and characterization of two anionic trypsins from the hepatopancreas of carp

SUMMARY: Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH₂-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively.