RSM优化大豆蛋白酶水解条件

用As1．398中性蛋白酶水解大豆分离蛋白,采用四因素三水平中心组合设计优化大豆分离蛋白酶水解条件,应用SAS分析软件对实验数据进行处理。得以最佳酶水解条件为：温度40．2℃,pH7．2,酶与底物浓度比0．87%(W/W)。底物浓度8．86%(W/W),水解时间3 h；在此条件下水解度预测值为11．28%,实际测定水解度值为11．24%。     

核桃蛋白最佳酶解条件研究

以水解度为指标,研究五种蛋白酶对核桃蛋白水解作用;结果表明,AS.1398中性蛋白酶为水解核桃蛋白最佳酶,最佳酶解条件为:底物浓度2.5%,E/S为7%,温度40℃,pH7.5,酶解3h;在该实验条件下,AS.1398中性蛋白酶酶解核桃蛋白水解度可达45.37%,水解液仅微苦。     

干酪乳杆菌6033蛋白酶降解肌肉蛋白质的作用研究

本试验将干酪乳杆菌(Lactobacillus casei，Lc)6033蛋白酶液按5％的比例分别加入肌浆蛋白和肌原纤维蛋白提取液，然后设定不同的pH值，于15℃培养7d，定时取样，测定反应后的pH值、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测蛋白质的变化。结果发现：不同设定的肌浆蛋白和肌原纤维蛋白试验组在反应初期pH值均稍稍降低，随着反应进程，各试验组的pH值呈现出逐步升高的趋势；其中，肌浆蛋白以pH5．0、4．5组的变化较为明显，肌原纤维蛋白则以pH6．0、5．0、4．5组的变化较为明显。经7d振荡培养后，电泳检测发现，各试验组肌浆蛋白均表现出分解现象，尤其以pH5．0组和pH4．5组更为明显；而肌原纤维蛋白也都表现出了明显的分解现象，尤以pH5．0组肌原纤维蛋白分解最明显。     

榛子仁乳液分离蛋白酶解提取油脂工艺的研究

研究了用酶解法提取榛子仁油的工艺,并通过正交试验对工艺进行了优化。结果表明,酶解提取榛子仁乳液油的最佳工艺为：酶用量1500 IU／g,pH7．0,温度45℃,在离心速率为3000r／min的搅拌下酶解2h,榛子仁油的提取率为89．70％。     

Thermostable Proteinase in Salted Anchovy Muscle

Salted anchovy fillets produced in Japan were severely degraded above 35°C and were solubilized completely in a few hours at 55°C. In contrast, raw anchovy fillets were not solubilized, but the solubility of fillets gradually increased during the salting process. In contrast, salted and raw anchovy fillets from southern Europe were relatively stable. Proteolytic activity that digested myosin heavy chain in vitro was found in muscle extracts from both types of salted fillets. Enzymes with activity were high-temperature-active serine proteinases with substrate specificities similar to trypsin. Optimal pH was 7.4 (Japan) and >8.5 (Europe). Neither enzyme showed collagen-degrading activity.     

Catalytic Activities of Crude Enzyme Fractions from Monterey Sardine

Catalytic activities in sarcoplasmic fluid of Monterey sardine (Sardinops sagax caerulea) were identified. Hydrolysis at pH 7.6 on hippuryl-L-phenylalanine and hippuryl-L-arginine suggested the presence of carboxypeptidase A and B. Proteolysis on glutaryl-L-phenylalanine-p-nitroanilide, and inhibition by CuSO₄, phenylmethylsulfonyl fluoride and N-p-tosyl-L-lysil chloromethyl ketone, confirmed presence of a chymotrypsin-like proteinase and other serine enzymes. No hydrolysis on α-N-benzoyl-D,L-arginine-p-nitroanilide occurred. Leucine aminopeptidase was detected by hydrolysis on L-leucyl-β-naplithylamide-HCl. Activity at pH 3 proved the presence of an acid proteinase but a slight inhibition by EDTA suggested the involvement of a cathepsin D-like enzyme. Cathepsins A and B and collagenase were not detected.     

Cloning and characterization of baker's yeast alpha-glucosidase: over-expression in a yeast strain devoid of vacuolar proteinases

Two alpha-glucosidase (maltase) genes, designated GLUCPI and GLUCPII, have been cloned from an industrial strain of baker's yeast (Saccharomyces cerevisiae) by complementation of a maltase-negative mutant strain. The different genes were identified according to their alternatively expressed isoenzymes PI and PII in transformants after isoelectric focusing and activity staining in separated cell lysates. The gene encoding alpha-glucosidase PI (GLUCPI), which was not present in laboratory strains of S. carlsbergensis with a defined MAL1, 2, 3, 4 or 6 locus, was sequenced and compared with the recently published MAL6S gene. This comparison revealed single amino acid deviations at three positions in the predicted polypeptide sequence. In addition, the divergent promoter region of GLUCPI differed from MAL6S by a triple repeated 147-bp DNA segment. Maltose induction and glucose repression of alpha-glucosidase PI were not affected by the deletion of the repeated DNA segment. However, the absolute expression of alpha-glucosidase PI increased two- to four-fold. In addition, a two-fold increase in the maltase synthesis occurred when the cloned positive regulator gene MAL2-8ep was on the same plasmid. Furthermore, stability of the alpha-glucosidase in cultures in the stationary growth phase was greatly enhanced using a host strain lacking the proteinases A and B and the carboxypeptidases Y and S. Promoter trimming, MAL2-8cp stimulation and the use of a host strain deficient in four vacuolar proteinases resulted in alpha-glucosidase PI expression of about 13% of the soluble protein.     

A FLUOROMETRIC ASSAY FOR PROTEINASE A IN BEER AND ITS APPLICATION FOR THE INVESTIGATION OF ENZYMATIC EFFECTS ON FOAM STABILITY

A previously developed fluorometric assay using synthetic substrate, Succinyl-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, for yeast proteinase A (PrA) was modified for the accurate and quick determination for the activity in unpasteurized beer. Employing simple HPLC for the determination of 7-amino-4-methylcoumarine (AMC), a final degradation product on this assay, the activity of PrA in beer was measured without the interference of the fluorogenic and photosensitive substance present in beer. The assay for common unpasteurized beers was completed within 5 hours without any concentration procedure. Its linearity and reproducibility were satisfactory for quantitative purposes. Using a purified PrA from brewer's yeast, the effect of the PrA activity on foam stability during storage was furthermore clarified. The exclusive effect of PrA on foam stability was also demonstrated by proteinase inhibitor test.     

Subtilisin inhibitors in Canavalia and Vicia faba seeds. A comparative study

Subtilisin isoinhibitors (SI) were isolated from jack bean (Canavalia ensiformis L) and broad bean (Vicia faba L) seeds. Jack beans contain three isoinhibitors (pI 6.6, 6.3 and 6.0) that constitute 0.021 g per 100 g of dry seeds, while the two active proteins from broad beans (pI 5.7 and 5.1) represent 0.028%. The molecular weight, determined by gel filtration, is around 8000 D in both legumes. Large variations in specific activity of SI against subtilisin Carlsberg were detected in six species of the Canavalia genus, whereas only slight changes were found among six cultivars of C ensiformis. Antibodies raised against SI isolated from jack beans are specific for SI from different varieties of this and other species of the Canavalia genus. However, they do not recognise SI from broad beans and other legume seeds tested. In broad beans the variability between cultivars is significant. Cv Canaria has two active bands and four are detected in the ?dark’? variety, which is 2.7 times less active than the former. SI are specific towards microbial serine proteases, showing high affinity for proteinase K. No appreciable activity against animal proteases or plant thiol enzymes can be detected. SI are also inactive against 4 alpha-amylases of different origins. Reversible limited proteolysis of the reactive site bond suggests that SI interact with subtilisin by the ?standard mechanism’? usually accepted for the inhibition of trypsin by its proteinaceous inhibitors.     

酶法制胶反应结束方法的研究

研究了利用AS1.398中性蛋白酶制备明胶工艺中，酶解反应的终止方法，比较了水洗法、pH调节法、温度调节法对酶法制胶工艺中结束酶反应的效果。结果表明，水洗法和温度调节法可以有效的降低体系的表观活力，并且能够保证明胶质量。