XV. Yeast sequencing reports. Isolation and DNA sequence of the STE13 gene encoding dipeptidyl aminopeptidase

We have isolated a mutant which exhibits partial constitutivity for a -specific gene expression in α cells. The wild-type gene was cloned and demonstrated to be allelic to the STE13 gene, which encodes the dipeptidyl aminopeptidase involved in processing of the α-factor prepropheromone. Thus, the mating defect of the ste13 mutations in α cells may result both from the production of incompletely processed α-factor and from the increased expression of a -specific genes. The STE13 open reading frame of 931 amino acids contains a putative membrane-spanning segment near its amino terminus and is 31% identical to a second yeast dipeptidyl aminopeptidase (DAP2). A null mutant of STE13 has been constructed: it is viable and sporulation-proficient. The sequence has been deposited in the GenBank data library under Accession Number L21944.     

氨肽酶脱苦效果的研究

目的研究从豆腐乳中筛选到的一种枯草芽孢杆菌所产的氨肽酶脱除大豆蛋白质水解物苦味的效果。方法用高效液相色谱法和感官评定。结果1%的大豆蛋白质溶液经胰蛋白酶水解,水解液呈苦味,添加氨肽酶可较好地脱苦,水解液中游离氨基酸的总量增加了28.8%,疏水氨基酸的量也明显增加。结论枯草芽孢杆菌氨肽酶具有较好的脱苦效果。     

Aminopeptidase Activities from Lactobacillus sake in Models of Curing Ingredients and Processing Conditions for Dry Sausage

The effect of curing agents and dry sausage processing parameters on four aminopeptidases (AP 1, AP 2, AP 3, and AP 4) of Lactobacillus sake was investigated. NaCl exerted a strong inhibitory effect against AP 1 but activated the remaining aminopeptidases. NaNO₃ caused inhibition of AP 3 and AP 4. Ascorbic acid inhibited AP 1 and AP 3 while glucose only reduced AP 3 activity. Water activities and processing temperatures affected activity of all aminopeptidases. Acid pH decreased aminopeptidase activity except for AP 4. The combined effects of curing agents and process parameters to simulate different stages during dry sausage processing indicated that AP 2 and AP 4 may remain active throughout the process.     

血清 AFU、LAP 及 GGT 联合检测在早期原发性肝癌患者诊断中的应用价值

目的：探讨血清α-L-岩藻糖苷酶（AFU）、亮氨酸氨基肽酶（LAP）、r-谷氨酰转移酶（GGT）联合检测在早期原发性肝癌患者中的诊断价值。方法对85例肝病患者（肝硬化组45例、早期原发性肝癌组40例）及40例健康体检者（健康对照组）的血清 AFU、LAP 及 GGT 含量进行检测。结果早期原发性肝癌组、肝硬化组血清 AFU、GGT 的含量均高于健康对照组（均 P ＜0．05）。早期原发性肝癌组的 LAP 含量明显高于健康对照组,血清 AFU、LAP、GGT 单项检测及 AFU＋LAP＋GGT 联合检测阳性率均明显高于肝硬化组及健康体检组（均 P ＜0．05）,其中 AFU＋LAP＋GGT 联合检测阳性率最高（92％）。肝硬化组与健康对照组 LAP 含量比较差异无统计学意义（P ＞0．05）。结论AFU＋LAP＋GGT 联合检测有利于原发性肝癌的早期诊断。     

Family M42 aminopeptidase from the syntrophic bacterium Symbiobacterium thermophilum: characterization using recombinant protein

The chromosomal DNA of the syntrophic thermophile Symbiobacterium thermophilum contains open reading frames of the genes encoding family M42 aminopeptidases, Pep1079, Pep1080, and Pep1081. To characterize these peptidases, the genes were cloned into Escherichia coli and overexpressed. Our experiments using the recombinant proteins confirmed that Pep1079, Pep1080, and Pep1081 are components of arginyl or lysinyl aminopeptidases that require Co2+ for enzymatic activity. Coexistence of Pep1079 and Pep1080 is necessary for expressing high peptidase activity. Pep1081 enhances the activity of Pep1079 and Pep1080.     

采用模糊综合评价法比较黑豆多肽的脱苦工艺

采用模糊数学综合评判法,分析比较了β-环糊精包埋脱苦和枯草杆菌氨肽酶水解脱苦等对黑豆多肽苦味值的影响,确定最佳脱苦工艺条件。结果表明,枯草杆菌氨肽酶的脱苦效果优于β-环糊精,枯草杆菌氨肽酶脱苦的最佳工艺条件为:加酶量1500LAPU、pH8.5、温度50℃、时间4h。     

A model to assess lactic acid bacteria aminopeptidase activities in Parmigiano Reggiano cheese during ripening

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid β-naphthylamide (βNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.     

Leptin up-regulates the lactogenic effect of prolactin in the bovine mammary gland in vitro

The ability of leptin to up-regulate prolactin action in the mammary gland is well established. We examined the effect of leptin and prolactin on traits associated with lactation. Leptin and prolactin enhanced proliferation (thymidine incorporation) of the mammary gland cells, elevated the cells’ proliferation in a dose-responsive manner, and synergized to elevate the expression of amino acid metabolism via a 90% increase in aminopeptidase N expression. Leptin and prolactin decreased apoptosis (decreased caspase-3 expression by 60%) in the same manner. Leptin enhanced the effect of prolactin on all of these processes in bovine mammary explants. Leptin and prolactin regulated mTOR (mammalian target of rapamycin) by increasing expression by 66%, which is one of the signal-transduction junctions involved in the regulation of proliferation, apoptosis, and protein synthesis. These findings support the hypothesis that leptin up-regulates prolactin action in the bovine mammary gland.     

氨肽酶与中性蛋白酶协同水解大豆分离蛋白的研究

以大豆分离蛋白为底物,游离氨基酸含量、多肽分布、水解度为指标,氨肽酶ProteAXG为对照,开展中性蛋白酶和氨肽酶双酶协同水解的研究。在底物质量浓度6 g/dL,中性蛋白酶添加量5000 U/g,氨肽酶添加量9.75×105U/g,pH 8.5,温度50℃,水解4 h后,水解液中游离氨基酸含量13.6 mg/mL,多肽相对分子质量在1100以下,其中600左右的多肽约占16%,二肽三肽约占75%,水解度达50.8%。氨肽酶与中性蛋白酶优化后,水解度达62%。结果表明:该氨肽酶无论单独或与中性蛋白酶组合水解,都能达到较为深度的水解效果。