Molecular and cellular studies on biofunctional proteins from microorganisms.

1. 我们分析了微生物产生的特定糖苷酶的功能，并试图利用这些酶生产有用的物质。(a)利用微生物糖苷酶的转糖基化活性，酶促合成各种生物活性糖肽。(b)利用微生物糖苷酶研究糖基化对蛋白质功能的影响。我们发现，具有广泛底物特异性的氨基肽酶A、B、N和二肽酶D是大肠杆菌K-12的四种半胱氨酸甘氨酸酶。测定了纯化后的氨基肽酶B的酶学特性。我们通过一种新的亲和标记剂鉴定出大肠杆菌γ-谷氨酰转肽酶的催化亲核试剂是位于小亚基n端的thrr残基。我们发现Gpr1是一种假定的g蛋白偶联受体，可以调节酵母中葡萄糖依赖的细胞cAMP水平。功能分析显示，高糖低氮条件下GPR1基因干扰物的假菌丝发育减少。我们还澄清了编码参与细胞粘附的细胞表面蛋白的FLO11基因转录水平的降低导致GPR1干扰物的假菌丝缺陷。

1. We analyzed the function of specific glycosidases produced by microorganisms and attempted to produce useful substances using these enzymes. (a) Enzymatic syntheses of various bioactive glycopeptides were carried out using transglycosylation activity of microbial glycosidases. (b) Effect of glycosylation on the function of protein was investigated using microbial glycosidases.2. We found that aminopeptidases A, B and N, and dipeptidase D with broad substrate specificity are the four cysteinylglycinases of Escherichia coli K-12. Enzymatic characteristics of purified aminopeptidase B were determined.3. We identified that the catalytic nucleophile of Escherichia coli γ-glutamyltranspeptidase is the Thrresidue at the N-terminal of the small subunit by a new novel affinity labeling agent.4. We found that Gpr1, a putative G-protein coupled receptor, regulated glucose dependent cellular cAMP level in yeast Saccharomyces cerevisiae. Functional analysis revealed that pseudohyphal development observed under high glucose and low nitrogen condition was reduced in the GPR1 gene disruptant. We also clarified that the reduced transcriptional level of FLO11 gene which encodes cell surface protein involved in cell adhesion caused pseudohyphal defect in the GPR1 disruptant.