生淀粉糖化酶催化位点氨基酸及酶合成调控的初步研究

Studies on amino acid residues in the active site of raw-starch enzyme and regulation of the enzyme synthesis of Rhizopus OR-1UVN

通过对Rhizopus OR-1UVN菌种所产生淀粉糖化酶在不同底物不同缓冲溶液条件下酶最适pH的测定,推测出该生淀粉糖化酶活力中心催化位点氨基酸是天冬氨酸(Asp)和谷氨酸(Glu)。实验证明5～50mg/mL浓度葡萄糖对生淀粉糖化酶没有抑制作用。分别以浓度<5mg/mL葡萄糖和淀粉为碳源的培养基进行不同碳源发酵实验,发现以淀粉为碳源的培养基Ⅰ发酵15h开始产生淀粉糖化酶,以葡萄糖为碳源的培养基Ⅱ发酵35h开始产酶(葡萄糖浓度<8mg/mL),而且前者菌体较后者少,由此可知葡萄糖对产酶有阻遏作用。实验还发现解阻遏熟淀粉糖化酶的葡萄糖浓度(15mg/mL)比生淀粉糖化酶的要高。由于葡萄糖的阻遏作用不发生在翻译水平,而发生在转录水平上,而且生淀粉糖化酶(G1)与熟淀粉糖化酶(G2)来自同一条DNA链,可以推测存在mRNA的拼接。通过以生淀粉为碳源的比较实验,发现生淀粉对生淀粉糖化酶形成的诱导作用可能主要是通过mRNA拼接的调节来实现的。

Amino acid residues( Asp,Glu) in the active site of the raw-starch-digesting enzyme from Rhizopus OR-1UVN were inferred from the optimum pH under the condition of various starch and buffer. It was verified by experiment that 5 -50mg/mL glucose could not inhibit the activity of the enzyme. The strain was cultivated respectively with glucose (< 50mg/mL) and starch as carbon source. The beginning of enzyme synthesis in a medium containing starch as the carbon source and in a medium containing glucose as the carbon source were at 15h and 35h (glucose<8 mg/mL) respectively. However the former biomass was less than latter. The results showed the repression of glucose. Moreover, the repression of glucose was presented at transcription level rather than at translation level. As a result, it suggested that the regulation of glucose was similar to operon. In addition, the derepression of the enzyme (G2)that can only hydrolyze dextrinated starch was at higher glucose concentration(15mg/mL) than that of raw-starch enzyme(Gl). mRNAs of Gl and G2 were copies from the same chromosomal DNA. The results suggested that there was the splicing event. The result of comparison of cultivating in the medium containing raw starch as the carbon source to that of containing dextrinated starch as the carbon source suggested that raw starch associated with the splicing event.