摘要
从５Ｌ罐发酵Ｌ－山梨糖的Ｇｌｕｃｏｎｏｂａｃｔｅｒ ＳＣＢ３２９和Ｂａｃｉｌｌｕｓ ｔｈｕｒｉｎｇｉｅｎｓｉｓ ＳＣＢ９３３混合菌株中差速离心收集ＳＣＢ３２９菌体，破碎，离心获得无细胞抽提液，硫酸铵分级沉淀蛋白后依次经ＤＥＡＥＣｅｌｌｕｌｏｓｅ ５２和Ｑ Ｓｅｐｈａｒｏｓｅ ＦＦ柱层析分离得到了Ｌ－山梨糖脱氢酶（ＳＤＨ），它能将Ｌ－山梨糖脱氢氧化为Ｌ－山梨酮，ＳＤＳ－ＰＡＧＥ电泳测得分子量约为６０ＫＤ。动力学性研究表明它为一个典型的Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ氏酶，对Ｌ－山梨糖作用的Ｋｍ值为 ５２．７ ×１０－３ｍｏｌ／Ｌ，对ＤＣＩＰ作用的Ｋｍ值为 ２．０６ × １０－３ｍｏｌ／Ｌ，最适作用ｐＨ和温度分别为７．０和４２℃。Ｍｇ２＋，Ｃａ２＋是酶的激活剂，Ｃｕ２＋是酶的抑制剂，ＥＤＴＡ对该酶也有明显的抑制作用。不同碳源对酶活的影响研究表明它在ＳＣＢ３２９中是组成型表达。
After the fractional precipitation by (NH4)2SO4 and the chromatography of DEAE Cellulose 52 and Q Sepharose FF, the L-sorbose dehydrogenase has been purified from the cell free extract of the mixed culture of Gluconobacler oxydans SCB329 and Bacillus thringiensis SCB933 in the fermentation of L- sorbose in 5L fermenter. It's MW is about 60an in the SDS-PAGE electrophoresis. Dynamic studies demonstrates that it is a typical Michalis-Menten enzyme with Km of 52.7 × 10 -3 -mol/ L for L-sorbose and 2.06 ×10-3mol/ L for DCIP. Its optimal pH and temperature are 7.0 and 42℃ respectively. Mg2+ and Ca2+ can activate the enzyme although Cu2+ and EDTA inhibit it. The L-sorbose dehydrogenase is conjectured a constitutive enzyme of Gluconobacter oxydans SCB329 by the analysis of the enzyme activity after the flask fermentation in the different carbon sources.
出处
《微生物学通报》
CAS
CSCD
北大核心
2000年第2期89-92,共4页
Microbiology China
