酮古龙酸菌WB0104中L-山梨糖脱氢酶的分离和性质研究  

Isolation and characteristics research of L-sorbose dehydrogenase in Ketogulonigenium sp.WB0104

作　　者：郝爱鱼 贾茜 吴洪涛 周华芮 耿文飞 高文利 赵军强 贺建功 

机构地区：[1]华北制药集团新药研究开发公司,石家庄050015 [2]华北制药集团维尔康公司,石家庄050015

出　　处：《工业微生物》2008年第1期10-14,共5页Industrial Microbiology

基　　金：国家863计划资助项目2001AA223071

摘　　要：从两步法生产维生素C重要前体2-酮基-L-古龙酸(2-Keto-L-gulonic acid,简称2-KLGA)的第二步转化菌酮古龙酸菌Ketogulonigeniumsp.WB0104的细胞质中,经SP-SepharoseFast Flow、DEAE-CL6B和凝胶过滤的方法,分离到了L-山梨糖脱氢酶(L-Sorbose Dehydrogenase,简称SDH),质谱测定该酶分子量为60.499kd,而用凝胶过滤法测定其分子量为139.053±4.96kd,由此推测该酶为具有两个相同亚基的二聚体;辅基分析表明SDH含有辅基pyrroloquinolinequinone(PQQ)。以L-山梨糖(L-Sorbose)为底物,2,6-Dichlorophenolindophenol(DCIP)为电子受体,SDH具有明显的脱氢酶活性;以L-Sorbose为底物的Km值为23.94mmol/L。在无细胞体系中,在phenazine methosulfate(PMS)存在的情况下,SDH能将底物L-Sorbose直接转化为2-KLGA。Ketogulonigenium sp. WB0104 is the second microorganism in the production of 2-keto-L-glulonate （2-KLGA）, which is an important procursor of Vintim C. L-Sorbose Dehydrogenase（SDH） was isolated from the cell free extract of the culture of WB0104 by centrifugation, ultrasonication, ultracentrifugation and chromatography on SP-Sepharose Fast Flow, DEAE-CL6B and gel filter. The molecular weight （MW）of SDH was 60.499 kd by mass-spectrum（MS） analysis and was 139. 053 ± 4.96kd by gel filter anylasis of superdex 200, which showed that SDH was a dimer incluing identical subunits. Prosthetic group was identified as pyrroloquinoline quinone（PQQ）. SDH activity was assayed according to the method of 2, 6-dichlorophenolindophenol as electron acceptor and L-sorbose as a substrate. Km value was 23.94 mmol/L by the analysis method above mentioned with L-sorbose as substrate. In vitro bioactivity anaylsis showed SDH could convert L-sorbose to 2-KLGA directly under the condition of phenazine methosulfate（PMS） as electron acceptor.

关 键 词：L-山梨糖 2-酮基-L-古龙酸 L-山梨糖脱氢酶 维生素C 

分 类 号：TQ925[轻工技术与工程—发酵工程]