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Analysis of sorbitol metabolism in genus Gluconobacter.

葡糖杆菌属山梨醇代谢分析。

基本信息

批准号：
18580078
负责人：
TOYAMA Hirohide
金额：
$ 2.45万
依托单位：
University of the Ryukyus (2007)Yamaguchi University (2006)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580078/
关键词：
Gluconobacter Acetic acid bacteria Sorbitol Oxidative fermentation

项目摘要

It is revealed that D-sorbitol, not L-sorbose, is the inducer of sldSLC, the gene for FAD-sorbitol dehydrogenase (SLDH), yielding L-sorbose from D-sorbitol. The obtained results were published.PQQ-glycerol dehydrogenase (GLDH), yielding also L-sorbose from D-sorbitol, connects efficiently to cytochrome bo 3 terminal oxidase and higher energy conservation ratio, thus it plays a major role in L-sorbose production. On the other hand, FAD-SLDH linked preferably to the cyanide-insensitive terminal oxidase, CIO. The obtained results were published.The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent L-sorbose reductase (NADPH-SR) activity. The obtained results are published. Now the enzyme is served for structural analysis by x-ray crystallographyReverse transcription-PCR experiments indicated that sboRA comprises an operon. It is expected that SboR is a transcriptional regulator which binds with L-sorbose. SboR was expressed and purified from E. coli transformant and used for gel-shift assay with the DNA fragment containing the promoter-operator region for sboRA, however, we failed to demonstrate that SboR has an ability to bind the DNA fragment with or without L-sorbose. The obtained results were published.The gene for FAD-gluconate dehydrogenase (GADH), yielding 2-ketogluconic acid from D-gluconate, was cloned and sequenced.For easy measurement for 5-ketogluconate (5KGA) and 2-ketogluconate (2KGA), genes for 5KGA reductase and 2KGA reductase were cloned and expressed in E. coli, Both enzyme preparations were easily obtained from E. coli transformants after one column chromatography.

结果表明，d -山梨醇而非l -山梨醇是fad -山梨醇脱氢酶（SLDH）基因sldSLC的诱导剂，sldSLC是由d -山梨醇生成l -山梨醇的基因。结果发表。pq -甘油脱氢酶（GLDH）也能从d -山梨醇中生成l -山梨糖，它与细胞色素bo3末端氧化酶的连接效率高，能量保存率高，在l -山梨糖的生成中起主要作用。另一方面，FAD-SLDH与氰化物不敏感末端氧化酶（CIO）结合较好。结果发表。从大肠杆菌转化中表达和纯化的sba酶显示nadph依赖性l -山梨糖还原酶（NADPH-SR）活性。并发表了所得结果。逆转录- pcr实验表明，sboRA含有一个操纵子。预计SboR是一种与L-sorbose结合的转录调节因子。从大肠杆菌转化中表达和纯化了SboR，并将含有sboRA启动子-操作子区域的DNA片段用于凝胶转移试验，然而，我们未能证明SboR具有与L-sorbose或不含L-sorbose的DNA片段结合的能力。结果发表。克隆了d -葡萄糖酸脱氢酶（GADH）基因，并对其进行了测序。为了方便测定5-酮葡萄糖酸盐（5KGA）和2-酮葡萄糖酸盐（2KGA），我们克隆了5KGA还原酶和2KGA还原酶基因，并在大肠杆菌中进行了表达。