Analysis of sorbitol metabolism in genus Gluconobacter.

结果表明，D-山梨醇而不是L-山梨糖是由D-山梨醇产生L-山梨糖的山梨醇脱氢酶基因sldSLC的诱导剂。PQQ-甘油脱氢酶也是从D-山梨醇中产生L-山梨糖的酶，它能有效地与细胞色素BO3末端氧化酶连接，具有较高的能量守恒率，在L山梨糖的生产中起着重要的作用。另一方面，FAD-SLDH最好连接到氰化物不敏感的末端氧化酶CIO。从大肠杆菌转化子中表达和纯化的SboA酶具有依赖于NADPH的L山梨糖还原酶(NADPH-SR)活性。所获得的结果已发表。逆转录-聚合酶链式反应实验表明，sboRA含有操纵子。推测Sbor是一个与L-山梨糖结合的转录调控因子。从大肠杆菌转化子中表达和纯化了Sbor，并将其与含有sboRA启动子-操纵区的片段进行了凝胶位移分析，但我们未能证明Sbor具有与或不与L山梨糖结合的能力。对D-葡萄糖酸转化为2-酮葡萄糖酸的FAD-葡萄糖酸脱氢酶(GADH)基因进行了克隆和测序，为了便于测定5-酮葡萄糖酸(5KGA)和2-酮葡萄糖酸(2KGA)，克隆了5KGA和2KGA还原酶基因并在大肠杆菌中表达，转化子经一次柱层析即可获得这两种酶制剂。

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.