葡萄糖酸氧化杆菌产木糖醇发酵条件优化及关键酶基因的克隆与表达
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摘要
本论文对Gluconobacter oxydans CGMCC 1.637 (G.oxydans CGMCC 1.637)转化D-阿拉伯醇产木糖醇的发酵条件进行优化并从G. oxydans CGMCC1.637中克隆得D-阿拉伯醇脱氢酶(D-arabitol dehydrogenase,ArDH)基因及木糖醇脱氢酶(xylitol dehydrogenase, XDH)基因,并对木糖醇脱氢酶的酶学性质进行研究。优化获得较适合的D-木酮糖发酵条件为：pH5.5, CaCO3 10g/L,装液量10%,接种量5%,30℃。在该发酵条件下,D-木酮糖的产率D-木酮糖的产量达到465mmol/L,得率(mmol/mmol)达到97%,转化效率为15.1 mmol/L h.研究表明,发酵液中D-木糖、柠檬酸、甘油、酒精、葡萄糖酸钠、葡萄糖等碳源的加入,会影响D-阿拉伯醇转化为D-木酮糖的效率。葡糖糖、D-果糖,D-甘露糖、D-木糖、甘油、D-山梨醇、乳糖、D-阿拉伯醇和木糖醇碳源对木糖醇脱氢酶活力和G. oxydans转化能力的影响的研究表明：D-山梨醇对G. oxydans CGMCC 1.637木糖醇脱氢酶的活性有明显的促进作用,而葡萄糖、果糖、木糖、木糖醇、D-阿拉伯醇对木糖醇脱氢酶活性有明显的抑制作用。在转化实验中,用D-甘露糖培养的Goxydans CGMCC 1.637的转化能力明显高于其他碳源培养的G. oxydans CGMCC1.637的转化能力,其中,用D-阿拉伯醇培养的G. oxydans CGMCC 1.637将D-木酮糖转化为木糖醇的能力最低,仅为对照(未加如上所述碳源的培养基)的35%,因此D-阿拉伯醇对G.oxydans CGMCC 1.637中木糖醇脱氢酶的D-木酮糖还原活性具有抑制作用。这一发现为克服一直以来G.oxydans发酵中D-木酮糖到木糖醇转化率低这一难题提供了线索。克隆自G.oxydans CGMCC 1.637的木糖醇脱氢酶与已报道的序列相似性仅为77%,是一个新的木糖醇脱氢酶基因。酶学性质研究表明,该酶在催化还原反应时,以D-木酮糖为底物,以NADH为辅酶,最适反应温度为30℃,最适反应pH为6.0；在催化氧化反应时,以木糖醇为底物,以NAD+为辅酶,最适反应温度为35℃,最适反应pH为10.0。在最适反应条件下,该酶氧化反应最高酶活为23.27 U/mg,而还原反应最高酶活约为氧化反应酶活的10倍(255.52U/mg)。
A highest yield of D-xylulose was achieved in the optimized fermentation conditions, including pH 5.5,30℃of culture temperature,100 mL fermentation medium in a 1000-mL flask of broth content and 5%(v/v) of inoculums size after 24 h fermentation. The investigation of various carbon sources on the bioconversion of D-xylulose to xylitol in G. oxydans CGMCC 1.637 after grown on them revealed that D-xylulose reductive activities of xylitol dehydrogenase was remarkably induced when G. oxydans CGMCC 1.637was cultivated on D-sorbitol. D-arabitol, which supported a high cell growth, remarkly inhibited the oxidative activity of xylitol dehydrogenase and the bioconversion ability of G. oxydans CGMCC 1.637. Oxidative activity of xylitol dehydrogenase in G. oxydans CGMCC 1.637 and the bioconversion ability of G. oxydans CGMCC 1.637 after grown on D-arabitol were inhibited, which provided a valuable clue for further study to increase xylitol yield from D-arabitol. Touch-down PCR was applied to clone the xylitol dehydrogenase gene from chromosomal DNA of G. oxydans CGMCC 1.637. The 798-bp open reading frame of xylitol dehydrogenase encoded a protein of 265 amino acids, with the molecular mass of 27.95 kDa. Sequence analysis of the putative protein revealed it to be a member of short-chain dehydrogenase/reductase family. Xylitol dehydrogenase showed oxidative activity with xylitol and sorbitol and no activity with other polyols, such as D-arabitol. Km and Vmax with xylitol was 78.97mmol/L and 40.17 U/mg, respectively. The highest oxidative activity of xylitol dehydrogenase for xylitol was only 23.27 U/mg at optimum conditions (pH 10.0,35℃). However, the activity of its reverse reaction, D-xylulose reduction, reached 255.55 U/mg at optimum conditions (pH 6.0,30℃), 10-times higher than that of xylitol oxidation.
A highest yield of D-xylulose was achieved in the optimized fermentation conditions, including pH 5.5,30℃of culture temperature,100 mL fermentation medium in a 1000-mL flask of broth content and 5%(v/v) of inoculums size after 24 h fermentation. The investigation of various carbon sources on the bioconversion of D-xylulose to xylitol in G. oxydans CGMCC 1.637 after grown on them revealed that D-xylulose reductive activities of xylitol dehydrogenase was remarkably induced when G. oxydans CGMCC 1.637was cultivated on D-sorbitol. D-arabitol, which supported a high cell growth, remarkly inhibited the oxidative activity of xylitol dehydrogenase and the bioconversion ability of G. oxydans CGMCC 1.637. Oxidative activity of xylitol dehydrogenase in G. oxydans CGMCC 1.637 and the bioconversion ability of G. oxydans CGMCC 1.637 after grown on D-arabitol were inhibited, which provided a valuable clue for further study to increase xylitol yield from D-arabitol. Touch-down PCR was applied to clone the xylitol dehydrogenase gene from chromosomal DNA of G. oxydans CGMCC 1.637. The 798-bp open reading frame of xylitol dehydrogenase encoded a protein of 265 amino acids, with the molecular mass of 27.95 kDa. Sequence analysis of the putative protein revealed it to be a member of short-chain dehydrogenase/reductase family. Xylitol dehydrogenase showed oxidative activity with xylitol and sorbitol and no activity with other polyols, such as D-arabitol. Km and Vmax with xylitol was 78.97mmol/L and 40.17 U/mg, respectively. The highest oxidative activity of xylitol dehydrogenase for xylitol was only 23.27 U/mg at optimum conditions (pH 10.0,35℃). However, the activity of its reverse reaction, D-xylulose reduction, reached 255.55 U/mg at optimum conditions (pH 6.0,30℃), 10-times higher than that of xylitol oxidation.
引文
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[1]Prust C, Hoffmeister M, Liesegang H, et al. Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans[J]. Nature Biotechnology,2005, 23:195-200.
[2]Sugiyama M, Suzuki S-I, Tonouchi N, et al. Cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol[J]. Bioscience Biotechnology Biochemistry,2003,67:584-591.
[3]Cheng HR, Jiang N, Shen A, et al. Molecular cloning and functional expression of D-arabitol dehydrogenase gene from Gluconobacter oxydans in Escherichia coli[J]. FEMS Microbiology Letters,2005,252:35-42.
[4]Kulhanek M. Microbial dehydrogenations of monosaccharides[J]. Advance in Appl Microbiology,1989,34:141-182.
[5]尤蓉,余晓斌,李正华.控制pH值对云芝发酵罐液态培养的影响[J].食品与发酵工业,2001,27(12)：20-23.
[6]王翠华,李友元,陈长华,李啸.温度对丙酮酸生物合成动力学、能荷和氧化-还原度的影响[J].生物工程学报,2006,22(2)：316-321.
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