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Vc二步发酵伴生菌巨大芽孢杆菌的选育 总被引:10,自引:2,他引:10
巨大芽孢杆菌经紫外线诱变,获得2株耐低pH、抗KGA的菌株:Bn,B5。在pH6.7~7.0的发酵培养基中与氧化葡萄糖酸杆菌发酵,Bn和B5的平均糖酸转化率分别提高3.5%,3.3%.在pH6.2的发酵培养基中,平均糖酸转化率提高11.4%,12.3%。在pH6.2和pH7.0含3%KGA的培养基中,2菌提前3~6h到达对数生长期,稳定期延长3~6h.经连续30代转接,特性稳定。 相似文献
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实验充分利用混合菌系氧化葡萄糖酸杆菌(Gluconobacter oxydans)和蜡状芽孢杆菌(Bacillus cereus)混合发酵的优良特性,通过在发酵过程中间歇流加L-山梨糖的方法,实现了在自动控制温度、pH和溶氧的条件下,高效发酵L-山梨糖生成2-酮基-L-古龙酸(2-KLG)的目的。结果表明:当将L-山梨糖的终浓度调高到14%(w/v)时,2-KLG产量为130mg/mL左右,转化率达90%,发酵周期40—60h之间。结论:发酵过程中间歇流加L-山梨糖可以解除高浓度糖对产酸的抑制作用,提高了糖的转化率,但是发酵周期略有延长。 相似文献
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实验通过紫外线两轮诱变的方法诱变选育氧化葡萄糖酸杆菌(Gluconobacter oxydans),以实现提高2-酮基-L-古龙酸(2-KLG)产量的目的,获得1株高产2-KLG的菌株G5。结果证明该突变菌株在pH6.5—6.7的发酵培养基中与蜡质芽孢杆菌(Bcillus cereus)混合发酵,G5的平均糖酸转化率提高了13.49%,酸量达到83.6mg/mL,发酵周期缩短了2—3h。经连续10代转接发酵实验,证明其产酸稳定性较好。结论:氧化葡萄糖酸杆菌(Gluconobacter oxydans)的突变体G5提高了糖酸转化率,缩短了发酵周期。 相似文献
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新组合菌系氧化葡萄糖酸杆菌SCB329-苏芸金芽孢杆菌SCB933能在较长时间内保持高的转化活力且具有极强的抗杂菌污染的特性。在一次投糖分批发酵的基础上,探索在控制溶氧、pH、温度等条件下,分批加入L-山梨糖发酵生产2-酮基-L-古龙酸新工艺。采用新工艺,既充分利用了菌系的优良特性,又避免了高糖浓度可能对菌系造成的不良影响。L-山梨糖最终浓度达到14%(w/v),产酸120—135g/l,转化率90%左右,发酵周期40—65h。 相似文献
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就维生素C微生物一步发酵方法进行了探索,构建了酮古龙酸杆菌、氧化葡萄糖酸杆菌和芽孢杆菌三菌混菌一步发酵的方法。研究发现,植物内生芽孢杆菌可以与酮古龙酸杆菌配合,促进酮古龙酸杆菌生长和产酸。在有山梨醇存在的条件下酮古龙酸杆菌及其伴生菌能够快速地生长增殖,植物内生芽孢杆菌在发酵的10h中不断消耗山梨醇。5L的发酵罐中,酮古龙酸杆菌、氧化葡萄糖酸杆菌和植物内生芽孢杆菌三菌混菌一步发酵在恒定的30℃温度,600r/min搅拌速度和1.5vvm通气条件下,补料发酵过程中醇酸质量转化率达到了81.89%,在分批发酵过程中,醇酸质量转化率达到了87.90%,进一步优化了维生素C生产工艺。 相似文献
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目前,国内维生素C主要采用二步发酵法生产,其中第一步为生黑葡萄酸杆菌(Gluconobacter melano-genus)将D-山梨醇转化为L-山梨糖。考察了该菌株在提高培养基中山梨醇浓度时的发酵特性和发酵条件。实验室摇瓶实验结果显示,通风量、发酵前期及后期pH值控制、接种种液类型都影响高浓度山梨醇摇瓶发酵的转化率。以35%山梨醇浓度发酵液做种子液明显优于生产上采用的三级种子液(12%~17%山梨醇浓度),培养基前期pH值5.0~6.0,后期pH值4.2~3.9,装液量180 mL,发酵周期在30 h之内,山梨醇转化率在98%以上。培养基山梨醇浓度由23%提高到35%,发酵周期延长8 h。上述实验结果对指导生产工艺优化具有重要意义。 相似文献
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碳源和氮源对5-酮基-葡萄糖酸生成的影响 总被引:1,自引:0,他引:1
氧化葡萄糖杆菌Gluconobacter oxydans可以将葡萄糖氧化成葡萄糖酸,并进一步氧化成2-酮基-葡萄糖酸(2KGA)和5-酮基-葡萄糖酸(5KGA),其中5KGA在催化剂的作用下能够转化为L(+)-酒石酸。为了提高5-酮基-葡萄糖酸产量,以仅生成5KGA的氧化葡萄糖杆菌Gluconobacter oxydans HGI-1为出发菌株,研究不同碳源(蔗糖、乳糖、麦芽糖、淀粉、葡萄糖)和有机氮源(酵母浸粉、鱼粉、玉米浆、黄豆饼粉、棉籽饼粉)对5KGA产量的影响。500 mL摇瓶试验结果表明,当葡萄糖浓度为100 g/L时,5KGA产量最高为98.20 g/L;当有机氮源为酵母浸粉、鱼粉和玉米浆,其添加量的蛋白含量为1.60%时,5KGA产量分别为100.20 g/L、109.10 g/L和99.83 g/L,其中,使用鱼粉的5KGA产量最高,使用玉米浆的5KGA产量比酵母浸粉略低。出于经济考虑,文中选择玉米浆作有机氮源,并在5 L发酵罐中进行分批发酵放大试验,5KGA的产量为93.80 g/L,最大生成速率为3.48 g/(L·h),平均生成速率为1.56 g/(L·h)。结果表明,葡萄糖和玉米浆分别为Gluconobacter oxydans HGI-1规模化生产5KGA的最适碳源和氮源,可利用葡萄糖几乎全部(85.93%)转化为5KGA。 相似文献
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Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans.
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Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob+) in Escherichia coli S17-1 (Tra+). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose. 相似文献
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Ano Y Shinagawa E Adachi O Toyama H Yakushi T Matsushita K 《Bioscience, biotechnology, and biochemistry》2011,75(3):586-589
Selective, high-yield production of 5-keto-D-gluconate (5KGA) from D-glucose by Gluconobacter was achieved without genetic modification. 5KGA production by Gluconobacter suffers byproduct formation of 2-keto-D-gluconate (2KGA). By controlling the medium pH strictly in a range of pH 3.5-4.0, 5KGA was accumulated with 87% conversion yield from D-glucose. The pH dependency of 5KGA formation appeared to be related to that of gluconate oxidizing activity. 相似文献
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Saichana I Ano Y Adachi O Matsushita K Toyama H 《Bioscience, biotechnology, and biochemistry》2007,71(10):2478-2486
For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA. 相似文献
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《Journal of Molecular Catalysis .B, Enzymatic》1999,6(3):341-350
Gluconobacter suboxydans IFO 12528 was selected as the best strain for 5-keto-d-gluconate (5KGA) production by oxidative fermentation. 5KGA was markedly accumulated by the strain during cultivation in a medium containing d-glucose and/or d-gluconate. The resting cells and the membrane fraction also catalyzed 5KGA formation with a minimal formation of 2-keto-d-gluconate (2KGA), an alternative keto-d-gluconate from d-gluconate. The membrane fraction of the organism was confirmed to contain a membrane-bound d-gluconate dehydrogenase (GADH) catalyzing d-gluconate oxidation to 5KGA of which optimum pH and temperature were found at pH 4 and 15°C, respectively. After treating the membrane fraction with EDTA allowing conversion from holo-GADH to the apoenzyme, 5KGA-forming GADH was confirmed to be a pyrroloquinoline quinone (PQQ)-dependent enzyme by the fact that the enzyme activity was restored by the addition of CaCl2 and PQQ. The 5KGA-forming GADH was totally distinct from 2KGA-forming GADH in which a covalently bound FAD functions as coenzyme. 5KGA-forming GADH was well solubilized from the membrane fraction with n-octyl-β-d-thioglucoside and 5KGA formation was favourably catalyzed at relatively lower temperature, while 2KGA-forming enzyme was solubilized with Triton X-100 and relatively higher temperatures was optimum for 2KGA formation. These results are completely discrepant from the conclusion proposed by Klasen et al. [R. Klasen, S. Bringer-Mayer, H. Sahm, J. Bacteriol., 177, 1995, 2637] claiming that 5KGA was produced by d-gluconate oxidation catalyzed by NADP-dependent cytoplasmic 5KGA reductase from Gluconobacter species at fairly alkaline pH such as 10. 相似文献
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为确定维生素C二步发酵中巨大芽孢杆菌(伴生菌)芽孢形成对氧化葡萄酸杆菌(产酸菌)产酸的影响,本研究通过对巨大芽孢杆菌生长特性分析,选取培养12h(未形成芽孢)和36h(芽孢大量形成)巨大芽孢杆菌B.m2980,检测其胞外液、胞内液以及混合液对产酸菌生成2-酮基-L-古龙酸的影响。结果表明,在未开始形成芽孢时,伴生菌胞外液、胞内液及混合液对产酸菌的生长和产酸有较低的促进作用,其中胞内液的促进能力大于胞外液;在芽孢生成后,胞外液以及混合液对产酸菌生长和产酸的促进能力显著提高。 相似文献