【作者】 徐静；

【导师】 高超；

【作者基本信息】 山东大学 ， 微生物学， 2017， 硕士

【摘要】 氧化葡萄糖酸杆菌(Gluconobacter oxydans)能够快速不完全氧化多种糖或醇制备相应化合物,是重要的生物催化剂。目前主要利用甘露醇或山梨醇有机培养基培养G.oxydans。但是多元醇有机培养基价格昂贵,较高的培养成本会影响后续生物转化过程的放大。因此,寻找低成本培养方法对G.oxydans在工业上的应用具有重要意义。本论文利用无机盐培养基,首先分析了G.oxydans在常见碳源中的生长能力,发现甘油能够有效支持G.oxydans的生长。进一步分析G.oxydans和G.oxydans衍生菌株在甘油无机盐培养基中的生长代谢时,获得一株双敲除菌株△1068A0854。△1068△0854在甘油无机盐培养基中菌体得率为0.20g DCW/g,是野生菌的6.7倍,而且具有更高的葡萄糖脱氢酶活性。因此,利用甘油无机盐培养基培养△1068△0854制备生物催化剂,不仅可以有效降低培养成本,而且具备生物催化优势。利用甘油无机盐培养基培养△1068A0854获得的静息细胞催化木糖生产木糖酸,产量为61.5 g/L,得率为1.05g/g。根据基因组注释,G oxydans胞内存在甘油代谢操纵子。该操纵子由glpD(gox2088编码的甘油-3-磷酸脱氢酶),glpF(gox2089编码的甘油易化蛋白),glpK(gox2090编码的甘油激酶)三个结构蛋白基因和转录调控蛋白基因glpR组成。论文第二部分主要分析了该操纵子的代谢调控机制。通过RT-PCR确定了该操纵子调控蛋白基因glpR和结构基因glpD、glpF、glpK共转录表达。GlpR为该操纵子的阻遏蛋白,其结合区域为glpR和glpD的基因间区Pgdh。虽然文献中GlpR阻遏蛋白的常见效应物为甘油-3-磷酸,但本文研究发现甘油或其中间产物并不是G oxydans甘油代谢操纵子的效应物。菊粉是一种线性直链多糖,由果糖通过β(2,1)糖苷键连接,末端常通过α(2,1)糖苷键连接一个葡萄糖。如何高效的水解菊粉是其作为微生物发酵碳源首要解决的问题。目前菊粉水解通常有酸解和酶解两种方法。本论文第三部分首次发现新的水解菊粉方式,Fe3+可以在高温条件下高效水解菊粉。利用Fe3+水解菊粉制备的高浓度糖液浓度达到854.16 g/L,Fe3+终浓度大约为1.35 mM。因此,Fe3+和菊粉在标准条件下灭菌获得的菊粉水解液可以为微生物发酵提供糖源,具有工业应用价值。更多还原

【Abstract】 Gluconobacter oxydans is an important biological catalyst,which can incompletely oxidize a variety of sugars or alcohols to produce corresponding compounds-G.oxydans is always cultured in sorbitol or mannitol organic medium,the price of which is very expensive.Therefore,it’s very significant for industrial application to find an inexpensive medium of the strain.In this study,the growth of G.oxydans on minimal mediums with different common carbon source was examined.It was found that glycerol could support the growth of G.oxydans in minimal mediums,and we got a derivative,△1068△0854.The cell yield of △1068△0854 from glycerol minimal medium was 0.20 g DCW/g,which was 6.7 times compared with wild type,and △1068△0854 had higher glucose dehydrogenase activity.△1068△0854 from glycerol minimal medium reduces production cost and has catalytic advantage,giving more values to the industry application.Xylose catalyzed by △1068△0854 resting cells from glycerol minimal medium produced 61.5 g/L xylonate,and the yield was 1.05 g/g.According to the genome annotation,G.oxydans contains a glycerol operon,which is composed of glpD(glycerol-3-phosphate dehydrogenase encoded by gox2088),glpF(glycerin facilitator encoded by gox2089),glpK(glycerol kinase encoded by gox2090)and glpR(transcriptional regulatory protein encoded by gox2087).The second part of this paper,the regulatory mechanism of this operon was explored.RT-PCR analysis indicated that the operon is composed of glpR,glpD,glpF and glpK.GlpR is the repressor protein of this operon,and its binding region is the intergenic region of and Although glycerol-3-phosphate is a common inducer of glycerol operon,glycerol and its intermediates can not induce the glycerol operon in G.oxydans.Inulin is a kind of linear polysaccharide.It is composed of fructoses connected by β(2,1)glycosidic bonds and a glucose connected to the end by an a(2,1)glycosidic bond.Hydrolysis of inulin is the first problem to be solved.Inulin is usually hydrolysed by enzyme or acid.In the third part of this paper,we found that Fe3+ can hydrolyse inulin under high temperature.In this way the final concentration of sugar solution is 854.16 g/L,and the concentration of Fe3+is about 1.35 mM.The hydrolysate can be used for microbial fermentation and has high economic value.更多还原