秸秆水解液五、六碳糖混合发酵产琥珀酸放线杆菌的酶活分析及定向选育

doi:10.7506/spkx1002-6630-200901045

食品科学 ›› 2009, Vol. 30 ›› Issue (1): 190-194.doi: 10.7506/spkx1002-6630-200901045

秸秆水解液五、六碳糖混合发酵产琥珀酸放线杆菌的酶活分析及定向选育

李兴江，姜绍通* ，潘丽军，魏兆军，陈晓晖

合肥工业大学生物与食品工程学院

收稿日期:2007-12-23 修回日期:2008-03-16 出版日期:2009-01-01 发布日期:2010-12-29
通讯作者: 李兴江 E-mail:lixingjiang1978@hfut.edu.cn
基金资助:
国家科技支撑计划项目(2007BAD34B01)；安徽省自然基金项目(070411024)；合肥工业大学校基金项目(073002F)

Enzyme Activity Analysis and Directive Breeding of Actinobacillus succinogenes Fermenting Crop Straw Hydrolysate Containing Pentose and Hexose for Production of Succinic Acid

LI Xing-jiang，JIANG Shao-tong*，PAN Li-jun，WEI Zhao-jun，CHEN Xiao-hui

(School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China)

Received:2007-12-23 Revised:2008-03-16 Online:2009-01-01 Published:2010-12-29
Contact: LI Xing-Jiang E-mail:lixingjiang1978@hfut.edu.cn

摘要/Abstract

摘要：

目的：在产琥珀酸放线杆菌主要酶活分析基础上选育出高产突变株对工业规模生物转化农作物秸秆制备琥珀酸具有重要意义。方法：采用酸解方法处理原料秸秆并利用高效液相检测降解糖分，在菌株主要酶活分析及调控的基础上，利用软X 射线诱变结合丙烯醇抗性平板进行定向选育以降低乙醇流量，并对代谢酶活进行了分析。结果：秸秆水解液的检测表明200g 的秸秆干粉酶解得约47g 的葡萄糖和约26g 的木糖，酶活分析表明，Pepck、Pc及Mdh 是琥珀酸合成重要代谢酶且酶活可调控，发现乙醇脱氢酶(Adh)的高酶活造成了大量副产物乙醇的生成。筛选出的丙烯醇抗性突变株S.JST01 的乙醇产量由8.9g/L 降为1.4g/L，降低了84%，而琥珀酸流量产量由54.2g/L 升至63.1g/L，升高了16%。酶活检测表明Adh 的酶活力单位从614U 降低到108U。结论：围绕产琥珀酸放线杆菌Adh酶活降低实施定向选育有助于提高琥珀酸的流量，突变株的末端产物琥珀酸累积达63.1g/L，具有工业应用潜力。

关键词: 农作物秸秆, 产琥珀酸放线杆菌, 乙醇脱氢酶, 丙烯醇, 定向选育

Abstract:

Objective: It is very important to obtain high yield mutant strain on the base of key enzyme activity analysis of A. succinogenes for the industrial bioconversion of succinic acid from crop straw. Methods: The pentose and hexose hydrolyzed from crop straw hydrolyzed by dilute sulfuric acid were determinated using HPLC. During the fermentation of the hydrolysate, the activities of key enzymes were determinated and regulated. In order to decrease ethanol yield, those strains mutated by soft X-ray of synchronous radiation were screened out on the plates with high concentration of allyl alcohol. Then the alcohol dehydrogenase activity of the mutant strain was compared with that of the parent strain. Results: Determination of the hydrolysate showed that about 47 g glucose and 26 g xylose are from 200 g hydrolyzed crop straw. Pepck , Pc and Mdh are identified as the key enzymes of succinic acid metabolism and the high activity of Adh causes the cumulation of ethanol. Determination of end products from the parent strain indicated that the concentration of ethanol is the highest among those byproducts and that the yield of succinic acid and byproduct ethanol are 54.2 g/L and 8.9 g/L, respectively. Compared with the parent strain, the ethanol concentration produced by anti-allyl alcohol mutant strain S.JST01 decreases by 84%，from 8.9 to 1.4 g/L. Meanwhile the yield of succinic acid increases by 16%, from 54.2 to 63.1 g/L. Enzyme determination showed that the activity unit of alcohol dehydrogenase (Adh)decreases from 614 to 108. Conclusions: The interdiction of metabolic pathway of Adh decreases the metabolism prosuced by ethanol accordingly, thus the succinic acid flux is strengthened by the redundant carbon flux from the byproduct. Furthermore, the mutant strain S.JST01 with the end product yield of 63.1 g/L succinic acid is worth applying to industrial fermentation.

Key words: crop straw, Actinobacillus succinogenes, alcohol dehydrogenase, allyl alcohol, directive breeding

中图分类号:

李兴江，姜绍通*,潘丽军，魏兆军，陈晓晖. 秸秆水解液五、六碳糖混合发酵产琥珀酸放线杆菌的酶活分析及定向选育[J]. 食品科学, 2009, 30(1): 190-194.

LI Xing-jiang，JIANG Shao-tong*，PAN Li-jun，WEI Zhao-jun，CHEN Xiao-hui. Enzyme Activity Analysis and Directive Breeding of Actinobacillus succinogenes Fermenting Crop Straw Hydrolysate Containing Pentose and Hexose for Production of Succinic Acid[J]. FOOD SCIENCE, 2009, 30(1): 190-194.

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秸秆水解液五、六碳糖混合发酵产琥珀酸放线杆菌的酶活分析及定向选育

Enzyme Activity Analysis and Directive Breeding of Actinobacillus succinogenes Fermenting Crop Straw Hydrolysate Containing Pentose and Hexose for Production of Succinic Acid

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