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