α-酮戊二酸半醛脱氢酶的定点突变及酶学性质变化

摘要

3-HP是一种性质优良的化学中间体,以甘油为底物生产3-HP的研究备受青睐,而限制3-HP产量的主要原因是AldH活力过低.对源于Azospirillam brasilense的 α-酮戊二酸半醛脱氢酶(KGSADH)基因同源建模和结构分析,采用定点突变方法得到高活力KGSADH,将突变酶表达纯化,探讨酶学性质的变化.结果表明,TU-KGSADH(E120D/P219A)酶活力达6.03 U/mg,比原始酶比酶活提高了322%,最适温度由35℃降低为30℃,且热稳定性降低,最适pH为8.0,在pH 6.0-8.0条件下酶活力有所提高.Zn2+对KGSADH的酶活有较强的抑制作用,而Co2+、Fe3+、Fe2+对KGSADH的酶活有较大的激活作用.在酶的动力学方面,TU-KGSADH对乙醛的Km由7.58降为6.28 mmol/L,Vmax由10.6提高为12.3 U/mg.对酶学性质改变的因素分析发现,120位突变与最适pH下降和pH稳定性向酸性偏移有关,219位突变可能是最适温度和热稳定性降低的原因,为醛脱氢酶进一步定向改造提供参考.%3-HP is an excellent chemical intermediate,and the study on the production of 3-HP with glycerol as substrate is favored, while the main reason for limiting 3-HP production is the low activity of AldH. A gene of α-ketoglutorate semialdehye dehydrogenase(KGSADH) from Azospirillam brasilense was constructed by homology modeling and structure analysis. The KGSADH with high activity was obtained by site-directed mutation. The KGSADH was expressed and purified for the investigation of enzymatic properties. The results revealed that the activity of TU-KGSADH(E120D / P219A)reached 6.03 U/mg,which was 322% higher than that of the original KGSADH. The optimal temperature of TU-KGSADH decreased from 35℃ to 30℃,the thermostability reduced,and the optimal pH was 8.0 and enzymatic activity was improved slightly under pH 6.0-8.0. Zn2+ had a strong inhibitory effect on the activity of KGSADH,while Co2+,Fe3+ and Fe2+ had a great effect on the activity of KGSADH. In the kinetic aspect of the TU-KGSADH,the Km with acetaldehyde as a substrate reduced from 7.58 to 6.28 mmol/L, and Vmax increased from 10.6 to 12.3 U/mg. Finally,the factors affecting the changes of enzymatic properties were analyzed,The 120 site mutation was associated with the decrease of the optimal pH and pH stability to acidic migration,and the 219 site mutation may be the reason for the decrease of the optimum temperature and thermal stability,providing a fine reference for further research in directional modification of aldehye dehydrogenase.