摘要
为在谷胱甘肽合成工艺中降低成本,同时能够实现酶的可存储性、商品化性,将谷胱甘肽合成酶系经过合适的沉淀剂沉淀,戊二醛交联制备成共固定交联酶聚集体。通过优化沉淀条件,交联条件,蛋白浓度及添加剂的使用,最终得到最优的制备方法为:在4℃,30 g/L蛋白浓度条件下,加入PEI-600(添加剂w∶酶液蛋白w=1∶5)与酶液进行充分混合,然后加入60%饱和度硫酸铵沉淀4 h,最后以0.5%戊二醛进行交联。最终蛋白沉淀率为94.9%,酶活回收率为48.2%,单位CLEAs的酶活为4.9 U/g,分别相比对照组的28.7%及2.57 U/g提高了19.5%和2.33 U/g,GSH最高产量达到11.26 g/L,在转化第12批次时GSH产量为9.03 g/L,相对酶活力为78.4%,4℃条件下储藏33 d后相对酶活力为92.12%。经优化制备条件后得到的谷胱甘肽合成酶系CLEAs有较高的初始酶活力,能稳定转化10批次以上,储藏稳定性佳,具有优良的工业化前景。
In order to reduce the cost in synthesis of glutathione（GSH）,and concurrently to realize the enzyme storage and thecommercialization,the glutathione synthetase was precipitated through appropriate precipitant,and immobilized crosslinked enzymeaggregates（CLEAs）were prepared by glutaraldehyde crosslinking. By optimizing the precipitation and crosslinking conditions,as well as theuses of protein concentration and additives,finally the optimal preparation condition was determined as ：4℃,30 g/L protein concentration,adding PEI-600（additive W∶enzyme protein w = 1∶5）fully mixed with the enzyme solution,then 60% saturated ammonium sulfate addedfor precipitation for 4 h,and finally crosslinked by 0.5% glutaraldehyde. The final protein precipitation rate was 94.9% and the recovery ofenzyme activity was 48.2%,enzyme activity per CLEAs was 4.9 U/g,respectively,compared to the control group 28.7% and 2.57 U/g,andincreased by 19.5% and 2.33 U/g. The highest yield of GSH reached 11.26 g/L,GSH yield was 9.03 g/L in the 12 th batches of conversion,and the relative enzyme activity was 78.4%;after 33 d storage at 4℃,the relative enzyme activity remained 92.12%. In conclusion,afteroptimizing the preparation conditions,the CLEAs of glutathione synthetases presented higher initial enzyme activity,which could be stablyconverted more than 10 batches,with favorable storage stability and solid industrialization prospects.
出处
《生物技术通报》
CAS
CSCD
北大核心
2016年第11期261-270,共10页
Biotechnology Bulletin
关键词
谷胱甘肽合成酶系
固定化
交联酶聚集体
添加剂
glutathione synthetases
immobilization
crosslinked enzyme aggregates
additives
