摘要
将南极假丝酵母脂肪酶A(cala)基因克隆至组成型表达载体pGAPZαA中,电激转入X-33,获得高效表达的CALA酵母工程菌株。发酵液上清经超滤浓缩、硫酸铵沉淀和阴离子交换层析等步骤,获得纯化的重组CALA,其比酶活达384.90 U/mg。该酶最适温度为70℃,最适pH值为8.0。经50℃保温2 h,仍含有60%水解酶活力;在pH7.0和8.0溶液中比较稳定。经DMSO处理1 h,仍保持90%的活性;非离子型表面活性剂能提高CALA的酶活,金属离子在不同程度上抑制CA-LA的酶活。
Lipase A gene from Candida antarctica （CALA）was cloned into pGAPZαA vector to generate pGAPZαA-CALA, and then the construct was used to transform Pichia pastoris X-33 cells. Crude enzyme was purified with 10 kD membrane ,50% ammonium sulphate precipitation and Anion-exchange chromatography. After purification, the specific activity reached 384.90 U/rag. The optimal temperature and pH for CALA were 70% and pHS. 0, respectively. It remained 60% of activity after incubation at 50% for 1 h and the lipase was relatively stable at pH range 7.0 - 8. 0 after incubated in different pH solution at room temperature. The lipase remained 90% activity after incubated in buffer containing 30% dimethyl sulfoxide for 1 h. The presence of non-ionic surface active agent enhanced the activity of the enzyme, while metal ions decreased lipase activity.
出处
《生物技术通报》
CAS
CSCD
北大核心
2011年第6期175-181,共7页
Biotechnology Bulletin
基金
酶工程关键技术研究与产业(2009A1-E021)
关键词
南极假丝酵母脂肪酶A
毕赤酵母
组成型表达
纯化
酶学性质
Candida antarctica Lipase A（CALA） Pichia Pastoris GAP promoter Purification Properties of enzyme
