摘要
为建立一种高效的粘红酵母ATP-柠檬酸裂解酶粗酶提取方法,对比了酶法、研磨法、超声波法和玻璃珠法对粘红酵母胞内粗酶提取的影响,通过可溶性蛋白浓度分析和ATP-柠檬酸裂解酶活性测定,对粗酶提取方法进行评价,并在此基础上从震荡时间和珠体装量方面对玻璃珠法粘红酵母ATP-柠檬酸裂解酶提取工艺进行了优化。可溶性蛋白浓度分析和ATP-柠檬酸裂解酶活性测定结果表明:玻璃珠法提取粗酶优于其他3种方法。对玻璃珠法进行进一步优化,得到的最优化的提取条件为细胞干重与珠体重量之比为1∶1,冰浴震荡时间为20min,在此条件下,ATP-柠檬酸裂解酶比活可达到0.98U·mg^(-1),分别是酶法、研磨法、超声波法测得酶活性的1.9,2.2,2.1倍,可溶性蛋白浓度为6.5mg·m L^(-1),分别是酶法、研磨法、超声波法测得酶活性的1.4,4.1,3.3倍。经过优化的玻璃珠法提取粘红酵母胞内粗酶液操作简便,且能够保持较高的ATP-柠檬酸裂解酶活性,是较理想的粘红酵母粗酶提取方法。
In order to provid e an efficient method of crude enzyme extraction, the extraction of ATP-citrate lyase(ACL) from Rhodotorula glutinis was studied. Four different broking methods including snailase enzymolysis, grinding crush, ultrasonic crush, and glass beads crush were compared. Soluble protein concentration and the specific activity of ACL analysis indicated that the method of glass beads crush exhibited high efficiency for the crude enzyme extraction. Based on the above results, the method of glass beads crush was further optimized. The optimal conditions were the ratio of dry cell weight and glass beads 1:1 with a 20 min vibrating time. The results suggested that the soluble protein concentration and specific activity of ACL were 6.5 mg·m L^(-1) and 0.98 U·mg^(-1), respectively.The ACL specific activity and protein concentration obtained from modified glass beads method were 1.9 fold, 2.3 fold, 2.1 fold and 1.4 fold, 4.1 fold, 3.3 fold as that of snailase enzymolysis, grinding crush and ultrasonic crush, respectively. The optimized method of glass beads crush can be operated easily and higher enzyme specific activity can be obtained. It can be used as an efficient enzymatic extraction method for Rhodotorula glutinis cells.
出处
《沈阳农业大学学报》
CAS
CSCD
北大核心
2016年第1期103-108,共6页
Journal of Shenyang Agricultural University
基金
清华大学自主科研计划项目(2012Z02144)
工业生物催化教育部重点实验室(清华大学)开放基金项目(2015302)
关键词
粘红酵母
粗酶提取
ATP-柠檬酸裂解酶
比活力
Rhodotorula glutinis
crude enzyme extraction
ATP-citrate lyase
specific activity
