反式-4-羟基-L-脯氨酸羟化酶基因密码子优化及表达

Improvement of Expression Level of Proline 4-hydroxylase by Codon Optimization

L-羟脯氨酸用途非常广泛,具有较好的应用价值,利用反式-4-羟基-L-脯氨酸羟化酶能将L-脯氨酸通过生物转化法生成反式-4-羟基-L-脯氨酸.对指孢囊菌(Dactylosporangiumsp.)RH1的反式-4-羟基-L-脯氨酸羟化酶基因密码子优化及蛋白可溶性表达条件进行了探索.基因p4hy的GC摩尔百分含量由73.63降至61.45;在23℃下,加入1mmol/L IPTG及分子伴侣pG-KJE8后可溶性蛋白的含量明显增多;密码子优化后的酶蛋白P4HY具有较好的催化生产羟脯氨酸的能力,反应40h后转化率达到72.8%.

Trans-4-proline hydroxylase can hydroxylate L-proline to trans-4-hydroxy-L-proline,which is a widely applied in medicine,food,biochemistry and the beauty industry.In order to improve the expression level of proline 4-hydroxylase of Dactylosporangiumsp.RH1,codon optimization and expression condition optimization have been performed.The GC content of gene p4hy was reduced from 73.65 to 61.45.The protein expression level and solubility of P4HY were improved when induced with 1mmol/L ITPG with the existence of chaperone plasmid pG-KJE8 for 6hat 23℃.The codon optimized P4HY has high catalytic activity for conversion of L-proine to trans-4-hydroxy-L-proline,with a conversion rate at 72.8%after 40h＇s reaction.