胱硫醚β-裂解酶的表达纯化及性质研究

Expression,Purification and Characterization of Cystathionine β-Lyase

通过PCR从大肠杆菌(E.coliK12)基因组DNA中扩增出胱硫醚β-裂解酶(Cystathionineβ-lyase,CBL)基因,构建了能高效表达CBL的重组菌E.coliBL21(pETDuet-1-CBL)。重组菌在30℃、0.5 mmol.L-1IPTG存在条件下诱导9 h,可溶性CBL的表达量达18 mg.L-1,占到菌体可溶性总蛋白的23%。将重组菌超声破碎后上清液经His-Trap Fast Flow亲和层析一步纯化得到CBL,回收率为71%,纯度达到94%,纯化后的CBL单位酶活为134.6 U.mg-1。为防止CBL溶液冻融后发生聚沉,向纯化后的酶液中加入5%甘油,-20℃保存。重组酶的最适反应温度为35℃,酶活性稳定的温度范围为20～35℃。重组酶的最适反应pH值为8.0,4℃下在pH值8.0的缓冲溶液中保温10 h酶稳定性最高。重组酶的KmL-cystathionine值为3.78 mmol.L-1,VmaxL-cystathionine值为0.928 mmol.L-1.h-1。本研究建立了基于CBL催化反应产物丙酮酸与2,4-二硝基苯肼显色原理测定CBL酶活性的新方法。

The cystathionine β-lyase（CBL） gene was cloned from the genome of Escherichia coli K12,and the recombinant E. coli BL21 （pETDuet-1-CBL） strata was constructed. After the recombinant E. coli was grown at 37 ℃ to an OD6oo of 0.4-0.6 ,it was induced with IPTG at a final concentration of 0.5 mmol · L^-1 for 9 h. The productivity of CBL reached 18 mg · L^-1 ,and was 23% of total soluble proteins of recombinant strain. The supernatant of the disrupted cells by sonication was directly loaded on HisTrap Fast Flow column. The CBL fraction was absorbed on the column and was eluted with imidazole at a final concentration of 250 mmol · L^-1. The purified CBL had a specific activity of 134.6 U · mg^-1 with a 71% activity recovery,and the purity reached 94% after purification by affinity chromatography. To prevent the protein aggregation, the fractions containing CBL added 50^ glycerol were stored frozen at -20 ℃. The optimum reaction temperature of recombinant enzyme was 35℃ ,and the purified CBL was relatively stable at the temperature of 20-35 ℃. The optimum reaction pH value of the purified CBL was 8.0,and the recombinant enzyme activity changed little when incubated in the buffer with pH value of 8.0 at 4 ℃ for 10 h. Km^L-cystathionine of recombinant CBL was 3.78 mmol · L^-1 and Vmax^L-cystathionine was 0. 928 mmol·L^-1·h^-1. In addition,the present study established a novel method for detemination of CBL activity based on the color reaction between pyruvate and 2,4-dinitrophenylhydrazine.