Klebsiella sp.GXK-1的α-L-鼠李糖苷酶的酶学性质

Enzymatic Characterization of α-L-rhamnosidase from Klebsiella sp.GXK-1

【目的】克隆、表达克雷伯氏菌Klebsiellasp.GXK-1菌株中的α-L-鼠李糖苷酶基因,并研究重组酶的酶学性质。【方法】比对分析GenBank数据库中克雷伯氏菌同属的α-L-鼠李糖苷酶基因序列,设计简并引物PCR扩增基因的保守区。扩增目的基因,以pSE380为表达载体构建重组质粒pSE-rha1,并在大肠杆菌E.coli XL-blue进行诱导表达,使用镍亲和层析纯化重组蛋白,研究目的蛋白RHA1的酶学性质。【结果】以pNPR为底物,进行重组酶酶学性质的研究。重组酶RHA1的最适pH值和最适温度分别为5.0和45℃,Km值为(0.223±0.030)mmol/L,V_(max)值为(1.272±0.121)μmol/(min·mg)。在pH值为6~10的缓冲液内酶活力仍保持在80%以上;在温度为40℃以下时,酶活较为稳定,但在温度高于40℃时酶活力迅速下降。RHA1能水解pNPR、橙皮苷和芦丁。【结论】RHA1具有良好的pH稳定性,不仅能够水解人工底物pNPR,还能够水解α-1,6键的天然底物橙皮苷和芦丁,具有一定的医疗应用价值。

[ObjectivelThe α-L-rhamnosidase gene in Klebsiella sp. GXK 1 strain was cloned and expressed,and the enzymatic properties of the recombinant enzyme were studied. [Methods]By searching GenBank database,the gene sequences coding α-L-rhamnosidase of klebsiella were an alyzed. The conservative sequence was amplified by degenerate primers. And a gene encoding L rhamnosidase was cloned by PCR. Then, the recombinant plasmid pSE rhal was constructed. And it was introduced and expressed in E. coli XL blue. The recombinant protein RHA1 was purified with Ni NTA. The enzymatic properties of the recombinant protein RHA1 were investi gated in detail. [Results] Using pNPR as a sub strate,the enzymatic properties of the recombi nase were studied. The optimum pH and optimum temperature of recombinase RHA1 were 5. 0 and 45℃ , respectively. Its Km and Vmax values were （0. 223±0. 030） mmol/L and （1. 272±0. 121） μmol/（mg · min） ,respectively. The enzyme activity remained above 80% in the buffer of pH 6-10;When the temperature was be low 40℃, enzyme activity was relatively stable. But when the temperature was higher than 40℃ , enzyme activity dropped rapidly. RHA1 could hydrolyze pNPR, hesperidin, rutin. [Conclusion]RHA1 has good pH stability,not only can hydrolyze artificial substrates but also can hy drolyze α-1 and 6 band natural substrates hesperidin and rutin,which has certain medical appli cation value.