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

关键词:  克雷伯氏菌  α-L-鼠李糖苷酶  克隆表达  酶学性质

Key words:  Klebsiella sp. GXK-1  α-L-rhamnosidase  cloning and expression  enzyme properties

摘要:

[目的]克隆、表达克雷伯氏菌Klebsiella sp.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℃,K_m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。

Abstract:

[Objective] The 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 analyzed.The conservative sequence was amplified by degenerate primers.And a gene encoding α-L-rhamnosidase was cloned by PCR.Then, the recombinant plasmid pSE-rha1 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 investigated in detail.[Results] Using pNPR as a substrate, the enzymatic properties of the recombinase 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 below 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 hydrolyze α-1 and 6 band natural substrates hesperidin and rutin, which has certain medical application value.