西黑冠长臂猿粪便微生物宏基因组来源的丙氨酸消旋酶的异源表达及性质研究

Heterologous expression and characterization of alanine racemase derived from fecal microbial metagenomics of Nomascus concolor

摘要:

丙氨酸消旋酶是异构酶家族中一类重要的酶,既能作为新型抗菌药物筛选的靶标,又是酶法合成D-氨基酸的关键酶之一,有巨大的研究价值。【目的】为挖掘动物胃肠道中未培养微生物的丙氨酸消旋酶基因资源,探究不同微生物来源丙氨酸消旋酶基因及其酶的功能和性质。【方法】本研究从西黑冠长臂猿粪便微生物宏基因组出发,基于序列分析和基因功能注释,克隆丙氨酸消旋酶基因并在大肠杆菌BL21(DE3)中成功异源表达,进行酶功能鉴定和性质研究。【结果】从宏基因组中获得2个丙氨酸消旋酶基因NCalr 1和NCalr 6并进行了重组表达,2基因全长均为1 170 bp,编码389个氨基酸;理论分子量分别为43.58 kDa和43.94 kDa。重组酶的最适pH均为12.0,最适温度为40和37。在pH 9.0-12.0条件下处理1 h,重组酶相对酶活性仍在90%以上,表明重组酶具有较好的耐碱性;在30-45处理1 h,相对酶活仍在85%以上,说明重组酶在30-45条件下稳定性好。重组酶的最适PLP浓度为10 μmol/L,添加10 μmol/L PLP时,可使得重组酶的相对活性提高30%和7%左右;但当PLP的结合位点由赖氨酸突变为丙氨酸后,相对酶活仅为初始酶活的1%和27%。两重组酶的K_m分别为(14.81±1.66) mmol/L和(25.87±1.95) mmol/L。10 mmol/L的Hg²⁺、Ag⁺、Zn²⁺和SDS对重组酶具有强烈的抑制作用;Fe³⁺对重组酶有极强的激活作用,10 mmol/L的Fe³⁺可将酶活性提高2.6-5.1倍。【结论】本研究首次利用宏基因组学技术从西黑冠长臂猿粪便微生物宏基因组中获得2个新型丙氨酸消旋酶,具有较好的应用前景。

关键词:丙氨酸消旋酶;粪便微生物宏基因组;异源表达;酶学性质

Abstract:

Alanine racemase belonging to the isomerase family has great research value.It can be used as a target for the screening of new antibacterial drugs and is one of the key enzymes in the enzymatic synthesis of D-amino acids.[Objective] To mine the alanine racemase gene resources from uncultured microorganisms in the gastrointestinal tract of animals,and clarify the functions and properties of alanine racemase genes from different microorganisms.[Methods] On the basis of sequence analysis and gene function annotation,alanine racemase genes were cloned from the metagenome of the fecal microorganisms of Nomascus concolor and heterologously expressed in Escherichia coli BL21(DE3) for the identification of enzyme functions and properties.[Results] Two alanine racemase genes,NCalr 1 and NCalr 6,were obtained,each of which had a length of 1 170 bp and encoded 389 amino acids.The deduced proteins of NCalr 1 and NCalr 6 had the molecular weights of 43.58 kDa and 43.94 kDa,respectively.The recombinant enzymes had the optimal performance at pH 12.0 and 40/37.The relative activities of the recombinant enzymes treated at pH 9.0-12.0 for 1 h were above 90%,indicating good alkali resistance.The recombinant enzymes had good stability at 30-45 relative activity above 85% fter being treated at 30-45 for 1 h.The optimal PLP concentration was 10 μmol/L for the two enzymes,and the incubation with 10 μmol/L PLP increased the relative activity of the enzymes by about 30% and 7%,respectively.However,the mutation from lysine to alanine at the PLP binding site reduced the relative activity of the two enzymes to only 1% and 27% of the initial activity,respectively.The K_m values of the recombinant enzymes were (14.81±1.66) mmol/L and (25.87±1.95) mmol/L,respectively.The recombinant enzymes were inhibited by 10 mmol/L Hg²⁺,Ag⁺,Zn²⁺,and SDS while activated by Fe³⁺,and 10 mmol/L Fe³⁺ increased their activities by 2.6-5.1 times.[Conclusion] The study obtained two new alanine racemases with good application prospects from the fecal microbial metagenomics of N. concolor for the first time.

Key words:alanine racemase;fecal microbial metagenomics;heterologous expression;enzymatic properties