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

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