| Sarcosine oxidase is a flavin oxidase and also has the function of N-methyl dehydration enzyme,which can complete the N-methyl dehydration reaction that is difficult to carry out by chemical methods.Sarcosine oxidas has a large role in many fields such as medicine and food application potential.The sarcosine oxidase gene derived from Thermomicrobium roseum(DSM 5159)was used as a research simulation,and it was expressed in Bacillus subtilis WB600.The non-conserved residues of the coenzyme-binding domain and the conserved sites near the coenzyme-binding domain were subjected to molecular evolution studies,further explore the influence of the non-conserved and conserved residues of the coenzyme-binding domain on the enzymatic properties,substrate suitability and chirality selectivity of Tr SOX.At the same time,strains with good catalytic performance were screened and the substrate spectrum of Tr SOX was further expanded.(1)Non-conservative site modification and enzymatic properties of Tr SOX coenzymebinding domain.According to the homologous sequence comparison,the coenzyme-binding domain of Tr SOX was modified by biological methods such as ancestral sequence reconstruction,and S46 H,S46T,I52 L,I52V,I52 M,V174M,V174 A,L209R,L209 D,L209V,L209 I,L209G,L209 P were obtained by site-directed mutagenesis design,in which V174 M could not get soluble protein.The enzymatic properties were measured respectively,and the results showed that the temperature,p H stability and secondary structure of the mutant did not change,and it had the same substrate chirality selectivity as the wild type.S46 T improved catalytic efficiency on substrates with large and hydrophobic side chains,I52 V and L209 V showed activity on L-proline,and L209 V showed a 67-fold boost on N-methyl-L-aspartate.V174 A showed catalytic activity towards(R)-2-methyl-pyrrolidine,broadening the spectrum of Tr SOX substrates.(2)Effects of the coenzyme-binding domain conserved site Arg54 on Tr SOX substrate fitness and enzymatic properties.Among the 19 mutants obtained by saturation mutation,a total of 15 mutants can be expressed and prepared,and the protein expression of almost all mutants is lower than that of wild-type Tr SOX,and only R54 D is increased by about 2.5times.All mutants maintained excellent thermal stability,except for R54 Q,R54N,R54 K,R54D and R54 V,the secondary structure composition of most other mutants was affected to some extent.The chiral selectivity of the mutant for substrates is the same as that of the wild-type Tr SOX,which can specifically recognize N-methyl-L-amino acid substrates and can remove methyl groups.The catalytic efficiency of R54 D and R54 K for N-methyl-L-amino acid-like substrates was significantly increased,while the catalytic efficiency of other mutants was decreased or even completely inactivated.In addition,some mutants including R54 P,R54K and R54 I were able to recognize novel non-amino acid related substrates such as 1,2,3,4-tetrahydroisoquinoline,1-methyl-1,2,3,4-tetrahydroisoquinoline,caprolactam,2-methylpiperidine,piperidine and(3S)-(+)-3-(methylamino)pyrrolidine,expanded the substrate adaptability of Tr SOX.(3)Covalent attachment of a coenzyme to the conserved residue Cys312 affects the substrate suitability and enzymatic properties of Tr SOX.The Cys312 site was subjected to site-directed saturation mutation,and 18 of the 19 mutants could be expressed and prepared.The mutants had the same chiral selectivity and excellent thermal stability as the wild-type Tr SOX.Analysis of the catalytic performance of the substrate,C312 W lost its catalytic activity to the substrate due to the loss of coenzyme;excellent mutants C312 A and C312 G were screened out,of which the catalytic activity of C312 A to N-methyl-L-leucine increased by 244.5 times,the catalytic performance of C312 G towards N-methyl-L-aspartate was enhanced by 24 times. |
