Overexpression,Characterization Of Arginase And Its Application In L-ornithine Production

L-ornithine is an important intermediary in the urea cycle and it is a non-essential amino acid in human body.L-ornithine,with a variety of physiological functions such as liver protection,bodybuilding,fatigue relieving,wound healing and bitterness inhibition,has been widely used in food,chemical and medicine industries.Arginase can hydrolyze L-arginine to L-ornithine and urea.Compared with other L-ornithine production methods,the use of arginase hydrolysis has several advantages including rapid production,few by-products and high specificity for substrate.Currently,the arginases applied in industrial L-ornithine production were almostly extracted from animal liver.Meanwhile,arginase is abundant in microorganisms and the use of microbial arginase has great application potential for its low cost.However,it still needs to further increase the conversion rate of L-arginine to reduce the substrate cost.In addition,the safety of engineered microorganisms and antibiotics resistance present growing concern of the public.Thus,the selection of safe hosts and construction of engineering strains without antibiotic resistance genes are the development direction of future researches.In the present study,a strain of Rummeliibacillus pycnus SK31.001 was screened from the strains that preserved in our laboratory.This strain was identified to have arginase producing capacity and the purified arginase was obtained.The arginase gene from R.pycnus was cloned,sequenced and overexpressed in Escherichia coli,and the recombinant arginase was characterized.A coupled system involving R.pycnus arginase and Jack bean urease was constructed for L-ornithine production.Furthermore,a plasmid-less and marker-less strain with multi-copy integration of the arginase gene was constructed using Bacillus subtilis 168 as a host.This food-grade expression could be a good candidate in L-ornithine production.The main results of this study are as follows:?1?During the strain screening,L-arginine was added into the culture medium to induce arginase.L-arginine conversion ability of these strains and the identification of L-ornithine were measured by HPLC.Finally,an arginase producing strain R.pycnus SK31.001 was obtained.R.pycnus arginase was purified with n-propyl alcohol precipitation method,DEAE-Sepharose FF column and Superdex 200 column.The specific activity of purified arginase was determined to be 256.2 U/mg.Bioinformatics analysis revealed that R.pycnus arginase was a hexamer with a subunit and native molecular mass of 34 k Da and 196 k Da,respectively.Primers for degenerate PCR were designed based on the conserved motifs found among amino acid sequences,and717 bp of R.pycnus partial arginase gene was sequenced.Inverse PCR primers were designed based on the sequenced fragment to amplify upstream and downstream of the known partial sequence.Combined with the results from the degenerate and inverse PCR,a 906 bp complete open reading frame?ORF?was obtained and then deposited in NCBI?Gen Bank ID:KP702726?.Protein sequence alignment showed that R.pycnus arginase had a conserved active site with 6amino acid residues binding to 2 central ions:D123,H125,D228,D230,H100 and D127.?2?Full-length R.pycnus arginase gene was amplified from genomic DNA and then inserded into p ET-28a?+?.Constructed recombinant plasmid p ET-28a?+?-arg was transformed into E.coli BL21?DE3?.The recombinant strain E.coli BL21?DE3?/p ET-28a?+?-arg was constructed to express arginase.Recombinant arginase was purified with Ni²⁺Sepharose 6 FF column and exhibited protein band of 34 k Da on SDS-PAGE,which was consistent with the wilde type.HPLC analysis of the enzyme reaction product revealed that the product was L-ornithine,confirming that the cloned gene encodes R.pycnus arginase.Mn²⁺could enhance R.pycnus arginase activity and the optimal concentration was 2 mmol/L.R.pycnus arginase showed optimal activity at p H 9.5 and most activity was at a p H range from 8.0 to 10.0.R.pycnus arginase showed p H stability from 8.0 to 9.0.The optimal temperature was 80°C and this enzyme was stable below 50°C.The specific activity was 914 U/mg and the K_m,V_max,k_cat,and k_cat/K_m values were determined to be 0.212 mmol/L,1111 U/mg,629.6 s^-1 and 2910mmol/L/s,respectively.The biosynthesis yield of L-ornithine with the purified arginase was144.4 g/L,and the molar yield was 95.2%.Inaddition,R.pycnus arginase was found to be actived by Ni²⁺.In order to further increase the conversion rate of L-arginine,a coupled system involving R.pycnus arginase and Jack bean urease was constructed and applied in L-ornithine production.Both enzymes showed robust activity at p H 6.5 and 40-50°C with the addition of Ni²⁺.Using this coupled system for L-ornithine bioconversion,a 99.7%molar yield was reached within 3 h without urea residue.?3?With the overlap extension PCR,the promoter P43 was fused with R.pycnus arginase gene.P43-arg expression cassette and lox71-spc-lox66 cassette were integrated into the chromosome of BSKA,which was derived from B.subtilis 168 with arg I knocked off,with site-directed and random integrations.The P43-arg expression cassettes were integrated into the amy E,thr C and some unknown loca with 2,2,2,3 copy numbers obtained.With the protoplast transformation technique and Cre/lox system,a total 9 copy insertions of P43-arg cassette strain BSKA-C3 was constructed without antibiotic resistance gene.The total enzyme activity was assayed to be 14.5 U/m L.Whole-cell conversion performed with BSKA-C3 led to a molar yield of 96.1%and 145.8 g/L L-ornithine was obtained within 8 h.BSKA-C3 was tested for 10 generations and there was no decrease of activity,which showed great segregational stability.