| Conjugated Linoleic Acid(CLA) is a kind of different position and geometric isomers with two conjugated double bonds of octadecadienoic acid. CLA widely exists in a variety of foods, with many biological activities, but different types of CLA present different physiological activity. The aim of this article is to express two kinds of Linoleic Acid Isomerase in E. coli expression system and compare with each other. The mutant library of lai from P. acnes was constructed by directed evolution of protein and the objective strains were screened by flow cytometry and label plasmid with green fluorescent protein. The fermentation condition of mutant strain was optimized to improve the t10, c12-CLA production, and LAI was purified to study the properties of enzyme. The mutation lai was expressed in Streptococcus thermophilus and used in ferment milk.The two kinds of lai were cloned, one was named lai-l, with ORF of 1776 bp, the other was named lai-p with ORF of 1275 bp. Blasting lai-l and lai-p showed that they were 100% same with linoleic acid isomerase gene in L. reuteri and P. acnes, and lai-l was 93% similarity with Lactobacillus helveticus and Lactobacillus gasseri. But lai-l and lai-p showed low sequence homology without mutation found in lai-l and lai-p. The lai-l encoded a protein named LAI-L with molecular weight of 67.9k D(591 amino acids), p I of which is 7.14. lai-l encoded a protein named LAI-P with molecular weight of 49.0k D(424 amino acids), p I of which is 4.81. LAI-L was a stable transmembrane and hydrophilic protein and has 11 Ser, 8 Thr, 8 Tyr, which may become protein tyrosine phosphorylation sites. It was predicted to consist of random coil(47.88%), alpha helix(34.84%) and β-sheet(17.26%) in LAI-L protein secondary structure. LAI-P is an unstable non-transmembrane and hydrophilic protein and has 3 Ser, 4 Thr, and 7 Tyr, which may become protein tyrosine phosphorylation sites. It was predicted to consist of random coil(45.28%), alpha helix(36.32%) and β-sheet(18.4%) in LAI-P protein secondary structure.Two kinds of lai were expressed in E. coli, which encoded two protein bands with molecular weight of 64 k D(LAI-L) and 55 k D(LAI-P) on SDS-PAGE. An expression vector p Cold-yclai-p-gfp was constructed, and lai-p was mutated by error-prone PCR. Mutantions were screened out by flow cytometry, while with 82.3% accuracy. There was only one amino acid changed in seven mutons, and two amino acids changed in four mutons and four amino acids and seven bases changed in M955. It was optimized to induct for 18 h by 0.2 mmol/L of IPTG. According to the result of GC 68.3±2.1μg/m L of t10, c12-CLA was produced by M955 identified by GC.The conditions of mutant linoleic acid isomerase production were optimized as follows: fermentation time of 18 h; 0.2 mmol/L of IPTG; 20% of liquid volume in flask; initial p H7 of medium; OD600 of biomass before induction was 0.4; concentration of LA was 0.5 mg/m L; concentration of ions was 0.02%. Conditions of mutant enzyme production were optimized by response surface method as follows: fermentation time of 19.5 h, OD600 of biomass of 0.47 before induction and 0.57mg/m L of LA. 143.33 μg/m L CLA was produced by mutant linoleic acid isomerase. In order to purify mutant linoleic acid isomerase, crude enzyme sample 10 m L was load on to affinity chromatography at flow rate of 0.25 m L/min. SDS-PAGE show a single band of mutant protein.Enzymatic properties of mutant linoleic acid isomerase were as follows: michaelis equation was y=0.0224x+0.0786, R2=0.9972, Km=0.285 mg/m L, Vmax=12.72 U/mg; optimal activities of the purified enzymes occurred at p H 7.0 and 37℃; enzyme was stable at 0~40 and p H 5.0~9.0; the concentration of LA was 0.5 ℃mg/m L; the enzyme activity was not significantly affected by the metal ions.Mutation and original linoleic acid isomerase gene were expressed in S. thermophilus. The recipient cell(treated by 1% of glycine) at OD600=0.8 was washed by electrotransformation bufferⅡ, and added by 1.0 μg/μL of plasmid as well as the 1.8 k V. Fermentation time of ferment milk from recombinant strain was 10% slower than that from normal bacteria. Texture, apparent viscosity and particle size were not significantly different from normal strain. Production of LA in ferment milk from transformed strains(lai and yclai) were 7.2 μg/m L and 30.2 μg/m L t10, c12-CLA, respectively. Mutant linoleic acid isomerase which was heterologous expressed had the higher ability to produce CLA and should be paid more attention and studied further. |
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