Effects Of Codon Optimization On The Enzymatic Properties And Conformation Of Tannase Expressed In Pichia Pastoris

Tannin acyl hydrolase?EC 3.1.1.20?,commonly called tannase,hydrolyzes ester and depside bonds of hydrolysable tannins releasing gallic acid,glucose and galloyl esters.It has been widely used in various industries such as food,leather,feed and pharmaceutical industries.At present,tannase is mainly produced by the deep liquid and semi-solid fermentation of fungi.However,there are so many deficiencies for tannase production,including low yield,complex purification process,high cost and low economic benefit.Given these disadvantages of tannase produced by the micro-organisms,cloning and over expression of tannase gene is the feasible process to solve the problem of industrial tannase production.The expression system of Pichia pastoris has been one of the most successful eukaryotic expression systems in recent years.But due to the existence of rare codons,it is difficult to the successful expression of gene sequence in Pichia pastoris.Meanwhile,the existence of rare codons will seriously inhibit the binding of m RNA with ribose,and prevent the translation of extraneous protein,which severely affects the expression level and structure of the heterologous protein.Codon optimization can improve the translation efficiency of heterologous protein and the expression level of protein.In this study,the gene sequence of tannase from Aspergillus oryzae NCU 002 was used as the research object,and its codon usage was optimized and converted to Pichia pastoris GS115.The effects of codon optimization on the expression level,enzymatic properties and spatial conformation of tannase in Pichia pastoris were further studied.The main results are as follows:?1?The genome DNA from Aspergillus oryzae NCU 002 was used as template,and was amplified by PCR.Through the analysis of the tannase gene sequence,the analysis results showed that CAI value?0.65?of the gene was lower.According to codon usage bias in Pichia pastoris,GC content and m RNA secondary structure,the codons of tannase gene were optimized by replacing all codons with codon usage bias without changing the original amino acid sequence of tannase.The optimized gene?Tanop?and wild-type gene?Tanwt?were constructed on the expression vector p PICZ?C,which formed recombinant plasmid p PICZ?C-Tanop and p PICZ?C-Tanwt.Furthermore,the two recombinant plasmids were converted to Pichia pastoris GS115.Two recombinant Pichia pastoris containing Tanop or Tanwt genes with a high tannase expression level were screened by tannase activity qualitative identification plate and PCR identification methods.They were named P.pastoris GS115/p PICZ?C-Tanop8 and P.pastoris GS115/p PICZ?C-Tanwt10,respectively.Compared with P.pastoris GS115/p PICZ?C-Tanwt10?maximum activity of 2.34 U/m L?,P.pastoris GS115/p PICZ?C-Tanop8 has a higher tannase expression level after methanol induction for 24 h with the maximum activity of 3.08U/m L.?2?The fermentation conditions of recombinant tannases were optimized,including the optimization of methanol concentration,p H value of potassium phosphate buffer,inoculation amount OD₆₀₀,induction temperature and fermentation time.The optimum fermentation conditions were as follows:methanol concentration was 0.5%,p H value of potassium phosphate buffer was 3.0,inoculation amount OD₆₀₀ was 1.0,induced temperature was 22?,fermentation time was 36 h.The maximum activity of tannase from P.pastoris GS115/p PICZ?C-Tanop8 and P.pastoris GS115/p PICZ?C-Tanwt10 reached 29.38 U/m L and 19.02 U/m L,which was 9.54 and 8.13 times of the strains before the optimization of fermentation conditions,respectively.After codon optimization,the maximum enzyme activity of P.pastoris GS115/p PICZ?C-Tanop8 was 1.54 times higher than that of P.pastoris GS115/p PICZ?C-Tanwt10.This indicated that codon optimization could effectively improve the tannase production of recombinant strain.?3?Two recombinant tannases?Tanop-tannase and Tanwt-tannase?were purified by ultrafiltration concentration,ammonium sulfate precipitation and DEAE-Sepharose Fast Flow chromatography.The SDS-PAGE showed that the molecular weight of the two recombinant tannases were 59.5 k Da.The specific activity of Tanop-tannase was 1362.67 U/mg with a purification fold of 10.65 with a recovery of 8.5%.The specific activity of Tanwt-tannase was 975.23 U/mg with a purification fold of 15.37 and recovery of 13.1%.The results suggested that codon optimization could improve the specific activity of recombinant tannase.?4?The enzymatic properties of Tanop-tannase and Tanwt-tannase were compared.The results were as follows:?1?Optimum p H and temperature for two recombinant tannases were 5.5 and 35?,respectively.The stability of p H and temperature were3.5?8.0 and 20??30?,respectively.?2?K⁺,Na⁺,Li⁺,Mn²⁺and Ca²⁺had little effect on the activity of two recombinant tannases.Co²⁺increased the activity of two tannases in high concentration.Mg²⁺,and Zn²⁺were found to be enzyme activators,which could effectively increase the activity of two recombinant tannases.The activity of two recombinant tannases was strongly inhibited by Cu²⁺.The low concentration of Hg²⁺,Fe³⁺and Al³⁺activated the activity of two recombinant tannases,but the high concentration?10 mmol/L?of Hg²⁺,Fe³⁺and Al³⁺inhibited the activity of two recombinant tannases.?3?The surfactants?Tween-80,Triton X-100,SDS?and inhibitors?EDTA,DMSO,DTT?had different inhibition on the activity of two recombinant tannases.The inhibition of SDS was the strongest.?4?Organic solvents?methanol,ethanol,glycerol,N-butyl alcohol,acetone,acetonitrile?had different inhibition on the activity of two recombinant tannases.With the increase of the organic solvents concentration,there are no significant change in the activity of two recombinant tannases.However,N-butanol,acetone and acetonitrile decreased the activity of two recombinant tannases,which the inhibition of acetonitrile was the strongest.Isoamyl alcohol inhibited Tanop-tannase,but activated Tanwt-tannase.?5?The K_m,V_max of Tanop-tannase and Tanwt-tannase were 8.84 mmol/L and 0.094?mol/min,11.12 mmol/L and 0.022?mol/min,respectively.?5?The spatial conformation of Tanop-tannase and Tanwt-tannase were compared.The results were as follows:?1?The results of circular dichroism spectroscopy showed that the secondary structure of Tanop-tannase was 14.4%?-helix,26.6%?-sheet,24.3%?-turn,34.8%random coil.Tanwt-tannase contained 18.6%?-helix,19.4%?-sheet,24.4%?-turn,37.6%random coil.The addition of Mg²⁺and Zn²⁺increased?-helix ratio of the two recombinant tannases and decreased the?-sheet ratio of the two recombinant tannases,which may cause changes of tannase conformation.?2?The results of fourier transform infrared spectroscopy showed that the characteristics absorption peaks of the tannase before or after the codon optimization did not change obviously,and there also were no obvious wavelength shift of the characteristic absorption peaks.But the addition of Mg²⁺and Zn²⁺,it caused a slight blue shift in the characteristic peaks near the amide-I band of tannases and the increase of absorption strength.?3?The fluorescence spectra showed that the maximum fluorescence intensity of Tanop-tannase was 1428.6 at334.4 nm,which higher than that of Tanwt-tannase?1187.5 at 335.2 nm?.It indicated that codon optimization caused the change of the microenvironment of tryptophan and tyrosine in the conformation of the two recombinant tannases.The addition of Mg²⁺and Zn²⁺had certain effects on the microenvironment of tryptophan and tyrosine of the two recombinant tannases.These results showed that the effect of codon optimization on the recombinant tannase were shown in the following aspects:?1?Codon optimization could improve the expression level and the specific activity of tannase.?2?Codon optimization had no significant influence on the optimum reaction p H and p H stability,the optimum reaction temperature and temperature stability,and the organic solvent tolerance stability of recombinant tannases.However,codon optimization reduced the inhibition of the surfactants and inhibitors on the activity of the recombinant tannases.Besides,Mg²⁺and Zn²⁺were potent activators for improving the activity of the recombinant tannases.?3?Codon optimization caused a little change in the spatial conformation of the recombinant tannases,such as the increase of secondary structure??-sheet?and fluorescence intensity,which was an important reason for improving the specific activity of Tanop-tannase.