Cloning And Expression And Enzymatic Properties Of Alginate Lyase And Optimization Of Fermentation Conditions

This study demonstrates that Pseudomonas putida can produce alginate lyase.Firstly,alginate lyase(Aly)gene of Pseudomonas putida KT2440 was screened and synthesized.The gene size is 1116 bp,encoding 372 Amino acid.Nde ? and EcoR ?were screened for restriction enzyme digestion,and the target gene was ligated into the same double-cut pET-28a(+)cloning vector to construct pET-28a(H+)-Aly recombinant plasmid was introduced into BL21 to construct a heterologous expression system.After induction with 1 mM IPTG,a higher yield of the target protein can be obtained.In terms of enzymatic properties,the optimum pH of the alginate lyase reaction was 7.5,the optimum temperature was 45?,and the optimum NaCl concentration was 0.5 M.The substrate specificity test proved that the alginate Lyase is a bifunctional enzyme that prefers poly M.Using the principle of Box-Behnken experimental design in Design Expert software,the three-factor and three-level response surface analysis experiments were carried out.The experimental results show that when the reaction temperature is 47?,the pH is 8.0,and the NaCI concentration is 0.6 M.When carried out,the maximum enzyme activity was 231 U/mL,and the specific enzyme activity was 189.3 U/mg,which was at a higher level in the specific activity of the currently reported alginate lyase.Based on the measured enzymatic conditions,the fermentation conditions of the recombinant bacteria were studied.The factors that significantly affected the total enzyme activity of the fermentation were found to be glucose concentration,inoculum size,initial pH and IPTG concentration.According to the principle of Box-Behnken experimental design,four-factor and three-level response surface analysis experiments were carried out.According to the optimization results given by RSM,the experiment was modified.The results showed that other factors were under optimal conditions when the fermentation of glucose was added.When the amount is 10 g/L,the inoculum is 1.3%,the initial pH is 8.2,and the IPTG-induced concentration is 1 mM,the maximum fermentation total enzyme activity is 225 U/mL.The enzymatic hydrolysate was mainly confirmed by TLC as disaccharide,which can be judged as the endonuclease;the antibacterial activity of the enzymatic hydrolysate showed that the alginate lyase degradation product had certain inhibition on Escherichia coli,lactic acid bacteria and Staphylococcus aureus.The antibacterial effect needs to be improved compared with the brown algae oligosaccharide degraded by hydrogen peroxide method;the anti-oxidation experiment shows that the alginate lyase enzymatic hydrolysate has the ability to scavenge free radicals;In DPPH·system,the IC50 of alginoglycan produced by enzymatic hydrolysis of this enzyme was 0.46 mg/ml.