Study On Fermentation Conditions Of Recombinant Bacterial Laccase And Its Application In Chitosan Modification

Laccase(E.C 1.10.3.2)can catalyze the oxidation of various phenolic,aromatic compounds,and some lignin and xenobiotics which are difficult to decompose.Laccase are widely applied in food,papermaking,decolorization,biological,pharmaceutical,chemical and other fields.At present,a variety of laccase genes have been cloned and expressed in different microorganisms,but most of their production is low.In this paper,the laccase gene from Bacillus vallismortis fmb-103 was cloned and heterologously expressed in Escherichia coli BL21(DE3)cells.The auto-induction strategy was applied during fermentation,and the process was controlled.The synergistic effect of heating and ammonium sulfate precipitation were studied to purify the recombinant laccase and determin the enzyme properties.To improve the antioxidant and antibacterial properties of chitosan,the laccase was used to catalyze the grafting of ferulic acid and chitosan.The chitosan and chitosan derivatives were used as main coating materials to experiment on mango preservation.The main results are summarized as follows:1.Heterologous expression of laccase and purification of recombinant laccaseFirstly,the laccase gene from Bacillus vallismortis fmb-103 was cloned and heterologously expressed in Escherichia coli BL21(DE3)cells.The auto-induction strategy was applied during fermentation,and the induction time,temperature control and fermentation time were studied,as follows:Cu2+ was added when the optical density at 600 nm(OD600)was 0.3,the fermentation temperature was adjusted to 16℃ when the OD600 was 0.9,and fermentation was stopped after 50 h.The yield of recombinant laccase was up to 3420 U/L,as assayed by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).Recombinant laccase was purified 4.47-fold by heating for 10 min at 70℃ and dialyzing against 50～60%ammonium sulfate,retained more than 50%activity after 10 h at 70℃,and demonstrated broad pH stability.2.Grafting of ferulic acid onto chitosan catalyzed by finb-rL103The effects of pH,temperature,time and concentration of ferulic acid of the grafting reaction were studied.The best grafting effect was achieved when the reaction system pH was 6.5,the time was 1 h,the temperature was 60℃ and the concentration of ferulic acid was 10 mM.Under the condition,the ferulic acid substitution degree of medium and high molecular weight chitosan was 0.194 and 0.225,respectively.UV/visible spectra and FT-IR spectra were used to characterize the chitosan grafted with laccase-catalyzed ferulic acid,proved that laccase-catalyzed oxidized ferulic acid can be covalently grafted onto chitosan.The antioxidant and antibacterial properties of grafted chitosan were analyzed.Compared with the original chitosan,the antioxidant activity of FA-chitosan was significantly improved.For the medium molecular weight chitosan,the DPPH radical scavenging rate reached 81.79%at a concentration of 2 mg/mL,the EC50(half inhibition rate)was 0.65 mg/mL.As for the high molecular weight chitosan,the DPPH radical scavenging rate reached 80.08%at a concentration of 2 mg/mL,the EC50 was 0.71 mg/mL.The antibacterial activity of grafted chitosan was aslo improved,and the antibacterial activity was higher at low concentration.At the concentration of 1 mg/mL,the bacteriostatic ring for Escherichia coli,Staphylococcus aureus,Bacillus subtilis and yeast of the medium molecular weight FA-chitosan increased by 2.09 mm、1.87 mm、1.38 mm and 1.44 mm,respectively.The bacteriostatic ring of the high molecular weight FA-chitosan increased by 2.19 mm、1.50 mm、1.68 mm and 1.48 mm,respectively.3.Application of chitosan and FA-chitosan in mango preservationThe chitosan and FA-chitosan were used for the preservation of "Mangifera indica Linn" after harvest at room temperature(25℃,RH 50%).The change of hardness,weight loss,yellowing index,desease incidence,respiration rate,titratable acidity,soluble solids,Vc,H2O2 and CAT、POD activity were studied.The physiological and biochemical indexes of mango were significantly improved by coating with chitosan and FA-chitosan.Mango with chitosan coating treatment reached respiratory peak after 12 days,which delayed more than 4 days compared with the control group.On the sixteenth days of storage,the weight loss rate of mango was as low as 12.85%,1.63%less than the control group;the fruit hardness was 0.62 N,0.10 N higher than the control group;the yellowing index and desease incidence were significantly lower;the highest content of Vc was 11.1 mg/100g,4.6 mg/100g higher than the control group;the maximum titratable acidity content was 0.45%,0.12%higher than the control group;the soluble solids content was up to 21.13%,1.31%higher than the control group.Moreover,coating with chitosan and FA-chitosan can decrease the content of H2O2 by keeping higher CAT and POD activity.Therefore,it can delay the ripening of mango during storage.Coating with FA-chitosan has better effect than coating with chitosan,with lower respiration intensity,higher Vc content,titratable acidity,soluble solid content,higher CAT and POD activity,lower H2O2 content in mango,and stronger inhibition of mango decay.On the sixteenth day,the desease incidence of mango coating with medium and high molecular weight FA-chitosan was 0 and 5%,respectively,which was lower than that of coating with chitosan by 10%and 5%,respectively.