Studies On The Effect Of Methanol Stress On The Production Of Laccase From Bacillus Recombinant And Enzymatic Modification Of Chitosan

Laccase is a multi-purpose green catalyst,which can catalyze the oxidation of a large number of aromatic compounds,producing only one by-product-water.Laccase is widely used as cross-linking agent,clarifier,restorative agent,decolorant and degradant in food,paper,textile,pharmaceutical and petrochemical industries.Laccase can be divided into fungal laccase,bacterial laccase,plant laccase and insect laccase.Compared with fungal laccase,bacterial laccase does not need glycosylation and has higher pH and temperature stability,which is more suitable for use in harsh industrial environment.However,natural laccase production is low.In order to increase yield,heterogenous expression technology is widely used.E.coli is regarded as the preferred expression host due to its clear background,mature research on gene expression regulation mechanism,rapid growth and reproduction,simple culture,low cost and high expression.However,proteins tend to be expressed intracellularly in Escherichia coli to form inclusion bodies,thus affecting protein folding and bacterial growth.It is of great significance to increase the production of recombinant bacterial laccase and extracellularly express recombinant bacterial laccase.In this paper,the laccase gene of Bacillus cerealis(fmb 103)was optimized,cloned and expressed heterologously in E.coli.Strategy for increasing yield and extracellular secretion of recombinant bacterial laccase(fmb-rL103)under methanol stress is used.The reasons for the increase of exocrine secretion and yield of recombinant laccase were studied through growth rate,methanol consumption level,acetic acid metabolism level,cell membrane integrity and expression level of fmb 103,msbB and waaF genes.Then,gallic acid was catalyzed to graft with chitosan by recombinant laccase in order to improve the antioxidant and bacteriostatic properties of chitosan.Gallic acid-chitosan derivatives(GA-g-CS)with higher antioxidant and bacteriostatic properties were used for chilled meat preservation.The results are summarized as follows:1.Effect of methanol stress on the yield of recombinant spore laccaseThe laccase gene of Bacillus cerealis(fmb 103)was optimized,cloned and expressed heterologously in E.coli(BL21(DE3)).The yield of recombinant bacterial laccase(fmb-rL103)was increased by adding methanol during fermentation.The amount of methanol,the timing of methanol addition,the induction point,the fermentation time,the growth state of bacterium,the level of methanol consumption,acetic acid metabolism and laccase gene expression were also studied.After adding 6%methanol at 0 h and inducing by IPTG at OD600=1.0,the yield of fmb-rL103 reached 35930.56±493.59U/L at the shaking flask level,10.5 fold-higher than before.At the fermenter level,the yield of fmb-rL103 reached 125500±2962.73U/L.During the whole process,methanol was gradually consumed,from the initial concentration of 5.59±0.03%to the end concentration of 1.19±0.03%.The acetic acid content gradually increased.Under methanol stress,the increase rate of acetic acid decreased,the growth of bacteria slowed down,and the expression level of fmb 103 increased 34.44 fold.2.Effect of methanol stress on the extracellular yield of recombinant spore laccaseBased on the results of Chapter 2,the effects of methanol on the extracellular production of fmb-rL103 were studied,including the amount and timing of methanol added,the induction point and induction time.In addition,the integrity of cell membrane was measured by transmission electron microscopy and PI dye kit.The expression levels of waaF and msbB genes related to extracellular expression were determined by q-PCR.After adding 6%methanol at 0 h and induction by IPTG at OD600=1.0 for 20 hours(i.e.fermentation time 52 hours),the yield of fmb-rL103 reached 10733.33±40 U/L(45?)at shaking flask leve.At the fermenter level,the yield of fmb-rL 103 reached 366666.67±1909(45?).At the end of fermentation,the results of TEM and flow cytometry showed that the cell membrane remained intact.The expression levels of waaF gene and msbB gene in the methanol stress treatment group were lower than those in the control group.3.Grafting gallic acid onto chitosan catalyzed by recombinant spore laccase for preservation of chilled meatGallic acid-g-chitosan(GA-g-CS)was prepared by laccase-catalyzed.The structural differences of chitosan(CS),Gallic acid-chitosan(GA-CS)and gallic acid-g-CS were compared by full wavelength scanning(UV-FWLS)and near infrared scanning(FT-IR).In addition,the functions of bacteriostasis and antioxidant were compared.The solution of CS,GA-CS and GA-g-CS were used to coat chilled meat at low temperature(4 ?,RH50%).During storage,the changes of color(brightness L*and redness a*),pH value,oxidation degree(T-BARS),freshness(TVB-N)and microorganisms(total colony count and coliform flora)of chilled meat were studied every two days.In Fourier transform infrared spectroscopy(FT-IR),the peak value of GA-g-CS at 1730 and 1640 cm-1(representing ester bond and amide bond respectively)changed.It was concluded that GA-g-CS formed ester bond and amide bond.The absorption peaks of GA-CS and GA-g-CS at 250 and 350 nm were observed in the full-wavelength scanning images.Compared with GA-CS,the absorption peak of GA-g-CS had a red shifted at 262 nm to 272 nm.All above proved that gallic acid could be covalently grafted onto chitosan catalyzed by fmb-rL103.When DPPH concentration was 2 mg/mL,the DPPH clearance rates of CS,GA-CS and GA-g-CS were 53.90±0.16%,85.85±0.28%and 80.60±0.16%,respectively.The EC50 values(half inhibition rate)were 1.84,0.08 and 0.43 mg/mL,respectively,which means that the oxidation resistance of the grafted chitosan is improved.Compared with CS,at the concentration of 1 mg/mL,GA-g-CS increased the diameters of bacteriostasis circles of Pseudomonas(isolated from pork by the Enzyme engineering laboratory of Nanjing Agricultural University),Acinetobacter(isolated from pork by the enzyme Engineering laboratory of Nanjing Agricultural University),Brochothrix thermosphacta(isolated from pork by the Enzyme engineering laboratory of Nanjing Agricultural University),Escherichia coli(ATCC 25922),Staphylococcus aureus(CMCC 26003),Salmonella acillus subtilis(CICC 21483)and Listeria monocytogenes(CICC 21662)by 34.02±0.05,33.84±0.09,35.71±0.17,36.09±0.12 26.70±0.20,27.92±0.01 and 21.20±0.16 mm,respectively.The results of chilled meat preservation showed that the chilled meat in control group,treated with CS,GA-CS and GA-g-CS reached the upper limit of pH value,T-BARS value,TVB-N value and microbial value on the 6th,10th,12th and 18th day,respectively.GA-g-CS can effectively improve the shelf life of 12 days with a promising fresh-keeping effect.