Plasma Mutagenesis Of Erwinia Rhubarb And Optimization Of Isomaltulose Production

Isomaltulose is a non-carcinogenic reducing disaccharide produced by intramolecular transglycosylation of sucrose isomerase（SIase,EC 5.4.99.11）.Isomaltulose has the functions of slow hydrolysis,anti-caries,low glycemic index（GI）,and obesity prevention.Atmospheric and room temperature plasma（ARTP）mutagenesis has been widely used in microbial breeding,in order to improve the catalytic performance of microorganisms due to its high efficiency and safety.Immobilized cell technology could further improve the stability and reusability of biocatalysts.In this study,the Erwinia rhapontici YS-1 that isomaltulose-producing was used as the study strain,ARTP mutagenesis of the strain was carried out,and mutant strains with high SIase enzyme activity were selected.The optimal growth and fermentation conditions of the mutant strains were studied to improve the SIase activity,and the conditions for the production of isomaltulose by free cells and immobilized cells of the mutant strains were optimized.This work laid the foundation for improving the production and industrial application of isomaltulose.The main contents were listed as follows:（1）The Erwinia rhapontici YS-1 was mutagenized by ARTP mutagenesis technology,and the mutagenesis conditions were determined at 300 W,28°C,irradiation distance of 2mm,working gas flow of 12.0 L/min,and irradiation treatment time of 50 s.The mutant target strain DXW-60 was finally obtained from 528 mutant strains after initial screening and shake flask re-screening.The SIase activity was 15.81 times that of the original strain,and the enzyme activity was stable after 8 generations of propagation.Compared with the SIase gene sequence of the original strain,the gene has not been mutated.We speculated that the regulatory sequence of SIase may be changed,which increased the expression level of the enzyme.（2）The growth and enzyme production conditions of the mutant strain were optimized.Based on the single factor experiment results of medium components and culture conditions,a response surface methodology was designed to further determine the optimal enzyme production conditions:sucrose concentration 77.6 g/L,peptone concentration 5.0 g/L,Na₂HPO₄ concentration 3.0 g/L,beef extract 3.0 g/L,p H 6.0,liquid volume 100 m L/250 m L,fermentation time 12.5 h.Under these condition,the strain grew vigorously,and the SIase enzyme activity reached the highest of 45.88 U/m L,which was 1.17 times higher than that of the mutant strain before optimization（21.18 U/m L）.（3）The conditions of converting sucrose into isomaltulose by DXW-60 free cells were studied,including cell dosage,substrate concentration,p H and temperature.The study showed that 85.86%isomaltulose yield can be obtained under the conditions of 0.30 g wet cells,700 g/L substrate concentration,natural p H,and temperature of 40°C.Compared with the original strain,the improvement of the enzyme activity made the transformation efficiency of DXW-60 higher and the transformation time shorter.However,the reusability of DXW-60 free cells was poor,and the yield of isomaltulose was significantly reduced after three batches（P<0.05）.Therefore,immobilized cells were considered to improve the reusability of DXW-60 free cells.（4）By comparing the mechanical strength,mass transfer performance,and the complexity of the preparation process of different immobilized cells.The sodium alginate-sodium carboxymethyl cellulose composite carrier was finally selected to immobilize DXW-60,and the immobilization conditions were studied.The optimal conditions for immobilizing cells were as follows:the addition of sodium alginate was 3%（W/V）,the addition of sodium carboxymethyl cellulose was 0.4%（W/V）,the concentration of crosslinkers Ca Cl₂ was 0.1 mol/L,the amount of cell embedding was 10%（W/V）,and the immobilization time was 30 min.Immobilized cells under this condition had the shortest transformation time（6 h）and the highest transformation rate.Compared with the free cells of DXW-60,the isomaltulose yield decreased to 69.53%after repeated use of three batches.The repeated utilization of DXW-60 immobilized cells was improved.The transformation ability after seven consecutive batches was stable,and the yield of isomaltulose could still reach 71.60%.