Cloning, Expression And Characterization Of L-arabinose Isomerase From Bacillus Cereus CZ: Bioconversion Of L-arabinose To L-ribulose Using The Enzyme

L-arabinose isomerase(L-AI;EC5.3.1.4) is an intracelluLar enzyme that catalyzes thereversible isomerization of L-arabinose to L-ribulose,a component of the pentose phosphateor the phosphoketolase pathways.The ability to chemoselectively covert L-arabinose to thecorresponding ketose L-ribulose would,therefore,be a powerful tool for the preparation ofpharmaceuticals.Since pure L-arabinose is expensive,an L-AI specific only for L-arabinoseamong sugers found in organic wasters could be extremely useful.In the present paper,wereport the cloning,expression,and characterization of an L-AI （BCAI）from B.cereus CZ andoptimize its enzymatic properties.The cloning experiments include four parts，they are：the amplification of L-arabinoseisomerase gene，the connection of L-arabinose isomerase gene and pMD18-T simple,theamplification of L-arabinose isomerase gene intoE. coli JM110, the sequence analysis andtesting of the target L-arabinose isomerase gene.The results showed thatL-arabinoseisomerase gene had successfullyconnected with T carrier into E. coli JM110.Sequencingresults show that the L-AI gene is1,824bp, encoding608amino acids, the same size as thereported L-AI molecular, which proves that the cloning DNA fragments is L-arabinoseisomerase gene.The expression of L-AI gene experiments include three parts，they are：the transformationand testing of plasmid pBE3-ara intoBacillus subtilisWB800, the determination of enzymeactivity,at the same time to test the connection transformationand the enzyme activity.Theresults showed that plasmid pBE3-ara had transformatedinto Bacillus subtilis WB800strainsand expressed the L-arabinose isomerase successfully.On the basis of the above experiments, single factor experiment include: carbon source,temperature, pH, time, metal ions, different surfactants, enzyme concentration (the reactionrate between enzyme and substrate).The experimental results show that the best conditions forsingle factor is carbon source L-arabinose, temperature85℃, pH7.0, time24h,surfactantCTAB, enzyme concentration(enzyme and substrate reaction ratio)0.2E+0.8S.Based on theexperimental data, the three biggest significance on the results are:temperature, pH,time.According to this conclusion,we use the Response surface analysis(Response surfacemethodology, referred to RSM) to optimize the properties of enzymology and get the bestcombination is: temperature84℃, pH7.0, time of25h. The predicted result of the conversion rate was10.53%.The validated result was11.65%as a result, the models developped wereconsidered to be accurate and reliable for improving the conversion of producting L-ribulose.