| Dextran has several important uses in the pharmaceutical, chemical and food industry. It is produced commercially by direct fermentation of Leuconostoc mesenteroides at present. Because of the introduction of chloridion in the process of hydrolysis, the quality of dextran has been reduced and it can pollute the environment. In our preceding research, dextransucrase genes had been cloned and expressed successfully in Escherichia coli. Based on this result, in this study, recombinant dextransucrase has been immobilized by enzyme engineering methods to synthesizing dextran and an initial study on controlling its molecular weight has been done.Five different preparations of immobilized recombinant dextransucrase were prepared: the native enzyme physically adsorbed onto Eudragit L-100 and hydroxyapatite bioceramics, the enzyme entrapped in alginate beads, the enzyme crosslinked with glutaraldehyde onto chitosan beads, and the enzyme affinity adsorbed on a Ni-NTA column. These preparations were used for dextran synthesis in 5% sucrose solutions at pH 5.4 and 30℃. The best results were achieved for dextransucrase entrapped in alginate beads. The maximum activity recovery was 45% and it can be used for several batches. Above all, its low cost was fit for industrial production.On the basis of the initial method, recombinant dextransucrase was immobilized by a novel entrapment method which uses the mixture of sodium alginate and agar. The immobilization conditions and properties of immobilized enzyme were investigated. The optimal immobilization conditions for dextransucrase were: 2ml of dextransucrase in the buffer of HAc-NaAc with 0.05g/L calcium chloride (0.02mol/L, pH5.4) embedded by the composition of 3ml 4% sodium alginate and 3ml 1% agar, then reacted with 1.5% calcium chloride at 0℃for 15min. The shape of immobilized enzyme was membrane. The immobilized recombinant dextransucrase had an optimum temperature of 40℃and an optimum pH of 5.4 with the sucrose concentration at 5%. Its Km value was 28.35mmol/L. Compared with native enzyme, the thermal and operational stabilities of the immobilized enzyme were enhanced.After this with hydroxyapatite as adsorbent, sodium alginate and agar as a combination carrier, recombinant dextransucrase was immobilized using the absorb-embedded method. The immobilization conditions and the partial properties of the immobilized enzymes were investigated. The optimal immobilization conditions for dextransucrase were: 2ml of dextransucrase reacted with 0.1g hydroxyapatite at 10℃for 10h, then embedded ut supra. The optimal temperature for immobilized enzyme was 30℃. The immobilized enzyme exhibited the maximal activity at pH4.2 and with the sucrose concentration at 5%. Its Km value was 30.56mmol/L. Compared with the enzyme immobilized by sodium alginate and agar only, the enzyme activity increased 15 times and the stability of pH was enhanced.Synthetizing dextran with the immobilized enzyme and controlling its molecular weight were studied eventually. According to the results, dextran's molecular weight can be controlled efficaciously by the introduction of dextranase. Through regulating the ratio of the two enzymes, the reaction time with sucrose, sucrose concentrations, temperature and pH dextran of a required size can be synthetized by one step. Since it can prevent foreign protein from introducing, the pharmacal security of the product was enhanced. Then dextransucrase and dextranase were immobilized together through the absorb-embedded method, it indicated that when the enzyme activity ratio of dextranase and dextransucrase was 1:60 and the reaction time was 4h, the molecular size of dextran reached to 20000. These researches may provide an important basis for dextran enzymic synthesis and industrial applications of the immobilized enzyme.
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