| Multifunctional protein which was constructed by gene fusion and expression can realize coenzyme regeneration. Not only can it overcome the mass transfer resistance of subatrates or products caused by whole-cell catalyst with using multi-gene co-expression, but also it reduces the purification cost of enzyme-coupled system as the catalyst. Immobilization technology makes fusion enzyme reusable and improves its stability. This research based on the model reaction of the asymmetric reduction of 4-chloro-3-oxobutanoate ethyl ester(COBE), which specifically includes:Two strains with cr2(Candida macedoniensis) and gdh(Bacillus sp.YX-1) genes fused by overlap extension PCR were constructed. The difference between them was that the position of cr2 and gdh genes was exchanged each other. The biological catalytic characteristics of fusion proteins CR2-GDH and GDH-CR2 were compared. The enzymatic properties of fusion protein CR2-GDH as the research object were studied. Diversities of the fusion protein were found: the optimal p H and temperature of asymmetric reduction were p H 4.5, 50℃, while that of glucose oxidation were p H 6.5, 40℃. Fusion protein was more sensitive to p H and temperature than parental enzymes. For the coenzyme NADPH, the Km values of fusion CR2-GDH and parental CR2 were 1.263 mmol/L and 0.626 mmol/L respectively. In the course of the asymmetric reduction catalyzed by fusion CR2-GDH, more amount of NADPH was added. Heavy metal ions(Cu2+、Fe2+、Fe3+) could inhibited fusion enzyme activities. 25 mmol/L Ca2+ improved the enzymatic activities of CR2 in CR2-GDH by 282.4% and GDH in CR2-GDH by 168.5% to the greatest extent.The asymmetric reduction of COBE for coenzyme regeneration was catalyzed by the fusion enzyme system CR2-GDH in aqueous phase with optimal reaction p H(5.5), temperature(30℃) and molar ratio of co-substrate/substrate(1.2:1). In order to compare the catalytic efficiency between fusion and parental enzymes, equal number of moles was added. The yields of the reaction were 100% and 37.3% within 3 h respectively. A substrate fed-batch strategy was introduced into fusion enzyme system for reduction COBE(50 g/L) in 5 h, and the optical purity and yield of(S)-CHBE were kept at >99% and 54%.Metal affinity magnetic nanoparticles were prepared to be the directed immobilization carrier of fusion protein. The FTIR spectra of nanoparticles indicates that IDA was coupled and transition metal ions Cu2+、Zn2+、Ni2+、Co2+ are chelated on the IMAN respectively. The results demonstrated that Zn2+ chelation was the best and the immobilized amount 10 mg/g, recovery of enzyme activity 60.1%.Mesoporous molecular sieves SBA-15 was the applicable one among 5 kinds of carriers by comparing carrier content, recovery of enzyme activity and cost. The remaining carriers were IMAN, MCM-41, modified silica, calcium alginate. Immobilization of fusion enzyme CR2-GDH on SBA-15 was realized. The results showed that adsorption capacity was 27.7 mg/g carrier under the condition of p H 5.5, enzyme concentration 1.4 mg/m L, reaction time one hour. The recovery of immobilized enzyme activity was increased by near 20% adding metal ion Ca2+ of 25 mmol/L. It was also found that the thermal and operational stabilities of the immobilized enzyme were higher than those of the free enzyme. The recovery was increased by 19.1% at the temperature of 40℃. Immobilized enzyme was not more sensitive to organic solvents especially alkanes than free enzyme. In biphasic system, the production yield obtained by Ca2+/SBA-15 immobilized enzyme was increased by 23.8%. The residual activity remained more than 70% after seven batches of repeated use. |
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