| Sucrose isomerase(EC 5.4.99.11), also known as isomaltulose synthase. Sucrose isomerase isomerizes sucrose to produce isomaltulose and trehalulose as the main products. Most of sucrose isomerases from microbial primarily produced isomaltulose(75%-85%) has been reported. Isomaltulose has many unique properties, such as low sweetness, lower insulin reaction and prolonged energy release. Thus, isomaltulose is very suitable for obesity, diabetics and athlete to eat. Therefore, isomaltulose has a broad market prospect as a new type of functional sweetener.In the present study, the sucrose isomerase from Serratia plymuthica AS9 was heterogeneous expressed in Escherichia coli and Brevibacillus brevis, respectively. Then the recombinant enzyme was purified and the enzymatic properties of the sucrose isomerase were investigated. Furthermore, the reaction conditions for isomaltulose production by sucrose isomerase was optimized in this study. Finally, in order to improve the thermal stability of sucrose isomerase, site-directed mutagenesis based on the B-factor combined with Rosetta Design was applied to enhance the thermostability of the enzyme. The main results were listed as follows:(1) The sucrose isomerase gene(pal Iori) was optimized and then the recombinant strain E. coli BL21(DE3)/p ET-24a(+)-pal I was constructed. In shake flask, the recombinant strain E. coli BL21(DE3)/p ET-24a(+)-pal I was cultured in TB medium at 25°C and the secreted enzyme activity was 253.1 U·m L-1 after cultivation of 24 h. Thus, recombinant strain E. coli BL21(DE3)/p ET-24a(+)-pal I was selected for further study. Different feeding rate of lactose was investigated in 3 L fermentor. The optimal fermentation conditions were as follows. The recombinant E. coli was cultured under 33°C, 30% dissolved oxygen concentration and p H 7.0. When OD600 reached 40, the induction was performed with 0.4 g·L-1·h-1 lactose, and the maximum activity in the supernatant was 1381.4 U·m L-1 after 27 h of induction, which was 5.46-fold of that in shake flask fermentation. The extracellular secretion rate was 64.5%.(2) The recombinant enzyme was purified by ammonium sulfate precipitation and SPSepharose FF cation exchange chromatography. The purified enzyme was exhibited a specific activity(total activity) of 957.7 U·mg-1. The optimum temperature and p H of the purified enzyme was 30°C and 6.0, and the half life of the enzyme was 100 h at 40°C and 39.2 min at 45°C, respectively. In addition, kinetic studies showed that the Vmax、Km、Kcat and Kcat/Km of the enzyme was 825.5 U·mg-1, 30.1 mmol·L-1, 992.8 s-1 and 33.0 mmol·s-1×L-1, respectively. In addition, the reaction conditions for isomaltulose production by sucrose isomerase was optimized in this study. The optimized conditions were as follows: 400 g·L-1 sucrose, p H 6.5, 30 °C, 20 U sucrose isomerase per gram sucrose and 8 h of incubation. Under the optimized conditions, the maximal yields of isomaltulose could reach 87.9%.(3) The B-factor indicates atomic displacement parameters obtained from X-ray data. It reflects the smearing of atomic electron densities with respect to their equilibrium positions as a result of thermal motion and positional disorder. Amino acid structure with higer B-factor is easy to be damaged under heating conditions. Thus lead to the decrease of the enzyme activity. Based on B-factor value, the Lys576 and Glu175 of sucrose isomerase with higher B-factor value were selected, Using Rosetta Design server to predict the stability mutation of the 175 and 576 site, the results showed that Glu175 and Lys576 was substituted with Asn175 and Asp576, respectively. Then three mutants(E175N, K576 D and E175N/K576D) were constructed by using site-directed mutagenesis. All mutants were purified and characterized in detail. The results indicated that the three mutants had a slight increase in optimal temperature from 30 to 35°C compared to that of the wild-type enzyme. In addition, the half-lives of the E175 N, K576 D and E175N/K576 D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the Km values for the E175 N, K576 D and E175N/K576 D mutants decreased by 6.6%, 2.0% and 11.0%, and Kcat/Km values were 1.38, 1.04 and 1.19 times greater than that of the wild-type enzyme. Moreover, the reaction conditions for isomaltulose production by mutants in 30°C was optimized in this study, the results showed that the E175 N, K576 D and E175N/K576 D mutants displayed a slight improved isomaltulose yield during sucrose catalytic.(4) The optimized sucrose isomerase gene(pal I) was linked to expression plasmid p SVEB. And then the recombinant plasmid p SVEB-pal ILSP was constructed and transformed into B. brevis. Then the recombinant strain B. brevis/p SVEB-pal ILSP was constructed. In shake flask, the extracellular enzyme activity of B. brevis/p SVEB-pal ILSP was 143.3 U·m L-1 in TM medium after cultivation of 48 h at 30°C. |
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