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Production of 4-α-glycosyltransferase by Cloned E.coli DH-5α

Author: JiXueXia
Tutor: JinZhengYu；XuXueMing
School: Jiangnan University
Course: Sugar works
Keywords: Cloned E.coli DH-5α 4-α-CGTase Fermentation optimization Specific 4-α-CGTase formation rate Specific cell growth rate
CLC: TQ925
Type: Master's thesis
Year: 2011
Downloads: 72
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Abstract


Cyclodextrin glycosyltransferase (CGTase) belongs to the group ofα-amylase. It could transfer the starch into cyclic oligosaccharides--cyclodextrins (CDs). Among the academic reports and commercial use, most of CDS areα-、β- andγ-CGTase, which have been exploited in the business market. Compared to those CDs, large-ring cyclodextrins (LR-CDs) composed of more than 9 glucopyranose units have specific cavity geometries and convenient functions. 4-α-Glycosyltransferase has the function of transdfering starch into large-ring cyclodextrin. Although there are many microorganisms reported to produce large-ring cyclodextrin, systematical investigations on production of 4-α-CGTase are rare.From the crude enzymes produced by cloned E.coli DH-5α(CGMCC No.3093), 4-α-GTase was separated and purified by high-temperature processing (65℃,20 min)、Ni-NTA and dialysis. The purified enzyme was demonstrated by SDS-PAGE to be a homogeneous protein and the molecular weight is estimated as 57KDa. It was proved to be with the high level of transglycosylation activity by HPLC and its smallest substrate was maltose. It was stable within the temperature range from 70℃to 85℃and the pH range from 6.0 to 8.5. The optimal temperature for the enzyme was 75℃and optimal pH was 7.5. The optimization of conditions for the production of 4-α-glucanotransferase (4-α-GTase) from recombinant E. coli DH-5α(CGMCC No. 3093) was carried out in shake flasks based on Plackett-Burman design and Box-Behnken design. It is clarified that the optimum conditions for the 4-α-GTase production were 31.52 g/L glycerol, 20 g/L beef extract, 5 g/L NaCl, 0.4 g/L CaCl2, 1.2 g/L MgSO4·7H2O, initial pH 7.19, incubation temperature 37.7°C, and 2% (v/v) inoculum size. The results showed that A (glycerol), E (initial pH), and G (incubation temperature) significantly affected the 4-α-GTase production and the order of the significance was as follows: G > A > E. Under these conditions optimized, a maximum yield of 251.3 U/mL was obtained in comparison with that (64.5 U/mL) obtained in basal medium. Box-Behnken experimental results indicated that there was no interaction between the three elements.4-α-CGTase production laws by cloned E.coli DH-5αwere investigated in a 5L fermenter. During the fermentation process, mycelium growth and enzyme production condition is not completely consistent. pH could be a indicator for the end fermentation. Because of oxygen seriously restricted the production of the enzyme, so it is necessary to optimize the DO supply. Taking the specific cell growth rate and specific 4-α-CGTase formation rate into consideration, two-stage DO control strategies were proposed. Those results suggest that two strategies were proved to be effective for 4-α-CGTase production. In comparison to the case (constant 400r·min-1), 4-α-CGTase production increased by 28.3% with two-stage DO control strategy. The highest specific activity was 602.2U/mL.

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