重組e.colidh5α發(fā)酵法生產(chǎn)4α葡萄糖基轉(zhuǎn)移酶

1、江南大學(xué)碩士學(xué)位論文重組E.coliDH-5α發(fā)酵法生產(chǎn)4-α-葡萄糖基轉(zhuǎn)移酶姓名：冀雪霞申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：制糖工程指導(dǎo)教師：金征宇;徐學(xué)明2011-03江南大學(xué)碩士學(xué)位論文 IIAbstract Cyclodextrin glycosyltransferase (CGTase) belongs to the group of α-amylase. It could transfer the starch into cyclic

2、 oligosaccharides--cyclodextrins (CDs). Among the academic reports and commercial use, most of CDS are α-、 β- and γ-CGTase, which have been exploited in the business market. Compared to those CDs, large-ring cyclodextrin

3、s (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 ther

4、e 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 w

5、as 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 w

6、as 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 70to 85and the pH range from ℃ ℃6.0 to 8.5. The optimal

7、temperature for the enzyme was 75and 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 f

8、lasks 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 MgSO

9、4·7H2O, 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 prod

10、uction 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-Beh

11、nken experimental results indicated that there was no interaction between the three elements. 4-α-CGTase production laws by cloned E.coli DH-5α were investigated in a 5L fermenter. During the fermentation process, myceli

12、um 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 optimiz

13、e 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

14、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. Key wor