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정보검색

소장정보 검색

국회도서관 홈으로 정보검색 소장정보 검색
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결과 내 검색 동의어 포함

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Contents

(Abstract) 6

I. Introduction 8

II. Materials and Methods 11

1. DsrE563 from Leuconostoc mesenteroides B-1299 constitutive mutant Organism, plasmids and growth conditions 11

DSRE563 gene from Leuconostoc mesenteroides B-1299CB4-BF563 11

Cloning of the truncated mutant gene ORFs 12

Expression of the Recombinant Enzymes 13

Purification of the recombinant enzymes 13

Enzyme activity assay 14

Electrophoresis and detection of active dextransucrase 15

Oligosaccharide synthesis with glucose, maltose and isomaltose as acceptors 15

Penicillium dextranase treatment of glucan synthesized by Dsr563-2 15

Nuclear Magnetic Resonance (NMR) 15

Acceptor reaction study by using Dsr563-2 16

2. Bifunctional fusion enzyme having dextranase and amylase acivities Dex2 and G6Amy gene cloning 16

Construction of dex2 :: G6amy-pRSET 18

Enzyme expression and purification 19

Protein concentration assay 21

Determination of optimum condition and stability for temperatures 21

Determination of optimum condition and stability for pH 21

Enzyme activity assay 22

Inhibition assay of polymer production by mutansucrase by adding 2DA 22

III. Results and discussion 23

Amino acid sequence of DsrE563 23

Construction and expression of DsrE563-1 and DsrE563-2 genes 23

Glucan produced by DsrE563-2 26

NMR analysis of DsrE563-2 polymer 26

Acceptor reaction by DsrE563-2 28

The construction of dex2-, G6amy- and de2::G6amy-pRSET 33

Expression and purification of the recombinant enzymes 39

Enzyme function of the recombinant enzymes 42

The optimum condition and stability for temperatures 43

The optimum condition and stability for pH 45

Inhibition of polymer synthesis of mutansucrase by the addition of 2DA 45

IV. Conclusion 48

V/VI. References 49

(국문초록) 52

감사의 글 54

List of Tables

Table 1. Designed primers for PCRs 17

Table 2. Relative amount of the acceptor products synthesized by using Dsr563-2 dextransucrase with glucose and isomaltose 31

Table 3. Comparison of acceptor reaction product component and amount produced by Dsr563-2 and 512FMCM dextransucrase 32

List of Figures

Figure 1. Designed construction of the Dex2::G6Amy gene 19

Figure 2. Schematic primary structure of DsrE563 and truncated forms constructed . 25

Figure 3. SDS-PAGE and PAS staining of the Dsr563-2 25

Figure 4. Penicillium dextranase treatment with the polymer produced by Dsr563-2 27

Figure 5. ¹³C- NMR spectra of glucan that synthesized by Dsr563-2 in DMSO. OS ; O-substitute carbons 27

Figure 6. Purified PCR products of the dex2 and G6amy 33

Figure 7. Constructed Dex2::G6Amy gene 34

Figure 8. Neucleic acids and amino acids sequence of 2DA 38

Figure 9. Expression of the recombinant enzymes 40

Figure 10. Purification of the recombinant enzymes 41

Figure 11. Enzyme activities 42

Figure 12. The effect of temperature on the enzyme activity and stability 44

Figure 13. The effects of pH on enzyme activity and stability 46

Figure 14. Inhibition of polymer production using mutansucrase and sucrose by the addition of 2DA 47

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