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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
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
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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