碩士 / 國立臺灣海洋大學 / 食品科學系 / 100 / The purpose of this thesis is using carrageenase carIII gene from Pseudomonas vesicularis MA103 as a reporter gene combine with plasmid pNZ8149 from nisin-controlled expression system and transgenic to Lactococcus lactis NZ3900 host cells, then evaluate the characterization of nisin-induced k-carrageenase crude enzyme (PV-Car-C), the composition of oligosaccharide (Car-F5) from Pv-Car-C hydrolyzed k-carrageenan of the antioxidant bioactivity and anticoagnlant bioactivity. The carIII forward primer encode NcoI restriction site and the reverse primer encode PstI restriction site. Then using the gene of carrageenase carIII as a template for PCR reaction, and this PCR amplified product as ligated into pGEM-TTM vector, and then transformed into the competent cells of E. coli DH5α. Then pGEM-TTM vector, which carrying the cloned carIII gene, was digested with NcoI and PstI, and ligated into the LAB vector pNZ8149. The recombined vector II (pNZ8149-palIII) was electroporated into the Lc. lactis NZ3900. And the lac+ yellow colony was picked and the punified and examined it’s plasmid profile 4 successful transformed carIII cloned were identified by designated. It was proved that the sequence of the cloned DNA fragments was completely identical to the DNA sequence of the carIII gene, and the succeed clone was designated as cloned strain NZ3900-rcarIII. The clones induced by 10 ng/mL nisin for 6 hr could not obtain extracellular carrageenase activity. The intracellular crude enzyme solution could obtain higher carrageenase activity (0.042 U/mg), which revealed a 106 kDa protein band by SDS-PAGE. The extracellular crude enzyme solution with molecular mass > 10 kDa, > 30 kDa, and > 100 kDa were collected by UF system. It could obtain higher carrageenase activity (0.034 U/mg). On substrate specificity, Pv-Car-C can hydrolyze k-Carrageenan. Analyzing the hydrolysate of Pv-Car-C by TLC, compared to the standards, discovered that the main hydrolysate were assumed to be DP5 oligosaccharide. Oligosaccharides hydrolysates with molecular mass < 3 kDa were collected by a UF system, then though GPC and HPLC to obtain DP5 oligosaccharide (Car-F5). FTIR chromatograms showed that was obvious signal enhancement in wave numbers 1066 cm-1 resulted from sulfate contents, and the sulfate contents of Car-F5 was 17.70%. In the evaluation of the antioxidation effect of Car-F5, soluble total phenol, DPPH free radical scavenging, Fe2+ chelating effect and of Car-F5 did not increase, but the reducing power of Car-F5 had significant increase (13.95 TE mg/mL). In the experiment of anticoagulant activities of rabbit plasma, prothrombin time analysis of Car-F5 (20 mg/mL) was delayed to 132 sec, and activated partial thromboplastin time analysis of Car-F5 (5 mg/mL) was delayed to 319 sec, and it was tendency for reduction on absorbance. This study provides optimal parameters for the NICE system in cloned strain NZ3900-rcarIII as well as a means to produce functional Car-F5, and serving as the basis for development of health food for preventing the high risk of thrombotic cardiovascular disease in the future.
| Identifer | oai:union.ndltd.org:TW/100NTOU5253086 |
| Date | January 2012 |
| Creators | Yi-Lang Feng, 馮一郎 |
| Contributors | Chorng-Liang Pan, 潘崇良 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 172 |
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