碩士 / 國立海洋大學 / 水產食品科學研究所 / 83 / The objectives of this research are to purify and characterize the transgultaminase (TGase) from Streptoverticillium ladakanum, and to apply it in minced mackerel meat. The cultural filtrate from S. ladakanum cultivated in a medium composed of 1% glycerol, 1.5% yeast extract, 0.1% MgSO4 and 0.2% K2HPO4 at 28℃ for 4 days, was harvested by centrifugation, followed by membrane filtration. The filtrate was subjected to ammonium sulfate fractionation and the precipitate at 55∼75% saturation of ammonium sulfate was collected. Then, the TGase was purified to electrophoretical homogeneity after Blue Sepharose Fast Flow chromatography. The recovery of total TGase activity was 63%. the molecular weights were 30.5 and 37.5 kDa estimated by Superdex 75 HR gel filtration and SDS-PAGE, respectively. Its pI value was 7.9 and the enzyme was shown to be calcium-independent. This enzyme was quite tolerant to the variation of pH and had an optimum pH at 6 for the enzyme activity. The optimal temperature for this enzyme activity was 50℃. Its rate constant for thermal inactivation at 50℃ was 6.2×10-4 sec-1. It was inhibited by PCMB, PMSF, NEM and IAA, which demonstrated that its active center might contain cysteine. It was also inhibited by Cu2+, Zn2+, Fe2+, Pb2+, Co2+ and Ni2+, but not by N1+, Ba2+, Mg2+, Mn2+ and Ca2+. The Km and Vmax values of TGase were 6.97 mM and 10.64 μmole/min/mg, respectively, using N-α-carbobenzoxy-glutaminyl-glycine as substrate. The percentage of the basic amino acids, including lysine, histidne, and arginine, was 16.9%, as shown from the amino acid analyses. It mainly caused the inter-(or intra-) molecular crosslinks of myosin monomer heavy chain of mackerel muscular proteins, as evidenced by SDS-PAGE. The gel strength of minced fish meat added with TGase at a ratio of 0.34 unit/g of meat could be increased by 3.9 folds, as compared with that of the control. The scanning electron microscopic photograph further demonstrated the moire compact structure of fish myofibrils due to the action of this enzyme.
| Identifer | oai:union.ndltd.org:TW/083NTOU3084002 |
| Date | January 1995 |
| Creators | 林香美 |
| Contributors | 蔡國珍 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 80 |
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