| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 97 === Starch branching enzyme (SBE, EC 2.4.1.18) is one of the enzymes in the starch biosynthetic pathway and it plays a vital role in the formation of amylopectin, the main component of the starch molecule. The chemical fine structure changed by SBE would affect the physical property and function of starch. The objective of the thesis was to optimize the expression of a potential recombinant SBEII clone and characterize the active recombinant enzyme to be used in modifying the starch molecular structure. The constructed pET21b-VrSBEⅡwas first transformed into BL21 (DE3) expression host and induced with 0.2 mM IPTG for 5 hours at 37℃, SDS-PAGE analysis showed the molecular size of the recombinant protein was 108 kDa. This rVrSBEⅡwas expressed as a soluble protein with crude enzyme activity of 0.25 U/mg and the activity was enriched 27 fold by Ni-NTA affinity chromatography. To increase the solubility and activity of the enzyme, the clone was further expressed in Origami B host and induced under the same induction condition. Improvement of rVrSBEⅡwas not only the solubility but the crude activity of 0.46 U/mg has increased to 14.9 U/mg after purification. The enzyme was most active at pH 7.0 at 30℃and relatively stable up to 30℃.More than 90% of the original activity remained after incubated at pH 7.0-9.5. When 1 mg/ml amylose substrate was used to react with 6�n�慊 of purified rVrSBEⅡat 30℃, there was a time-dependent decrease of the absorbance at OD660 and became steady after 90 minutes. GPC-HPLC analysis of the enzyme products showed the peak eluted at an earlier elution time as reaction proceeded, indicating the formation of branching and size enlargement by rVrSBEⅡin the product. The present study shows that the pET21b-VrSBEⅡclone was able to be expressed as a biologically functional enzyme in E. coli system and the protein solubility and activity were improved by expressing in Origami B host. Basic characteristics of rVrSBEⅡand the product were established.
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