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Cloning and Expression of Maltooligosyltrehalose Trehalohydrolase in E.coli from Micrococcus Roseus QS412

Author: XuYaQin
Tutor: YuanQiPeng
School: Beijing University of Chemical Technology
Course: Pharmaceutical Engineering
Keywords: Rose Micrococcus Trehalose Maltooligosyl trehalose hydrolase Protein expression Enzyme activity
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 70
Quote: 2
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Abstract


Trehalose (Trehalose, α-D-glucopyranosyl-α-D-glucopyranoside) is the hemiacetal hydroxyl group from two glucose molecules by a combination of a non-reducing disaccharide, having the unique biological characteristics can protect biological macromolecules resistance to adverse environmental pressure. Over the past decade, its broad application prospects aroused great interest in their research of scientists from various countries. Our laboratory previous Micrococcus QS412 in rose production trehalose enzyme system genes have been cloned. This article focuses on the means of using genetic engineering to construct genetically engineered bacteria, maltooligosyl trehalose hydrolase (maltooligosyltrehalose trehalohydrolase, MTHase) expression and enzyme activity in E. coli. First Primers were designed to clone the trehalose hydrolase gene treZ of Rose Micrococcus QS412 maltooligosyl, inserted into the expression vector pET28a () to construct the recombinant plasmid pET-MTH, makes recombinant strain E.coli BL21 (DE3) (pET-MTH ) in LB medium, 28 ° C with 0.1mmol/LIPTG induced 8h, expression fusion protein 6His-MTHase, 30% of the exogenous protein accounted for the total bacterial protein. Collect the fermentation broth of bacteria, washing broken, the determination of the crushing of the whole bacterium enzyme activity, resulting enzyme activity obtained per liter of fermentation broth 44U. Fusion protein vector pET-MTH the His-MTHase ??expressed in E.coli BL (DE3) most of the form of inclusion bodies. The temperature is the most important factor for the level of protein expression and cell growth, induced after 4 ℃ ~ 37 ℃ temperature range, and 0.04 to 1 mmol / L IPTG inducer concentration range, and found both to improve the enzyme activity had no significant effect finalized fermentation of recombinant bacteria induced conditions of 28 ℃, 0.1 mmol / L IPTG, induced 6h. Broken bacterial inclusion bodies obtained, washed, dissolved, try to dialysis, and refolded protein enzyme activity detected, but undetectable enzyme activity, spatial structure may renaturation wrong. Taking into account the T7 promoter is too strong leading to the expression of most of the exogenous protein His-MTHase ??form of inclusion bodies, attempts to treZ gene is inserted into the carrier with a weak promoter. The Department of Chemical Engineering, Tsinghua University Li Qiang teachers to build their own constitutive expression vector was constructed the the recombinant plasmid pGEMKT-HPf-MTH, was transformed into E. coli host strain, but no significant expression, undetectable enzyme activity. In summary, this work makes QS412 comes from Rose Micrococcus maltooligosyl trehalose hydrolase genes in E. coli active expression of the expression vector and host strain for further replacement genetically engineered bacteria to express recombinant protein enzyme living laid the foundation.

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