Cloning And Expression Of Gene Encoding MTHase From Corynebacterium Glutamicum ATCC13032

Trehalose is a very stable non-reducing disaccharide which was linked by two glucopyranose units in anα,α-1,1-glucosidic bond. This sugar is present in a wide variety of organisms in nature. As a kind of natural nonspecific protection of living matter, trehalose can protect cells and biomacromolecules from inactivation or denaturation caused by a variety of stress conditions, including dehydration, cold, heat, desiccation, oxidation, radiation and toxic organic compounds. At present, trehalose has been used in areas of medicine, cosmetics, food industry and so on. The market prospect of trehalose is favorable.Today, trehalose is mainly produced using enzymes from organisms, especially from bacteria. Among these methods, the TreS pathway which convert maltose to trehalose in one step and the TreY-TreZ pathway which convert starch to trehalose in two steps are most economic. In this study, we cloned and expressed the TreZ protein of TreY-TreZ pathway ,which called maltooligosyl-trehalose trehalohydrolase (MTHase) in microbe systems and researched the characteristics of the protein.First,we cloned treZ encoding MTHase from Corynebacterium glutamicum ATCC13032. We got a target fragment of 1764bp. It was inserted into prokaryotic expression vector pRSET-B and then was transformed into the host Escherichia coli BL21 (DE3) pLysS. A recombinant enzyme was obtained by IPTG induction. In the optimum expressing conditions, the soluble recombinant enzyme accounted for about 40% of the total cell soluble proteins and reached to 0.27mg per fermentation liquor. But still, there were some of the recombinant protein expressed as inclusion bodies. The activity of the recombinant enzyme was analyzed by detecting the product of it using Thin Layer Chromatography and Ion Chromatography. It was proved that the soluble enzyme together with maltooligosyl trehalose synthase was capable of decreasing dextrin to produce trehalose.In order to find whether the MTHase can be secreted expressed, we constructed recombinant plasmid of pPIC9K-treZ and pHT43-treZ, then transformed them into Pichia pastoris GS115 and B. subtilis WB800N, respectively. We tried some different cultural conditions, but still could not see target proteins in culture medium.