| Trehalose(α,α-trehalose) is a disaccharide formed byα,α-1,1 linkage of two D-glucose molecules. It occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant against the effects of freezing of dehydration. It also possesses physical and or chemical properties that are different than other sugars,which may make trehalose an attract ive ingredient in food, health and beauty and pharmaceutical products. This paper briefly reviews recent advances in the manufacture of trehalose, according to its biosynthetic pathways, and provides an overview of the maior applications of trehalose in food industry, biology, cosmetics,medical industry and agriculture.Trehalose synthase play the key role in enzymatic process produced trehalose. Maltose can be transfered from anα,α-1,4-glycosidic linkage into a trehalose withα,α-1,4-glycosidic linkage. There were some disadvantages in the existing trehalose synthase which the transform effciency is too low;,such as the thermostability is too low and the reaction time is long. Therefore, there is much interest in researches on the biological catalyse method. It is necessary for the yeild to perform mutagenesis on trehalose synthase so that the high levels of total trehalose synthase activity to improve the industrial capability.First of all,thermus aquaticus FL-03 chromosome genome DNA as a template, the trehalose synthase gene were cloned and high-expressed in E.coli by nested PCR amplification, was done with by PCR mediate genomic library screening. And more,trehalose synthase was evolved by PCR mediate and half-rational design. The three-dimensional model of TreS was from Pyrococcus horikoshii. It was constructed basis on the crystal structure of trehalose synthase from on the SWISS-MODEL Internet. United into analyzing the conserved region of trehalose synthase amio sequence from several different origin code and Site-directed Mutagenesis completely.In addtion, to improve the cellular permeability to better shadow, a greater degree of damage to treatment the cell, to promote endocrine cells of the trehalose synthase released into the extracellular by extracellular enzymes, through the centrifuge to separate this enzyme extracted from the supernatant, raising the yield of trehalose. The productivity was improved, the cost decreased and the technological process which offer the basis of commercial production.The experimenta 1 results indicate that:(1) the trehalose synthase gene TresC01 was cloned and high-expressed, and the 3D structure of the trehalose synthase was successful constructed by homology modeling,furthermore, the probable active site was forecasted.(2)The relationship between the function groups and catalytic activity of enzyme revealed that acquired the 3D structure information of the homology modeling, A197, H100, H305 and S547 in 4 amino acids remnants radicle of combining with the domains related to the catalyst activity of S547 and passing the 3D model, the initial analyzes 4 amino acidses remnants the radicle change may effect the TresS catalize activity.(3) T-TersC01 for mold plate, lie to lead through PCR the method matches TresC01 progress saturation to mutate a search to seaweed sugar, result display the A197 and H100 distinguish a saturated mutation for other of 19 amino acidses after, totally acquired 13 mutation, only wild-type pEA 197 H to have higher of activity other all have no activity; wild-type pEA197H lived to raise than TresC01 was 1.52 times.(4) High throughput screening method of expressing.The results pointed out hat the enzyme activity was lost after A197and H100 which were converted to other 19 kinds of amio acids,and that the mutants A197X and H100Q were also indicated no activities expect of A197H. The reason of activity lost probably was that these stucture positions were important to hold on the structure stability of the protein domains, and they possibly were crucial residues or active sites of the enzyme. The specific activity of the mutants H305 and S547 (His→Cys and Ser→Cys) were decreased largely and reduced to 66%,99%, the wild-type pEH305C respectively was shown that the activity was increased Compared with the wild-type,four degree approxately,It can not only give aid for useful data providing and information in studying the relation of the sturcture and function by Studying and analysis, these changes about the properties of the mutants but also can afford the theoretic basis to improve the molecule evolution on trehalose synthase in the future.(5) Directed evolution of trehalose synthase.The cloned that adoption higher-copies carries the body XL10-gold to successfully acquire 41 cloned to carry a body, according to the keeping of blue white spot view reaction, created the colon vector screening system; Adopt DNS-ELISA (diastatic enzyme) sieving at high enzyme activity, the method can consumedly lower the impact of background quickly and efficiently. Belong to this laboratory to found..(6) Use of permeabilized cells allows the conventional enzyme purification and immobilization steps to be omitted and simplifies the synthesis procedure. The study on recombinant escherichia coli cell wall permeability was carried out by different surfactants (Triton-X100, DMSO,cetyltrimethy-lammonium bromide (CTAB)) of water, the results show that of Triton-X-100 to improve the cellular permeability to better shadow, a greater degree of damage to treatment the cell, to promote endocrine cells of the trehalose synthase released into the extracellular by extracellular enzymes, through the centrifuge to separate this enzyme extracted from the supernatant, raising the yield of trehalose.The article is the first systematic study to improve the property of trehalose synthase successfully by rational and irrational design evolution. This studies beneficial to understanding the relationship between 3D structures and properties of trehalose synthase,as well as the researches of producing threhalose highly by the gene engineering strain.
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