The Research Of Trehalose Production Technology

In recent years, Escherichia Coli (E.coli) has been widely used in industrial production of heterologous protein and it is considered to be one of the most effective protein expression systems. Because it has clear genetic engineering bacteria system background and its characteristics of cultivate easily, high density, high quantity of protein expression, the cost is low, and hard to dye the mixed bacterium. It become the great choice of the enzyme for the industrial production of high quality.This topic systematic studied and explored the system through expressing different promoter and different copy number of plasmid and different sources of trehalose synthase in E. coli to screen recombinant E. coli which had the ability of effective expression of trehalose synthase, and then increase production of trehalose. Three kinds of room temperature source and two kinds of thermophilic source of trehalose synthase genes could be chose to express in E. coli respectively. And we used the appropriate method of enzymology properties for production of trehalose determination.This work attempted to knock out encoding membrane protein gene lpp of E. coli, fused express secretion signal peptide and trehalose synthase gene3 and truncate trehalose synthase gene for purpose of exocytosis expression of trehalose synthase. Reducing induced expression temperature, replacing weaker promoter or RBS sequence on the recombinant plasmid and molecular chaperone coexpression could been adopted to reduce inclusions generation of trehalose synthase and then improve the solubility of trehalose synthase expression. According to the homologous model of trehalose synthase structure, we used some methods such as exploring key amino acids in the enzyme catalytic activity center or around the substrate specificity center and using site-directed mutation technology to improve the trehalose synthase activity. Utilizing these technologies, we realised reformation of trehalose synthase and layed the groundwork for trehalose industrialized production. Obtained the experiment results as followed:1. Cloned the trehalose synthase gene treS respectively from three kinds of room temperature sources, such as Pseudomonas putida NBRC 14164 genome, Corynebacterium glutamicum ATCC 13032 genome and Streptomyces coelicolor ATCC 23899 genome, and two kinds of thermophilic source, such as Thermus thermophilus HB27 genome and Thermotoga maritime MSB 8 genome, and used four plasmids which have different promoter and different copy number to construct twenty engineering E.coli containing different trehalose synthase gene. Successful expression of trehalose synthase and measured the enzyme activity through the in vitro experiment. The highest enzyme activity could reached 2614 U and the specific activity could reached 1.996 U·mg-1. The induced fermentation conditions and the enzymatic optimized reaction conditions also be carried on. To determine the best fermentation condition:fermentation time was 30 h, fermentation temperature was 16 ?, IPTG concentration was 0.5 mM·L-1, lactose concentration was 1.5 mM·L-1. Optimal enzymatic reaction conditions: enzyme reaction temperature was 50 ?, reaction time was 10 h, the substrate concentration of maltose was 10%, the enzyme reaction pH was 9.0. Metal ions Ca2+ supplementation was 15 mM·L-1, the enzyme activity increased by 19%, reducing agent Vc supplemengtation and DTT supplementation was 4 mM·L-1, the enzyme activity increased by 9.4% and 41%, respectively.2. Knocking out E. coli which had encoding membrane protein gene lpp by Red homologous recombination made porous membrane structure. It made trehalose synthase secretive and trehalose concentration increased by 1.48-fold after reaction, Trehalose concentration obtained from reaction of broken bacteria with maltose was decreased. Secretion signal peptide and trehalose synthase gene were fusion expressed on the recombinant plasmid, trehalose synthase also had ability of secretion. It made trehalose synthase secretive and trehalose concentration increased by 77.91% after reaction, Trehalose concentration obtained from reaction of broken bacteria and fermentation broth with maltose was increased by 23.45%. To a small extent, the secretion has been realised. Reducing induced temperature and changing weaker promoter or RBS sequence strength on the recombinant plasmid could effectively reduce the formation of inclusion body. Three kinds of molecular chaperone sigma32, GroEL/S and DnaK/J were used respectively or in the form of random combination with trehalose synthase gene co-expression. Further improve the solubility of trehalose synthase expression, the highest soluble expression of the combination of three kinds of molecular chaperone with trehalose synthase co-expressing increased 12-fold than the original strain. According to the homologous model of trehalose synthase structure, we explored key amino acids in the enzyme catalytic activity and around the substrate specificity center. Utilizing site mutation fought for improving the activity of trehalose synthase.3. Trehalose synthase of other four sources and the C-terminal of tttreS from Thermus thermophilus HB27 fused expressed in E.coli. C-terminal of tttreS played an important role on the structure of the catalytic center of trehalose synthase. The other kinds of trehalose synthase activity had been obviously improved, the activity of trehalose synthase from Pseudomonas putida NBRC 14164 was 2.53-fold than original strain without C-terminal of tttres from Thermus thermophilus HB27. The activity of trehalose synthase from Corynebacterium glutamicum ATCC 13032 was 5.58-fold than original strain without C-terminal of tttres from Thermus thermophilus HB27. The activity of trehalose synthase from Streptomyces coelicolor ATCC 23899 was 2.74-fold than original strain without C-terminal of tttres from Thermus thermophilus HB27 and the activity of trehalose synthase from Thermotoga maritime MSB8 was 4.57-fold than original strain without C-terminal of tttres from Thermus thermophilus HB27.