Gene Cloning, Expression And Characterization Of A Novel Trehalose Synthase Isolated From A Marine Pseudomonas

Trehalose is a stable and non-reducing disacarides showing widespread occurrencein many organisms. Trehalose has many applications in the pharmaceutical, food andcosmetics industries due to its special characteristics. However, its applications arelimited by the high cost of production. Until now, At least five biological pathwayswere found in organisms for trehalose production. Among them, Trehalose synthase(TreS) utilizes the low cost substrate of maltose and convert it to trehalose in one step.This pathway is prospective for the industrial production of trehalose.The ocean is a complicated environment which has the characteristics of salinity,high pressure, cold and hypoxia. In addition, trehalose can protect microorganismsagainst environmental pressures. Marine microorganisms, to achieve tolerance, willincrease the level of cytosolic trehalose to cope with external stress. Therefore,screening TreS-producing strains from the ocean and isolating the effective enzyme isprespective for the production of trehalose.Although cloning, expression and characterization of several tres genes from manydifferent strains have been reported, tres genes from marine microorganisms have notbeen investigated until now. In this study, a novel tres gene was obtained from a marinePseudomonas. This new gene was cloned and expressed in Escherichia coli.(E.coli)and the recombinant enzyme was purified and characterized. Several importantresidues that might be involved in catalysis or substrate binding were identified bysite-directed mutagenesis. In addition, possible mechanism concerning the aglyconerearrangement during the conversion between maltose and trehalose catalyzed by thisenzyme was also investigated in this study. Section I: Isolation, cloning, expression and purification of this novel tres genePseudomonas sp. P8005, which demonstrated the highest activity of trehaloseproduction, was isolated from hundreds of marine bacteria collected by this laborary. Acore region of648bp was obtained by degenerate PCR. Subsequently, an open readingframe (ORF) with a length of3369bp, encoding1122amino acids and with a predictedmolecular weight of126kDa was obtained by thermal asymmetric interlaced PCR（TAIL-PCR）. The amino acids sequence identities between this TreS and otherreported TreS was relatively low (29%-35%). The new sequence was submitted toGenbank database (Genbank Accession No: JQ951963). The novel tres gene wasnamed G526.Then the recombinant expression vector pET-32a-G526was constructed andtransformed into E. coli strain BL21(DE3). The recombinant TreS (rG526) wasproduced after induction. After ultrasonication and centrifugation, the purified rG526was obtained by using the Ni sepharose affinity chromatography. After purification,one major protein band at about141kDa which corresponded to the theoretical valuecould be found in SDS-PAGE.Section II Enzymatic characterization of rG526The recombinant enzyme can catalyze the interconversion between maltose andtrehalose. The ratio of maltose to trehalose at equilibrium point is about3:7. In additionto trehalose, a small amount of glucose (about5%of the total yield) is produced duringthe reaction. The novel enzyme has a higher affinity and a higher catalytic efficiency(Km/kcat) towards maltose than towards trehalose. The optimal pH and temperature ofrG526was pH7.2and37℃, respectively. It can remain over80%of its initial activityafter pre-incubated at temperature below40℃for1h. When the pre-incubationtemperature is over50℃，the residual activity is less than10%of its initial activity.Reagents such as Cu2+and SDS had strong inhibition on the enzymatic activity ofrG526. Adequate concentration of K+was necessary for the enzymatic activity. This enzyme demonstrated the highest activity at20-40mM of K+.Section III Identification of functional important residues and investigation ofpossible catalytic mechanisms of rG526By searching the PDB database, the amino acids sequence was shown to have highidentities with the structural solved protein, trehalulose synthase MutB (PDB:1_ZJA).Several important residues involved in catalysis or substrate binding have already beenidentified in the crystal structure of MutB. By sequence alignment, severalcorresponding residues were also found in G526(D78, Y81, H121, D219, E261, H331and D332). These residues were individually replaced by alanine. The dramaticallydecrease of each mutant enzyme implied that these residues might be important incatalysis or substrate binding. By using isotope-labeled substrate, it was demonstratedthat [2H2] trehalose combined with unlabeled trehalose could not convert to [2H]trehalose or [2H] maltose and no incorporation of [2H7] glucose into maltose ortrehalose happened during the reaction catalyzed by rG526.ConclusionA novel TreS was obtained from marine Pseudomonas in this study. This TreS cancatalyze the interconversion between maltose and trehalose and the equilibrium point istowards production of trehalose indicating its potential application in industry. Severalresidues that might be important in catalysis or substrate binding was also identified inthis study, including: D78, Y81, H121, D219, E261, H331and D332. Isotope-labeledsubstrate experiments demonstrated that this enzyme involved a completelyintramolecular mechanism.