Characterization And Directed Evolution Of Trehalase From Marine Bacterium Zunongwangia Sp.

Trehalose is widely distributed in nature and isolated mainly from bacteria, fungi, insects, invertebrates, and plants. Trehalases(EC 3.2.1.28) are the enzymes that specifically hydrolyze trehalose to glucose. Trehalases show potential applications in food additives, design of insecticide, and agriculture with great prospect. In the present study, we targeted a trehalase from marine bacterium Zunongwangia sp., and its enzymatic properties were analyzed. Meanwhile, we improved the catalytic efficiency of trehalase by random mutagenesis. The results were as follows:1.A novel gene, tre Z(1590 bp,G+C 36.98%) encoding an α, α-trehalase of 529 amino acids was cloned from the deep-sea bacterium Zunongwangia sp. and expressed in Escherichia coli BL21(DE3) with an estimated molecular mass of ~ 61 k Da by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 37, with the highest identity(44%) to a trehalase from E. coli among the characterized trehalases. The recombinant Tre Z displayed its optimal activity at 50 oC and p H 6.5. It retained approximately 141 and 147% of its initial activity after 48 h of incubation(25 oC) at p H 7.5 and 8.0, respectively. In addition, it had a salt-tolerance range of 0–5 M Na Cl with a salt optimum at 1 M(136% of its initial activity), more than 100% of its original activity in 0-3.5 M Na Cl, the Tre Z can also retain 55% activity in 5 M Na Cl. It showed that Tre Z was inhibited by Fe3+, Cu2+, Zn2+, ADP, Co2+, EDTA, and ATP, and enhanced by K+, Ni+, Mg2+, Ca2+, and Ba2+. The Km, Vmax and catalytic turnover number(kcat) of wild-type Tre Z were 0.99 mmol/L, 0.176 mmol l-1 min-1 and 263.2s-1, respectively. Mutant C4 showed a 61% decrease in Km and a 65% increase in catalytic turnover number(kcat), and approximately 230% increases in kcat/Km.2. Tre Z with improved catalytic efficiency were obtained using error-prone PCR and a 96-well plate with DNS high-throughout screening system. Three variants B6, C4 and D9 were screened from the mutant library containing 8000 colonies. B6, C4 and D9 exhibited the optimal p H at p H 6.0; the three variants retained more than 60% of the orginal activity after 48 h incubaction over a p H range from 6.0-7.0. The temperature optima for mutants B6 and D9 were 45 oC, downed 5 oC compared with wild type; C4 exhibited optimal temperature at 50 oC, which is same with wild type. Compared with wild type, mutant B6 showed a 56.7% decrease in Km(0.4197 mmol/L) and a 41.3% decrease in catalytic turnover number(kcat)(154.4 1 s-1), and approximately 38.9% increases in kcat/Km; mutant C4 showed a 61.7% decrease in Km(0.3793 mmol/L) and a 64.7% increase in catalytic turnover number(kcat)(433.69 s-1), and approximately 230% increases in kcat/Km; mutant D9 showed a 31% decrease in Km(0.6811 mmol/L) and a 1.5% decrease in catalytic turnover number(kcat)(259.15 s-1), and approximately 43.1% increases in kcat /Km.3. The muatant C4 with improved catalytic efficiency is remarkable, which has two mutation sites: substitution 227(Yâ†'H) and substitution 442(Râ†'G). To research the effect of the two sites in catalytic efficiency, single site mutants about the two sites were constructed by directed mutagenesis. Mutant Y227 H showed a 23.7% decrease in Km(0.7194 mmol/L) and a 13.9% increase in catalytic turnover number(kcat)(299.83 s-1), and approximately 57% increases in kcat/Km; mutant R442 G showed a 56.4% decrease in Km(0.4317 mmol/L) and a 21.5% increase in catalytic turnover number(kcat)(319.88 s-1), and approximately 178% increases in(kcat/Km). The sum of Y227 H and R442 G catalytic efficiency(kcat/Km) increase is about 230%, which is equal to C4 catalytic efficiency(kcat/Km) increase. That indicated the two sites(227,442) both contributed for variant C4 increasing in catalytic efficiency(kcat/Km), it is providing useful information of trehalase protein structure and function.