Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution

Abstract

The glycoside hydrolase LXYL-P1–2 from Lentinula edodes can specifically hydrolyze 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes for the semi-synthesis of paclitaxel. In order to improve the catalytic properties of the enzyme, we performed error-prone PCR to construct the random mutant library of LXYL-P1–2 and used the methanol-induced plate method to screen the mutants with improved catalytic properties. Two variants, LXYL-P1–2-EP1 (EP1, S91D mutation) and LXYL-P1–2-EP2 (EP2, T368E mutation), were obtained from the library and exhibited 17% and 47% increases in their catalytic efficiencies on 7-β-xylosyl-10-deacetyltaxol. Meanwhile, compared with LXYL-P1–2, EP1 and EP2 showed elevated stabilities in the range of pH ≥ 6 conditions. After treatment at pH 12 for 48 h, EP1 and EP2 retained 77% and 63% activities, respectively, while the wild-type only retained 33% activity under the same condition. Molecular docking results revealed that the S91D mutation led to a shorter distance between the R-chain and the substrate, while the T368E mutation increased negative charge at the surface of the enzyme, and may introduce alterations of the loop near the active pocket, both of which may result in improved stabilities and catalytic activities of enzymes. This study provides a practical directed evolution method for exploring catalytically improved glycoside hydrolase.

Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

“…Moreover, the activities of the purified enzymes were measured through hydrolysis of the chromogenic substrates PNP-Xyl and PNP-Glu, respectively 30 . As shown in Fig.…”

“…Subsequently, the resultant strains ( Fig. 5 A) were further cultivated for 7 days prior to the determination of the volumetric and biomass enzyme ( β -xylosidase) activities described previously 30 . The results indicated that, except for UPC, both PDI and VHB could apparently enhance the volumetric and biomass enzyme activities compared with those of control; typically, VHB had exerted the highest effect on day 7 ( Figure 5 , Figure 7 ).…”

“…The recombinant yeasts harboring the Lxyl-p1 − 1 variants were firstly inoculated in 500-mL shake flasks containing 100 mL buffered minimal glycerol complex medium (BMGY) medium (containing 10 g/L yeast extract, 20 g/L tryptone, 13.4 g/L YNB, 0.4 mg/L biotin, 10 g/L glycerol, 100 mmol/L potassium phosphate buffer, pH 6.0) at 30 °C with shaking at 200 rpm for 48–60 h. Then methanol was added every day to maintain 1% ( v / v ) for the induction of the gene expression. After 7 days of induction, the recombinant enzymes were isolated and purified according to the method described in our previous report 30 .…”

Site-directed Mutagenesis of a β-Glycoside Hydrolase from Lentinula edodes

“…In order to investigate the effect of I368E mutation on the enzyme activity, the recombinant yeast GS115-3.5K-P1−1 I368E was constructed, and its volumetric and biomass enzyme activities were detected as described previously [32]. After induction by methanol for 4 days, the enzyme activities of GS115-3.5K-P1−1 I368E had exceeded those of GS115-3.5K-P1−1 (Figure 1).…”

“…In our previous study, the directed evolution of LXYL-P1−2 had been conducted. From the random mutant library created by error-prone PCR, we obtained a mutant LXYL-P1−2-EP2 (LXYL-P1−2 T368E ) that harbored the T368E mutation, which exhibited a 47% increase in its catalytic efficiency on XDT and elevated stability in the range of pH ≥ 6 compared with LXYL-P1−2 [32]. Recently, we found that the mutant LXYL-P1−1 I368T also exhibited similar β-xylosidase and β-glucosidase activities compared with the high-active LXYL-P1−2, although the activities were lower than those of LXYL-P1−1 A72T , LXYL-P1−1 V91S (the most active single mutant), and the double mutant LXYL-P1−1 A72T /V91S [33].…”