Dataset Information

Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived ?-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside.

ABSTRACT: Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived ?-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM-1 s-1) toward ?-nitrophenyl-?-d-glucopyranoside (?NPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to ?-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel ?-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

SUBMITTER: Yin B

PROVIDER: S-EPMC6940790 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside.

Yin Bangqiao B Hui Qinyan Q Kashif Muhammad M Yu Ran R Chen Si S Ou Qian Q Wu Bo B Jiang Chengjian C

International journal of molecular sciences 20191210 24

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM-1 s-1 ...[more]

PMID: 31835569

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Project description:Thermal stable ?-glucosidases with transglycosylation activity could be applied to the industrial production of oligosaccharides as well as conjugation of sugars to biologically useful materials. Therefore, ?-glucosidases isolated from thermophiles have gained attention over the past decade. In this study, the characterization of a highly thermostable ?-glucosidase and its thermostability improved mutant from newly isolated strain Thermus thermophilus TC11 were investigated.The recombinant ?-glucosidase (TtAG) from Thermus thermophilus TC11 was expressed in Escherichia coli BL21 (DE3) and purified. The purified enzyme had a molecular mass of 184 kDa and consisted of 59-kDa subunits; it showed hydrolytic activity for pNP-?-D-glucopyranoside (pNPG), sucrose, trehalose, panose, and isomaltooligosaccharides and very low activity for maltose. The highest specific activity of 288.96 U/mg was observed for pNPG at 90 °C and pH 5.0; Pb(2+) provided a 20 % activity increase. TtAG was stable at 70 °C for more than 7 h and had a half-life of 195 min at 80 °C and 130 min at 90 °C. Transglycosylation activity was also observed with sucrose and trehalose as substrates. TtAG showed differences on substrate specificity, transglycosylation, multimerization, effects of metal ions and optimal pH from other reported Thermus ?-glucosidases. One single-substitution TtAG mutant Q10Y with improved thermostability was also obtained from random mutagenesis library. The site-saturation mutagenesis and structural modelling analysis indicated that Q10Y substitution stabilized TtAG structure via additional hydrogen bonding and hydrophobic interactions.Our findings indicate that TtAG is a highly thermostable and more acidic ?-glucosidase distinct from other reported Thermus ?-glucosidases. And this work also provides new insights into the catalytic and thermal tolerance mechanisms of ?-glucosidases, which may guide molecular engineering of ?-glucosidase and other thermostable enzymes for industrial application.

Project description:BACKGROUND: The thermostable ?-glucosidase (TnBgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e.g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of TnBgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-?-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3. RESULTS: Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on ?-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as Tm by differential scanning calorimetry (101.9°C for wt), was kept in the mutated variants and significant decrease (?T of 5-10°C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in KM and turnover. The KM-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 × increase for pNPGlc, while the KM decreased a corresponding extent for Q3.Turnover was only significantly changed using pNPGlc, and was decreased 2-3 × in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 × compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in KM for this substrate. CONCLUSION: These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both KM and turnover. An affinity change, leading to a decreased KM, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site.

Dataset Information

Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived ?-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside.

Publications

Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside.

Similar Datasets

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