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Improved transferase/hydrolase ratio through rational design of a family 1 ?-glucosidase from Thermotoga neapolitana.

ABSTRACT: Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable ?-glucosidase from the well-studied glycoside hydrolase family 1 from Thermotoga neapolitana, TnBgl1A, was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three out of four studied mutations increased the hydrolytic reaction rate in an aqueous environment, while none displayed this property in the presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the presence of an organic solvent has a great influence. We could also show that a single amino acid replacement in a less studied part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential yield of alkyl glycoside from 17% to 58%.

SUBMITTER: Lundemo P

PROVIDER: S-EPMC3648050 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Improved transferase/hydrolase ratio through rational design of a family 1 β-glucosidase from Thermotoga neapolitana.

Lundemo Pontus P Adlercreutz Patrick P Karlsson Eva Nordberg EN

Applied and environmental microbiology 20130322 11

Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 from Thermotoga neapolitana, TnBgl1A, ...[more]

PMID: 23524680

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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

Improved transferase/hydrolase ratio through rational design of a family 1 ?-glucosidase from Thermotoga neapolitana.

Publications

Improved transferase/hydrolase ratio through rational design of a family 1 β-glucosidase from Thermotoga neapolitana.

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