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Combination of oxyanion Gln114 mutation and medium engineering to influence the enantioselectivity of thermophilic lipase from Geobacillus zalihae.

Wahab RA, Basri M, Rahman MB, Rahman RN, Salleh AB, Leow TC - Int J Mol Sci (2012)

Bottom Line: The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced.Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M.The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.

View Article: PubMed Central - PubMed

Affiliation: Enzyme and Microbial Technology Group, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia; E-Mail: basya@science.upm.edu.my ; Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor 81310, Malaysia.

ABSTRACT
The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.

No MeSH data available.


The location of the oxyanion Gln114 that was selected for site-directed mutagenesis in the catalytic pocket of T1 lipase with respect to the catalytic triad, Ser113, His358 and Asp317.
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f5-ijms-13-11666: The location of the oxyanion Gln114 that was selected for site-directed mutagenesis in the catalytic pocket of T1 lipase with respect to the catalytic triad, Ser113, His358 and Asp317.

Mentions: The oxyanion Q114 in T1 lipase selected for site-directed mutagenesis is located next to the catalytic Ser, and is important in the stabilization of the substrate intermediate during catalysis (Figure 5). Mutations near the active site have been proposed to enhance enantioselectivity more strongly than distant mutations [13]. The differences in enantioselectivities between the lipase variants could have been attributed to the more hydrophobic Leu and Met residues at the as compared to the hydrophilic Gln in T1 lipase. In terms of hydrophobicity, Met (Q114M) is more hydrophobic, followed by Leu (Q114L), and finally Gln (T1 lipase). According to reports, high enantioselectivity in enzymes have been associated with conformational rigidity of enzyme structure [31]. Active sites that are more rigid will result in an enzyme that is more enantioselective, due to formation of a more rigid binding pocket that can permit only one enantiomer to properly enter the active site [32]. With regards to this, Met being the most hydrophobic was expected to reduce the flexibility in the binding pocket in Q114M: hence, enhancing its rigidity. Thus, it might explain the observed moderate improvement in enantioselectivity of the lipase. The proponent that enhanced enantioselectivity in Q114M could be the increased rigidity in its active site. In the case of Q114L, substitution with the hydrophobic Leu clearly did not improve selectivity of the enzyme. Apart from modulation of the rigidity in the binding pocket, replacement of Gln in T1 lipase could have also invoked other substantial changes, such as altering the shape and/or size of the active site [33]. Substitution of the bulky Gln residue with a much smaller Leu residue would logically free up more space at the substrate binding site and alleviate steric hindrance, allowing entrance of both enantiomers of ibuprofen acid. Hence, arrangements of residues in the vicinity of the mutation are disrupted, altering active site topology of Q114L that compromised enantiorecognition or chirality in the active site of the lipase.


Combination of oxyanion Gln114 mutation and medium engineering to influence the enantioselectivity of thermophilic lipase from Geobacillus zalihae.

Wahab RA, Basri M, Rahman MB, Rahman RN, Salleh AB, Leow TC - Int J Mol Sci (2012)

The location of the oxyanion Gln114 that was selected for site-directed mutagenesis in the catalytic pocket of T1 lipase with respect to the catalytic triad, Ser113, His358 and Asp317.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472768&req=5

f5-ijms-13-11666: The location of the oxyanion Gln114 that was selected for site-directed mutagenesis in the catalytic pocket of T1 lipase with respect to the catalytic triad, Ser113, His358 and Asp317.
Mentions: The oxyanion Q114 in T1 lipase selected for site-directed mutagenesis is located next to the catalytic Ser, and is important in the stabilization of the substrate intermediate during catalysis (Figure 5). Mutations near the active site have been proposed to enhance enantioselectivity more strongly than distant mutations [13]. The differences in enantioselectivities between the lipase variants could have been attributed to the more hydrophobic Leu and Met residues at the as compared to the hydrophilic Gln in T1 lipase. In terms of hydrophobicity, Met (Q114M) is more hydrophobic, followed by Leu (Q114L), and finally Gln (T1 lipase). According to reports, high enantioselectivity in enzymes have been associated with conformational rigidity of enzyme structure [31]. Active sites that are more rigid will result in an enzyme that is more enantioselective, due to formation of a more rigid binding pocket that can permit only one enantiomer to properly enter the active site [32]. With regards to this, Met being the most hydrophobic was expected to reduce the flexibility in the binding pocket in Q114M: hence, enhancing its rigidity. Thus, it might explain the observed moderate improvement in enantioselectivity of the lipase. The proponent that enhanced enantioselectivity in Q114M could be the increased rigidity in its active site. In the case of Q114L, substitution with the hydrophobic Leu clearly did not improve selectivity of the enzyme. Apart from modulation of the rigidity in the binding pocket, replacement of Gln in T1 lipase could have also invoked other substantial changes, such as altering the shape and/or size of the active site [33]. Substitution of the bulky Gln residue with a much smaller Leu residue would logically free up more space at the substrate binding site and alleviate steric hindrance, allowing entrance of both enantiomers of ibuprofen acid. Hence, arrangements of residues in the vicinity of the mutation are disrupted, altering active site topology of Q114L that compromised enantiorecognition or chirality in the active site of the lipase.

Bottom Line: The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced.Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M.The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.

View Article: PubMed Central - PubMed

Affiliation: Enzyme and Microbial Technology Group, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia; E-Mail: basya@science.upm.edu.my ; Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor 81310, Malaysia.

ABSTRACT
The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.

No MeSH data available.