Lipases from Rhizomucor miehei and Humicola lanuginosa : Modification of the lid covering the active site alters enantioselectivity

• The homologous lipases from Rhizomucor miehei and Humicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed the S- enantiomer of 1-heptyl 2-methyldecanoate (R. miehei: E(S) = 8.5; H. lanuginosa: E(S) = 10.5), but the R-enantiomer of phenyl 2-methyldecanoate (E(R) = 2.9). Chemical arginine specific modification of the R. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E(R) = 2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E(S) = 2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E(S) = 1.9) and increased the enantioselectivity with the aromatic ester (E(R) = 4.4) as substrates. The mutation, Glu 87 Ala, in the lid of the H. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E(S) = 17.4) and a decreased enantioselectivity with the phenyl ester (E(R) = 2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.

Subject headings

NATURVETENSKAP  -- Kemi -- Organisk kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences -- Organic Chemistry (hsv//eng)

Keyword

alanine

arginine

decanoic acid derivative

ester

guanidine

heptanol

phenylglyoxal

triacylglycerol lipase

article

chemical modification

enantiomer

enzyme activation

enzyme active site

enzyme conformation

enzyme substrate

esterification

gas chromatography

hydrolysis

lipid metabolism

nonhuman

site directed mutagenesis

Comparative Study

Cyclohexanones

Decanoates

Enzymes

Immobilized

Kinetics

Lipase

Mitosporic Fungi

Mucorales

Stereoisomerism

Substrate Specificity

Support

Non-U.S. Gov't

Humicola

Rhizomucor miehei

Thermomyces lanuginosus

Publication and Content Type

ref (subject category)

art (subject category)