Studying the Active Site Pocket ofStaphylococcus hyicusLipase by Site-Directed Mutagenesis

Chang, Rey-Chang; Chen, Jack Chien; Shaw, Jei‐Fu

doi:10.1006/bbrc.1996.1749

Cited by 10 publications

(3 citation statements)

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“…These results are consistent with the observations that mutations at the equivalent positions do not seriously affect the enzymatic activity, unless the size of the amino acid side chain is seriously altered by the mutation [26][27][28], and the lipases from Bacillus species often have an A-X-S-X-G motif, instead of a G-X-S-X-G motif [29 31].…”

Section: Role Of a G-d/e-s-a-g Motifsupporting

confidence: 86%

Identification of catalytically essential residues in Escherichia coli esteraseby site‐directed mutagenesis

et al. 1999

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Section: Role Of a G-d/e-s-a-g Motifsupporting

confidence: 86%

Identification of catalytically essential residues in Escherichia coli esteraseby site‐directed mutagenesis

et al. 1999

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“…Finally, kinetic analyses using p-nitrophenyl butyrate as substrate showed K m , k cat and k cat /K m were 0.90 mM, 25.1 s −1 and 28.2 s −1 mM −1 (Table 5), respectively. In comparison, the K m , k cat and k cat /K m of S. hyicus lipase were 2.07 mM, 0.53 s −1 and 0.257 s −1 mM −1 , respectively (25). The S. epidermidis lipase had much higher substrate-binding affinity and catalytic efficiency than the S. hyicus lipase.…”

Section: Resultsmentioning

confidence: 87%

Site‐directed mutagenesis of a highly active Staphylococcus epidermidis lipase fragment identifies residues essential for catalysis

Chang

Chou

Shaw

2000

J Americ Oil Chem Soc

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A fragment of Staphylococcus epidermidis lipase gene (Lys-303 to Lys-688) was inserted into plasmid pET-20b(+). The resulting C-terminal His-tagged recombinant protein (43 kDa) was overexpressed in Escherichia coli BL21(DE3) as a highly active lipase and was purified with nickel-coupled resin. Putative catalytic sites were determined by site-directed mutagenesis. Mutant enzymes (S418C and H648K) lost enzyme activities, which strongly suggests that the proposed residues of Ser-418 and His-648 are involved in catalysis. Site-directed mutagenesis showed that in comparison with wild-type enzyme, the M419A and V649I enzymes showed a 2.0-and 4.0-fold increase in the k cat /K m , respectively, but the M419l, M419Q, V649A, and V649L variants lost enzyme activities. The wildtype enzyme and the V649I mutant favored the hydrolysis of pnitrophenyl esters of butyrate, but the M419A favored decanoate. The results suggested that the amino acid residues (Met-419 and Val-649), following the catalytic triad, could affect the substrate specificity and/or catalytic efficiency.

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“…The same conditions as above were used, except that 4 mL of hexane was added to the reaction mixtures and the reactions were extended for 48 h at 45 °C. One unit of the lipase activity was defined as the quantity of enzyme that liberates 1 µmol of p-nitrophenyl/min under the routine assay conditions (15). After concentration, the hydrolyzed GPC was isolated by a silica gel TLC and quantitatively analyzed by HPLC in comparison with standard lysophosphatidylcholine as described by Virto and Adlercreutz (16).…”

Section: Methodsmentioning

confidence: 99%

Influence of Acyl Chain Lengths in Mono- and Diacyl-sn-glycerophosphatidylcholine on Gelatinization and Retrogradation of Starch

Siswoyo

Morita

2001

J. Agric. Food Chem.

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The influence of starch with 1- or 2-monoacyl-sn-glycerophosphatidylcholine (GPC) having various chain lengths of fatty acids on gelatinization and retrogradation of starch was studied by the measurement of starch-GPC complex formation, complexing index, and differential scanning calorimetry. The addition of GPC to the starch sample slightly increased the blue value and lambda(max) with increasing chain length of GPC but decreased the phosphorus content and complexing index. The gelatinization onset and peak temperatures of starch complexes increased significantly with increasing chain length, but the enthalpies were statistically lower, except for the treatment with 1,2-distearoyl-sn-GPC when compared with that of the control. Among GPC (di and mono), 1- and 2-monomyristoyl-sn-GPC showed the highest complexing ability, whereas the complexing ability of the GPC decreased with the increasing chain length. According to the Avrami equation, the retrogradation rate (k, day(-1)) of starch was slower than that of the control, whereas the retrogradation rates of 1- and 2-monomyristoyl-sn-GPC were slowest among the GPCs. The positive linear relationship between k and the number of acyl groups of GPC suggests that a GPC with a shorter chain length could retard the retrogradation of starch during storage.

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Studying the Active Site Pocket ofStaphylococcus hyicusLipase by Site-Directed Mutagenesis

Cited by 10 publications

References 0 publications

Identification of catalytically essential residues in Escherichia coli esteraseby site‐directed mutagenesis

Identification of catalytically essential residues in Escherichia coli esteraseby site‐directed mutagenesis

Site‐directed mutagenesis of a highly active Staphylococcus epidermidis lipase fragment identifies residues essential for catalysis

Influence of Acyl Chain Lengths in Mono- and Diacyl-sn-glycerophosphatidylcholine on Gelatinization and Retrogradation of Starch

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