Identification of a Calcium Binding Site in <i>Staphylococcus hyicus</i> Lipase:  Generation of Calcium-Independent Variants

Simons, Jan-Willem F. A.; Kampen, Muriel D. van; Ubarretxena-Belandia, Iban; Cox, Ruud C.; Santos, Cristina M. Alves dos; Egmond, Maarten R.; Verheij, Hubertus M.

doi:10.1021/bi981869l

Cited by 71 publications

(41 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In addition, an extreme C-terminal motif consisting of a negatively charged amino acid followed by four hydrophobic residues, proven necessary for the secretion of metalloprotease PrtG from Erwinia chrysanthemi (15), was found near the C terminus of KB-Lip. We also found multiple GXXGXD motifs, which are supposed to be involved in Ca 2ϩ binding in proteases and lipases (7,16,28).…”

Section: Resultsmentioning

confidence: 74%

Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A

Rashid

Shimada

Ezaki

et al. 2001

Appl Environ Microbiol

158

View full text Add to dashboard Cite

We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at ؊5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca 2؉ . Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca 2؉ -dependent lipase. Addition of Mn 2؉ and Sr 2؉ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca 2؉ . The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C 10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.Lipases (glycerol ester hydrolases) are hydrolases acting on the carboxyl ester bonds present in acylglycerols to liberate fatty acids and glycerols. Lipases are versatile enzymes that are distributed throughout living organisms. A vast number of bacterial lipases with different enzymological properties and substrate specificities have been found (20). They have a wide range of potential applications in the hydrolysis, esterification, and transesterification of triglycerides and in the chiral selective synthesis of esters (17,19

show abstract

Section: Resultsmentioning

confidence: 74%

Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A

Rashid

Shimada

Ezaki

et al. 2001

Appl Environ Microbiol

158

View full text Add to dashboard Cite

show abstract

“…Calcium ions function as ligands to a number of adjacent residues at the active site. The loss of the calcium ion through either pH change or mutation to a residue that affects the calcium interactions has been proposed to disrupt the enzyme structure and decrease its thermal stability, as observed in Staphylococcus hyicus (49). However, the addition of calcium ions neither enhanced nor reduced the activity and/or stability of LipA or LipB.…”

Section: Fig 4 Effect Of Phmentioning

confidence: 99%

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases fromThermosyntropha lipolytica

Salameh

Wiegel

2007

Appl Environ Microbiol

View full text Add to dashboard Cite

Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH 25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH 25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C 18:1 ) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca 2؉ and one Mn 2؉ ion per monomeric enzyme unit. The addition of 1 mM MnCl 2 to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.

show abstract

“…Also, the role of Ca 2+ was thought to 395 stabilize the lipase tertiary structure. It has also been theorized that there is a direct 396 involvement of Ca 2+ in the catalysis for the activation of water molecules; however, this 397 theory is highly unlikely since the calcium-binding site was found to be far from the active 398 site in the Burkholderia glumae lipase crystal structure Noble et al, 1993;399 Rosenstein & Gotz, 2000;Verheij et al, 1980); (Simons et al, 1999 (Gupta et al, 2004).…”

mentioning

confidence: 99%