The synthetic rate of dipeptide catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in the presence of water-soluble organic solvents

Ogino, Hiroyasu; Gemba, Yuichi; Yamada, Mitsuharu; Shizuka, Michiko; Yasuda, Masahiro; Ishikawa, Haruo

doi:10.1016/s1369-703x(00)00062-0

Cited by 22 publications

(9 citation statements)

References 6 publications

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“…The increase in activity was only 1.2-fold for CHMO and maximal at 2% MeOH. It has to be noted that activation of enzymes at low co-solvent concentration, is a phenomenon already observed for other enzymes (Kudryashova et al, 2003;Ogino et al, 2000;Olofsson et al, 2006;Park et al, 2005;Tan and Lovrien, 1972). Interestingly, Tó th et al (2010) found a remarkable increase in activity of the flavoenzyme NADH oxidase from Thermus thermophilus in the presence of several alcohols (with MeOH the optimum was at about 40%).…”

Section: Activity and Stability Of Pamo And Chmo In Waterorganic Solvmentioning

confidence: 57%

Effects of water miscible organic solvents on the activity and conformation of the baeyer–villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: A comparative study

Secundo

Fiala

Fraaije

et al. 2010

Biotech & Bioengineering

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A broader exploitation of enzymes in organic synthesis can be achieved by increasing their tolerance toward organic solvents. In this study, the stability and activity of Baeyer-Villiger monooxygenases from Thermobifida fusca (PAMO) and Acinetobacter sp. (CHMO) in the presence of water miscible organic solvents were compared. PAMO was more stable than CHMO. The concentration of solvent (v/v) at which it halved its activity (C(50) ) was 4- to 16-fold higher than that observed for CHMO. For PAMO, the C(50) varied from 16% to 55% of solvent and followed the destabilizing order methanol < ethanol < 1,4-dioxane < acetonitrile < trifluoroethanol. In the case of CHMO, the maximal C(50) was 7% with methanol and even lower with the other solvents. Therefore, methanol was the most tolerated solvent. In the case of PAMO, methanol induced a significant increase of enzyme activity (up to fivefold), which was optimal at 20% (v/v) solvent. Only minor spectral variations were observed with PAMO in 20% methanol, suggesting that the increase of activity observed in this condition is not due to marked conformational changes. Fluorescence and circular dichroism analyses showed that the lower stability of CHMO toward organic solvent correlates with a more pronounced destructive effect on its secondary and tertiary structure. A possible rationale for the higher stability of PAMO could be inferred from inspection of the PAMO and CHMO (two enzymes of similar size) structure, which revealed a higher (up to twofold) number of ionic bridges in PAMO with respect to CHMO.

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Section: Activity and Stability Of Pamo And Chmo In Waterorganic Solvmentioning

confidence: 57%

Effects of water miscible organic solvents on the activity and conformation of the baeyer–villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: A comparative study

Secundo

Fiala

Fraaije

et al. 2010

Biotech & Bioengineering

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“…The half-lives of the PST-01 protease produced by P. aeruginosa PST-01 were very long in the presence of organic solvents. Furthermore, the peptide synthesis catalyzed by the PST-01 protease exhibited high reaction rate and high equilibrium yield (23)(24)(25). The amino acid sequence of the PST-01 protease and the overall three-dimensional structure of the PST-01 protease were very similar to those of thermolysin (26).…”

Section: Introductionmentioning

confidence: 91%

Stabilities and Conformational Transitions of Various Proteases in the Presence of an Organic Solvent

Ogino

Gemba

Yutori

et al. 2007

Biotechnology Progress

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The half-life of the activity of the PST-01 protease that was secreted by organic solvent-tolerant Pseudomonas aeruginosa PST-01 was very long in the presence of methanol as compared to that in the absence of methanol. The conformational transitions of the PST-01 protease, alpha-chymotrypsin, thermolysin, and subtilisin in the presence and absence of methanol were monitored by measuring the CD spectra. The conformational stabilities of the PST-01 protease and subtilisin in the presence of methanol were higher than those in the absence of methanol. This resulted in high stability of these proteases in the presence of methanol. Furthermore, it was suggested that the organic solvent stabilities of enzymes were closely related to the secondary structure by monitoring the conformational transitions of polyamino acids, which form the particular conformations, in the presence and absence of methanol.

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“…L-Leu-NH 2 was prepared from L-leucineamide hydrochloride (L-Leu-NH 2 ÁHCl) from Nacalai Tesque Inc. (Kyoto, Japan) according to the method of Ogino et al (2000). Z-L-Ala-L-Leu-NH 2 was not commercially available, therefore it was synthesized using the carbodiimide (N,NV-dicyclohexylcarbodiimide) (DCC) condensation method described in the Appendix.…”

Section: Enzymementioning

confidence: 99%

“…The product formation was followed by the same method of Ogino et al (2000). Fifty-microliter aliquots of the liquid sample were taken at appropriate time intervals, and each was mixed promptly with 450 AL of carrier liquid (acetonitrile/50 mM sodium phosphate buffer pH 3.0, 80/20 (v/v)) to stop the reaction.…”

Section: Initial Rates Of Synthetic Reaction In the Absence Of Productmentioning

confidence: 99%

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Kinetics and mechanism of a reaction catalyzed by PST‐01 protease from Pseudomonas aeruginosa PST‐01

Bobe

Abdelmoez²,

Ogino³

et al. 2004

Biotech & Bioengineering

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The initial rates of carboxybenzoyl-alanyl-l-leucyl-amide (Z-L-Ala-L-Leu-NH(2)) synthesis from carboxybenzoyl-L-alanine (Z-L-Ala) and L-leucineamide (L-Leu-NH(2)) and Z-L-Ala-L-Leu-NH(2) hydrolysis in a homogeneous dimethyl sulfoxide-aqueous buffer solution [1:1 (v/v)] system catalyzed by PST-01 protease from Pseudomonas aeruginosa were measured under a wide range of Z-L-Ala, L-Leu-NH(2) and Z-L-Ala-L-Leu-NH(2) concentrations. The initial rates of the synthetic reaction, in which Z-L-Ala-L-Leu-NH(2) was produced from Z-L-Ala and L-Leu-NH(2), were inhibited by the substrates. Furthermore, the initial rates of the synthetic reaction were not inhibited by the product Z-L-Ala-L-Leu-NH(2), and those of the hydrolytic reaction were inhibited by Z-L-Ala and L-Leu-NH(2). All the initial rate data of the synthetic and hydrolytic reactions were well correlated with the rate equation derived based on the proposed reaction scheme.

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The synthetic rate of dipeptide catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in the presence of water-soluble organic solvents

Cited by 22 publications

References 6 publications

Effects of water miscible organic solvents on the activity and conformation of the baeyer–villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: A comparative study

Effects of water miscible organic solvents on the activity and conformation of the baeyer–villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: A comparative study

Stabilities and Conformational Transitions of Various Proteases in the Presence of an Organic Solvent

Kinetics and mechanism of a reaction catalyzed by PST‐01 protease from Pseudomonas aeruginosa PST‐01

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