Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an <i>Escherichia coli</i> expression system

Kitazume, Tatsuya; Tanaka, Akinori; Takaya, Naoki; Nakamura, Akira; Matsuyama, Shigeru; Suzuki, Takahisa; Shoun, Hirofumi

doi:10.1046/j.1432-1033.2002.02855.x

Cited by 53 publications

(53 citation statements)

References 30 publications

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“…Substrate inhibition has previously been investigated with CYP505 from Fusarium oxysporum, and inhibition has been reported at concentrations above 0.2 mM of saturated fatty acids, longer than 13 carbons, during in vitro experiments (Kitazume et al 2002). Substrate inhibition of the artificial fusion construct by the substrate dodecanoic acid has been observed in this study, both in vitro and in vivo.…”

Section: Discussionsupporting

confidence: 51%

Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

Lundemo

Notonier

Striedner

et al. 2015

Appl Microbiol Biotechnol

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Cytochrome P450s are interesting biocatalysts due to their ability to hydroxylate non-activated hydrocarbons in a selective manner. However, to date only a few P450-catalyzed processes have been implemented in industry due to the difficulty of developing economically feasible processes. In this study, we have used the CYP153A heme domain from Marinobacter aquaeolei fused to the reductase domain of CYP102A1 from Bacillus megaterium (BM3) expressed in Escherichia coli. This self-sufficient protein chimera CYP153A-CPRBM3 G307A mutant is able to selectively hydroxylate medium and long chain length fatty acids at the terminal position. ω-Hydroxylated fatty acids can be used in the field of high-end polymers and in the cosmetic and fragrance industry. Here, we have identified the limitations for implementation of a whole-cell P450-catalyzed reaction by characterizing the chosen biocatalyst as well as the reaction system. Despite a well-studied whole-cell P450 catalyst, low activity and poor stability of the artificial fusion construct are the main identified limitations to reach sufficient biocatalyst yield (mass of product/mass of biocatalyst) and space-time yield (volumetric productivity) essential for an economically feasible process. Substrate and product inhibition are also challenges that need to be addressed, and the application of solid substrate is shown to be a promising option to improve the process.

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Section: Discussionsupporting

confidence: 51%

Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

Lundemo

Notonier

Striedner

et al. 2015

Appl Microbiol Biotechnol

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“…The heme-domain is fused with a diflavin-reductase domain, similar as for BM3, rendering the enzyme self-sufficient. CYP505A1 from Fusarium oxysporum (P450 foxy ) facilitates the hydroxylation of C6:0 to C16:0 fatty acids at the positions ω-1 to ω-3 (for C10:0 and C12:0 position ω-1 is most favored, for C11:0 position ω-2) and suffers in contrast to BM3 from substrate inhibition with fatty acid substrates longer than C13 [121]. Recently, CYP505A30 from the thermostable Myceliophthora thermophila was characterized as fatty acid hydroxylase.…”

Section: Cyp505mentioning

confidence: 99%

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

2017

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“…It has been demonstrated that the K m and k cat values for hydroxylation by other cytochrome P450s can be extremely variable (15,18). P450 bisd exhibited a low k cat value and appeared to be an ineffective enzyme for the hydroxylation of BPA.…”

mentioning

confidence: 99%

Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1

Sasaki¹,

Akahira²,

Oshiman³

et al. 2005

Appl Environ Microbiol

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In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450 bisd ), ferredoxin (Fd bisd ), and ferredoxin reductase (Red bisd ). We demonstrated that P450 bisd and Fd bisd are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd bisd revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450 bisd , in the presence of Fd bisd , Red bisd , and NADH, was able to convert BPA. The K m and k cat values for BPA degradation were 85 ؎ 4.7 M and 3.9 ؎ 0.04 min ؊1 , respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450 bisd might exhibit strict specificity. Two BPA degradation products of the P450 bisd system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.Bisphenol A (BPA) [2,2-bis(4-hydroxyphenyl)propane] is one of the industrially important compounds for production of polycarbonates, epoxy resins, and other plastics, and worldwide annual consumption of this compound is increasing. However, BPA is strongly suspected to be an endocrine disruptor (7,16,26) and exhibits slight or moderate toxicity in aquatic organisms (39). recently also reported that BPA is immunotoxic and reduces the nonspecific host defense to a level that causes acute toxicity in mice. Furthermore, Colborn and colleagues (4) reported that BPA is widely distributed in rivers, seas, streams, and soils. These reports have proposed that information about the biological conversion of BPA is useful for understanding the fate of this compound in the environment and also for establishing techniques to purge and remove BPA from the environment.Biological transformation of BPA has been reported by many researchers. In mammals, it has been established that BPA is converted to a glucuronide conjugate and a sulfate conjugate in rats (30, 31). In plants, cells of Eucalyptus perriniana and tobacco BY-2 metabolize BPA to its hydroxyl and/or glycosyl products (8, 24). However, it has been proposed that BPA in the environment is decomposed mainly by microorganisms (5, 39). Yim et al. (48) showed that in fungi BPA was metabolized to BPA-O-␤-D-glucopyranoside. Several investigators have also reported that lignin-degrading enzymes, including lignin peroxidase, manganese peroxidase, and/or laccase from basidiomycetes, react with BPA (6,9,34,44,45,46). However, i...

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Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system

Cited by 53 publications

References 30 publications

Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1

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