Functional expression in Escherichia coli of the tyrosine-inducible tyrosine ammonia-lyase enzyme from yeast Trichosporon cutaneum for production of p-hydroxycinnamic acid

Biochemical and Engineering Sciences, DuPont CR&D, Wilmington, DE 19880, United States
Enzyme and Microbial Technology (Impact Factor: 2.97). 09/2007; DOI: 10.1016/j.enzmictec.2007.03.013

ABSTRACT Deamination of aromatic amino acids phenylalanine or tyrosine is accomplished by the phenylalanine/tyrosine ammonia-lyase (PAL/TAL) enzyme. TAL enzyme is of interest since deamination of tyrosine produces p-hydroxycinnamic acid (pHCA), which has potential for a variety of applications. Among nine microorganisms tested for their ability to produce tyrosine-inducible TAL activity, the yeast, Trichosporon cutaneum showed the highest TAL catalytic activity and the lowest PAL/TAL catalytic efficiency ratio (0.8). The enzyme was purified to near homogeneity and its kinetics studied. The native enzyme appears to be a homo-tetramer with a calculated MW of 294 kDa, subunit MW of 73.5 kDa, and a pI of 5.8. When phenylalanine was used as substrate, the Vmax, Kcat and Km were ∼4.0 ± 0.2 μg/min/mg purified enzyme), 588 ± 29 per min and 4.9 ± 0.9 mM, respectively. However, when tyrosine served as the substrate the Vmax and Kcat were 0.59 ± 0.02 μg/min/mg purified enzyme), and 86.7 ± 29 per min, and substrate binding was apparently cooperative (nH ∼ 2.6 ± 0.4), with S0.5 ∼ 0.6 mM. This is the first reported positive cooperativity for a TAL enzyme. Based on the NH2-terminal and partial internal peptide sequences, the cDNA encoding the enzyme was cloned. Sequence analysis of TcTAL showed 56–62% similarity to other fungal PAL/TAL enzymes. High-level expression (∼30% of total soluble protein, based on SDS-PAGE analysis) of the cDNA in Escherichia coli was achieved using the arabinose inducible araB promoter. The recombinant enzyme possessed both PAL and TAL activities, as evident from the presence of both pHCA and CA in the culture medium.

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    ABSTRACT: Microbes constitute important platforms for the biosynthesis of numerous molecules of pharmaceutical interest such as antitumor, anticancer, antiviral, antihypertensive, antiparasitic, antioxidant, immunological agents, and antibiotics as well as hormones, belonging to various chemical families, for instance, terpenoids, alkaloids, polyphenols, polyketides, amines, and proteins. Engineering microbial factories offers rich opportunities for the production of natural products that are too complex for cost-effective chemical synthesis and whose extraction from their originating plants needs the use of many solvents. Recent progresses that have been made since the millennium beginning with metabolic engineering of microorganisms for the biosynthesis of natural products of pharmaceutical significance will be reviewed.
    BioMed research international. 01/2013; 2013:780145.
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    ABSTRACT: p-Hydroxycinnamic acid (pHCA), which serves as the starting material for production of a number of industrial chemicals, can be produced by deamination of tyrosine by phenylalanine/tyrosine ammonia lyase (PAL/TAL) enzyme. In this study, we characterized the PAL/TAL enzymes from the red yeast Rhodotorula glutinis (RgTAL) and a novel thermostable PAL/TAL from the wood rotting fungus, Phanerochaete chrysosporium (PcTAL). Both enzymes were expressed at ∼50% level of total soluble proteins under the control of the araB promoter. At 25 °C and pH 9.5, the RgTAL enzyme showed kcat and Km values of 0.93 s−1 and 68 μM for tyrosine and 1.5 s−1 and 126 μM for phenylalanine, while these values, for PcTAL, at the same pH and at 35 °C, were 1.3 s−1 and 44 μM for tyrosine and 3.3 s−1 and 161 μM for phenylalanine. The purified PcTAL was thermostable and retained its full activity at 60 °C for up to 3 h, while RgTAL lost most of its activity at this temperature. Thermostability of PcTAL allowed increasing the reaction temperature which, in addition to accelerating the reaction rate, improved solubility of the tyrosine substrate, thus, allowing production of significantly higher amounts of pHCA.
    Enzyme and Microbial Technology 12/2007; 42(1):58-64. · 2.97 Impact Factor
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    ABSTRACT: Trichosporon cutaneum phenylalanine ammonia lyase was selected as a model to investigate the dual substrate activity of this family of enzymes. Sequencing of the PAL gene identified an extensive intron region at the N-terminus. Five amino acid residues differing from a prior report were identified. Highest Phe : Tyr activities (1.6 ± 0.3 : 0.4 ± 0.1 μ mol/h g wet weight) were induced by Tyr. The enzyme has a temperature optimum of 32°C and a pH optimum of 8-8.5 and shows no metal cofactor dependence. Michaelis-Menten kinetics (Phe, K m 5.0 ± 1.1 mM) and positive allostery (Tyr, K' 2.4 ± 0.6 mM, Hill coefficient 1.9 ± 0.5) were observed. Anion exchange chromatography gave a purification fold of 50 with 20% yield. The His-Gln motif (substrate selectivity switch region) indicates the enzyme's ability to act on both substrates.
    Enzyme research. 01/2013; 2013:670702.


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Jun 11, 2014