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Purification and Characterization of Recombinant Porcine Aminoacylase I Expression in Pichia pastoris

Deng Bei

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Abstract

Recombinant porcine Aminoacylase Ⅰ(pACY1) was expressed in Pichia pastoris,and had been charactered.The recombinant strain GS115-pacy1 was constructed.Recombinant pACY1 were purified from its culture after methanol inducing by Phenyl-Sepharose and Q-Sepharose.Then the kinetic parameters of the purified enzymes were determined.The purified enzyme was homogenicity.Its purity was 98.6%.Specific activity(Vmax) and Michaelis constant(Km) of it were 280U/mg and 0.91mM,similar to 287U/mg and 0.84mM of natural porcine enzyme individually.The Tm value of recombinant pACY1 was 74℃,higher than 70℃ of natural enzyme.The activity of recombinant enzyme expressed in Pichia pastoris was similar with natural enzyme,but the heat stablity of former was better than the latter's.It will provide abundant and high-grade enzyme for chiral aimino acid industry.

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What this paper is about

Recombinant porcine Aminoacylase Ⅰ(pACY1) was expressed in Pichia pastoris,and had been charactered.The recombinant strain GS115-pacy1 was constructed.Recombinant pACY1 were purified from its culture after methanol inducing by Phenyl-Sepharose and Q-Sepharose.Then the kinetic parameters of the purified enzymes were determined.The purified enzyme was homogenicity.Its purity was 98.6%.Specific activity(Vmax) and Michaelis constant(Km) of it were 280U/mg and 0.91mM,similar to 287U/mg and 0.84mM of natural porcine enzyme individually.The Tm value of recombinant pACY1 was 74℃,higher than 70℃ of natural enzyme.The activity of recombinant enzyme expressed in Pichia pastoris was similar with natural enzyme,but the heat stablity of former was better than the latter's.It will provide abundant and high-grade enzyme for chiral aimino acid industry.

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Available abstract

Recombinant porcine Aminoacylase Ⅰ(pACY1) was expressed in Pichia pastoris,and had been charactered.The recombinant strain GS115-pacy1 was constructed.Recombinant pACY1 were purified from its culture after methanol inducing by Phenyl-Sepharose and Q-Sepharose.Then the kinetic parameters of the purified enzymes were determined.The purified enzyme was homogenicity.Its purity was 98.6%.Specific activity(Vmax) and Michaelis constant(Km) of it were 280U/mg and 0.91mM,similar to 287U/mg and 0.84mM of natural porcine enzyme individually.The Tm value of recombinant pACY1 was 74℃,higher than 70℃ of natural enzyme.The activity of recombinant enzyme expressed in Pichia pastoris was similar with natural enzyme,but the heat stablity of former was better than the latter's.It will provide abundant and high-grade enzyme for chiral aimino acid industry.

Key concepts: Pichia pastoris, Recombinant DNA, Enzyme, Pichia, Sepharose, Biochemistry, Chemistry, Enzyme assay

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Purification and Characterization of Recombinant Porcine Aminoacylase I Expression in Pichia pastoris — Research Paper | ScholarLens