Regulating substrate preference of phosphatase by reshaping the "cap domain" for multi-enzymatic biosynthesis of high-purity monosaccharides.

Phosphatases are a class of enzymes catalyzing the cleavage of monophosphate ester bonds from the phosphorylated substrates. They have important applications in construction of in vitro multi-enzymatic system for monosaccharides. However, the enzymes generally show substrate ambiguity, which has become a bottleneck for efficient biosynthesis of target products with high purity.

Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2-chloronicotinic acid.

Nitrilases are promising biocatalysts to produce high-value-added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2-chloronicotinonitrile (2-CN), very few robust nitrilases have been reported for efficient production of 2-chloronicotinic acid (2-CA) from 2-CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties.

Engineering of reaction specificity, enantioselectivity, and catalytic activity of nitrilase for highly efficient synthesis of pregabalin precursor.

Simultaneous evolution of multiple enzyme properties remains challenging in protein engineering. A chimeric nitrilase (BaNIT ) with high activity towards isobutylsuccinonitrile (IBSN) was previously constructed for biosynthesis of pregabalin precursor (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA). However, BaNIT also catalyzed the hydration of IBSN to produce by-product (S)-3-cyano-5-methylhexanoic amide.

Constitutive expression of nitrilase from for efficient biosynthesis of 2-chloronicotinic acid.

Unlabelled: 2-chloronicotinic acid (2-CA) is a key precursor for the synthesis of a series of pesticides and pharmaceuticals. Nitrilase-catalyzed bioprocess is a promising method for 2-CA production from 2-chloronicotinonitrile (2-CN). In this study, a mutant of nitrilase from (NIT/W167G) was constitutively overexpressed with as host, which exhibited a onefold increase in enzymatic activity compared with inducible expression.

High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions.

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of l-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction.

Rational Regulation of Reaction Specificity of Nitrilase for Efficient Biosynthesis of 2-Chloronicotinic Acid through a Single Site Mutation.

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for the efficient synthesis of 2-chloronicotinic acid (2-CA). The development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potential. Herein, a nitrilase from Rhodococcus zopfii (NIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN.

Recent advancements in enzyme engineering via site-specific incorporation of unnatural amino acids.

With increased attention to excellent biocatalysts, evolving methods based on nature or unnatural amino acid (UAAs) mutagenesis have become an important part of enzyme engineering. The emergence of powerful method through expanding the genetic code allows to incorporate UAAs with unique chemical functionalities into proteins, endowing proteins with more structural and functional features. To date, over 200 diverse UAAs have been incorporated site-specifically into proteins via this methodology and many of them have been widely exploited in the field of enzyme engineering, making this genetic code expansion approach possible to be a promising tool for modulating the properties of enzymes.

Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase.

Objective: To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase.

Results: The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed.

Synergetic degradation of waste oil by constructed bacterial consortium for rapid in-situ reduction of kitchen waste.

Traditional composting of kitchen waste (KW) is cost- and time-intensive, requiring procedures of collection, transport and composing. Consequently, the direct in-situ reduction of KW via treatment at the point of collection is gaining increasing attention. However, high oil content of KW causes separation and degradation issues due to its low bioavailability and the hydrophobicity, and therefore greatly limiting the direct application of in-situ methods for mass reduction.

TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Members of harbor a number of genes encoding putative aminotransferase class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon The TK1211 gene was expressed in under the control of the strong, constitutive promoter of the cell surface glycoprotein gene TK0895 (P ). The purified protein did not display aminotransferase activity but exhibited racemase activity.

Highly Efficient Chemoenzymatic Synthesis of l-Phosphinothricin from -Phenylacetyl-d,l-phosphinothricin by a Robust Immobilized Amidase.

A chemoenzymatic strategy was developed for the highly efficient synthesis of l-phosphinothricin employing a robust immobilized amidase. An enzymatic hydrolysis of 500 mM -phenylacetyl-d,l-phosphinothricin resulted in 49.9% conversion and 99.

Purification and Biochemical Characterization of a Tyrosine Phenol-lyase from Morganella morganii.

Tyrosine phenol-lyase (TPL) is a valuable and cost-effective biocatalyst for the biosynthesis of L-tyrosine and its derivatives, which are valuable intermediates in the pharmaceutical industry. A TPL from Morganella morganii (Mm-TPL) was overexpressed in Escherichia coli and characterized. Mm-TPL was determined as a homotetramer with molecular weight of 52 kDa per subunit.

Development of a robust nitrilase by fragment swapping and semi-rational design for efficient biosynthesis of pregabalin precursor.

Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi-rational design was developed for the engineering of nitrilase.

Highly regio- and enantioselective synthesis of chiral intermediate for pregabalin using one-pot bienzymatic cascade of nitrilase and amidase.

Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. In this study, a robust nitrilase from Arabis alpina (AaNIT) was screened and engineered. The N258D mutant was obtained with high catalytic activity and excellent enantioselectivity (E > 300) towards IBSN at a high substrate concentration of 100 g L.

Statistical medium optimization and DO-STAT fed-batch fermentation for enhanced production of tyrosine phenol lyase in recombinant Escherichia coli.

Tyrosine phenol lyase (TPL) is a robust biocatalyst for the production of L-dihydroxyphenylalanine (L-DOPA). The improvement of TPL production is conducive to the industrial potential. In this study, the optimization of culture medium of recombinant Escherichia coli harboring TPL from Fusobacterium nucleatum (Fn-TPL) was carried out.

Structure-Based Engineering of Amidase from sp. for Efficient 2-Chloronicotinic Acid Biosynthesis.

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring.

Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor.

To develop a highly efficient method for aprepitant chiral intermediate (S)-4-fluorophenylglycine, a continuous reaction system was established in packed bed bioreactor using amidase covalently immobilized on epoxy resin as biocatalyst. The epoxy resin was firstly modified by metal-chelate method and functional groups (Cu-IDA) generated were able to rapidly adsorb amidases, which were further covalently bound onto the modified resin with 90.1% immobilization yield and 80.

Efficient Biocatalytic Synthesis of Chiral Intermediate of Pregabalin Using Immobilized Lipase.

A mutant L206F/P207F/L259F of lipase (TTL) exhibited high hydrolytic activity towards 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) for synthesis of ()-2-carboxyethyl-3-cyano-5-methylhexanoic acid (-CCMA), a key chiral intermediate of pregabalin. However, low conversion at high CNDE concentration and unreusability of the free TTL mutant restricted its industrial applications. In this study, the TTL mutant was immobilized onto epoxy resin and its catalytic properties for kinetic resolution of CNDE were investigated.

Identification and engineering of the key residues at the crevice-like binding site of lipases responsible for activity and substrate specificity.

Objective: Rational engineering of the crevice-like binding site of lipases for improvement of lipases' catalytic properties.

Resuts: The residues located at the crevice-like binding site of four representative lipases including Thermomyces lanuginosus lipases (TLL and Lip), Rhizopus oryzae lipase (ROL), and Rhizomucor miehei lipase (RML) were identified through structural analysis. The residues at the bottom of the crevice-like binding site recognizing the substrates with short/medium carbon chain length and those located at the right-hand wall of the surface crevice region affecting the product release were changed by site-directed mutagenesis.

An efficient colorimetric high-throughput screening method for synthetic activity of tyrosine phenol-lyase.

Tyrosine phenol-lyase (TPL) naturally catalyzes the reversible β-elimination of l-tyrosine to phenol, pyruvate and ammonium. With its reverse reaction (synthetic activity), l-tyrosine and its derivatives could be synthesized with high atom economy, which are widely used in pharmaceutical industries. In this study, a high-throughput screening method for synthetic activity of TPL was developed.

Efficient chemoenzymatic synthesis of (S)-α-amino-4-fluorobenzeneacetic acid using immobilized penicillin amidase.

An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of racemic N-phenylacetyl-4-fluorophenylglycine. The optimum temperature, pH and agitation rate of the reaction were determined to be 40 °C, 9.5 and 300 rpm, respectively.

Process development for efficient biosynthesis of L-DOPA with recombinant Escherichia coli harboring tyrosine phenol lyase from Fusobacterium nucleatum.

The tyrosine phenol lyase (TPL) catalyzed synthesis of L-DOPA was regarded as one of the most economic route for L-DOPA synthesis. In our previous study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was exploited for efficient biosynthesis of L-DOPA. However, the catalytic efficiency decreased when the reaction system expanded from 100 mL to 1 L.

Rate-limiting steps in the Saccharomyces cerevisiae ergosterol pathway: towards improved ergosta-5,7-dien-3β-ol accumulation by metabolic engineering.

Ergosterol is the predominant nature sterol constituent of plasma membrane in Saccharomyces cerevisiae. Herein, the biosynthetic pathway of ergosterol was proposed to be metabolically engineered for the efficient production of ergosta-5,7-dien-3β-ol, which is the precursor of vitamin D4. By target disruption of erg5, involved in the end-steps of post-squalene formation, predominantly accumulated ergosta-5,7-dien-3β-ol (4.

Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA.

Tyrosine phenol-lyase (TPL) catalyzes the reversible cleavage of l-tyrosine to phenol, pyruvate and ammonia. When pyrocatechol is substituted for phenol, l-dihydroxyphenylalanine (l-DOPA) is produced. The TPL-catalyzed route was regarded as the most economic process for l-DOPA production.

Engineering of Talaromyces thermophilus lipase by altering its crevice-like binding site for highly efficient biocatalytic synthesis of chiral intermediate of Pregablin.

The scissile fatty acid binding site of lipases is divided into different sub-groups and plays an important role in the catalytic properties of the enzymes. In this study, the Talaromyces thermophilus lipase was engineered by altering its crevice-like binding site for efficient synthesis of chiral intermediate of Pregablin through kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE). The substitution of residues located at the crevice-like binding site with phenylalanine (Phe) resulted in significantly increased hydrolysis activity.