semi rational mutagenesis

BACKGROUND: Carboxypeptidase is an exopeptidase that hydrolysis amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization. RESULTS: The study has elevated the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, specific activity was 2...

Chitooligosaccharides (COS) possess versatile functional properties that have found extensive applications across various fields. Chitosanase can specifically hydrolyze β-1,4 glycosidic bonds in chitosan to produce COS. In this study, Csn-PD, a glycoside hydrolase family 46 chitosanase from Paenibacillus dendritiformis, which produces (GlcN)2 as its main product, was rationally redesigned aiming to improve its catalytic performance. Based on the results of molecular docking analysis and multiple sequence alignment, four amino acid residues in Csn-PD (I101, T120, T220, and Y259) were pinpointed for targeted mutations...

Phenylalanine ammonia-lyase (PAL) has various applications in fine chemical manufacturing and the pharmaceutical industry. In particular, PAL derived from Anabaena variabilis (AvPAL) is used as a therapeutic agent to the treat phenylketonuria in clinical settings. In this study, we aligned the amino acid sequences of AvPAL and PAL derived from Nostoc punctiforme (NpPAL) to obtain several mutants with enhanced activity, expression yield, and thermal stability via amino acid substitution and saturation mutagenesis at the N-terminal position...

As a key synthetic intermediate of the cardiovascular drug diltiazem, methyl (2R,3S)-3-(4-methoxyphenyl) glycidate ((2R,3S)-MPGM) (1) is accessible via the ring closure of chlorohydrin (3S)-methyl 2-chloro-3-hydroxy-3-(4-methoxyphenyl)propanoate ((3S)-2). We report the efficient reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (3) to (3S)-2 using an engineered enzyme SSCRM2 possessing 4.5-fold improved specific activity, which was obtained through the structure-guided site-saturation mutagenesis of the ketoreductase SSCR by reliving steric hindrance and undesired interactions...

Adenosine 5'-triphosphate (ATP) is a substrate for the biological synthesis of various valuable compounds, such as pharmaceuticals and agrochemicals. To make these biocatalytic processes more economically valuable, several ATP regeneration systems comprising enzymes and phosphate donors were developed. Due to their ability to synthesize ATP from both 5'-diphosphate and adenosine 5'-monophosphate, polyphosphate kinase 2 class III (PPK2-III) enzymes such as Cytophaga hutchinsonii PPK ( Ch PPK) have attracted considerable attention in the construction of ATP regeneration systems...

Tyrosol (2-(4-hydroxyphenyl) ethanol) is extensively used in the pharmaceutical industry as an important natural product from plants. In previous research, we constructed a recombinant Escherichia coli strain capable of de novo synthesis of tyrosol by integrating the phenylpyruvate decarboxylase ARO10 derived from Saccharomyces cerevisiae. Nevertheless, the insufficient catalytic efficiency of ARO10 required the insertion of multiple gene copies into the genome to attain enhanced tyrosol production. In this study, we constructed a mutation library of ARO10 based on a computer-aided semi-rational design strategy and developed a high-throughput screening method for selecting high-yield tyrosol mutants by introducing the heterologous hydroxylase complex HpaBC...

Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports...

As an important dietary supplement, S-adenosylmethionine (SAM) is currently synthesized by methionine adenosyltransferase (MAT) using ATP and methionine as substrates. However, the activity of MAT is severely inhibited by product inhibition, which limits the industrial production of SAM. Here, MAT from Bacteroides fragilis (BfMAT), exhibiting relatively low product inhibition and moderate specific activity, was identified by gene mining. Based on molecular docking, residues within 5 Å of ATP in BfMAT were subjected to mutagenesis for enhanced catalytic activity...

Proteases have been widely employed in many industrial processes. In this work, we aimed to improve the thermostability of the serine protease PB92 from Bacillus alcalophilus to meet the high-temperature requirements of biotechnological treatments. Eight mutation sites (N18, S97-S101, E110, and R143) were identified, and 21 mutants were constructed from B-factor comparison and multiple sequence alignment and expressed via Bacillus subtilis . Among them, fifteen mutants exhibited increased half-life (t1/2 ) values at 65 °C (1...

Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme...

Chitin deacetylase (CDA) catalyzing the deacetylation of crystal chitin is a crucial step in the biosynthesis of chitosan, and also a scientific problem to be solved, which restricts the high-value utilization of chitin resources. This study aims to improve the catalytic efficiency of AsCDA from Acinetobacter schindleri MCDA01 by a semi-rational design using alanine scanning mutagenesis and saturation mutagenesis. The quadruple mutant M11 displayed a 2.31 and 1.73-fold improvement in kcat/Km and specific activity over AsCDA, which can remove 68 % of the acetyl groups from α-chitin...

Regio- and stereo-selective hydroxylation of bile acids is a valuable reaction but often lacks suitable catalysts. In the research, semi-rational design in protein engineering techniques had been applied on cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, and a mutation library had been set up for the 1β-hydroxylation of lithocholic acid (LCA) to produce 1β-OH-LCA. After four rounds of mutagenesis, a key residue at W72 was identified to regulate the regio- and stereo-selectivity at C1 of LCA...

Diterpene synthase VenA is responsible for assembling venezuelaene A with a unique 5-5-6-7 tetracyclic skeleton from geranylgeranyl pyrophosphate. VenA also demonstrates substrate promiscuity by accepting geranyl pyrophosphate and farnesyl pyrophosphate as alternative substrates. Herein, we report the crystal structures of VenA in both apo form and holo form in complex with a trinuclear magnesium cluster and pyrophosphate group. Functional and structural investigations on the atypical 115 DSFVSD120 motif of VenA, versus the canonical Asp-rich motif of DDXX(X)D/E, reveal that the absent second Asp of canonical motif is functionally replaced by Ser116 and Gln83, together with bioinformatics analysis identifying a hidden subclass of type I microbial terpene synthases...

D-pantolactone (D-PL) is one of the important chiral intermediates in the synthesis of D-pantothenic acid. Our previous study has revealed that ketopantolactone (KPL) reductase in Saccharomyces cerevisiae (SceCPR) could asymmetrically reduce KPL to D-PL with a relatively weak activity. In this study, engineering of SceCPR was performed using a semi-rational design to enhance its catalytic activity. Based on the computer-aided design including phylogenetic analysis and molecular dynamics simulation, Ser158, Asn159, Gln180, Tyr208, Tyr298 and Trp299 were identified as the potential sites...

Orthogonal translation is an efficient tool that provides many valuable spectral probes capable of covering different parts of the electromagnetic spectrum and thus enabling parameterization of various structural and dynamic phenomena in proteins. In this context, nitrile-containing tryptophan analogs are very useful probes to study local electrostatics and hydrogen bonding in both rigid and dynamic environments. Here, we report a semi-rational approach to engineer a tyrosyl-tRNA synthetase (TyrRS) variant of Methanocaldococcus jannaschii capable of incorporating 5-cyanotryptophan (5CNW) via orthogonal translation...

The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has becoming increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1-ium (BANA+). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis...

In this study, engineered imine reductases (IREDs) of IRED M5, originally from Actinoalloteichus hymeniacidonis , were obtained through structure-guided semi-rational design. By focusing on mutagenesis of the residues that directly interact with the ketone donor moiety, we identified two residues W234 and F260, playing essential roles in enhancing and reversing the stereoselectivity, respectively. Moreover, two completely enantio-complementary variants S241L/F260N ( R -selectivity up to 99%) and I149D/W234I ( S -selectivity up to 99%) were achieved...

A higher specific activity of microbial transglutaminase (mTGase) is desirable for a broad range of applications ranging from food industry to biotechnology. Three-dimensional docking simulation of mTGase revealed that residues V65, W69, and Y75 were critical for substrate recognition. A semi-rational mutagenesis approach was applied to each residue to generate three separate mini mutant libraries. A high-throughput screening process identified five mutants that demonstrated improved specific activities than the wild type (WT) mTGase were isolated from the Y75 mini mutant library...

Cytochrome P450 enzymes play important roles in the biosynthesis of macrolide antibiotics by mediating a vast variety of regio- and stereoselective oxidative modifications, thus improving their chemical diversity, biological activities, and pharmaceutical properties. Tremendous efforts have been made on engineering the reactivity and selectivity of these useful biocatalysts. However, the 20 proteinogenic amino acids cannot always satisfy the requirement of site-directed/random mutagenesis and rational protein design of P450 enzymes...

Glycosynthases are mutant glycosyl hydrolases that can synthesize glycosidic bonds between acceptor glycone/aglycone groups and activated donor sugars with suitable leaving groups (e.g., azido, fluoro). However, it has been challenging to rapidly detect glycosynthase reaction products involving azido sugars as donor sugars. This has limited our ability to apply rational engineering and directed evolution methods to rapidly screen for improved glycosynthases that are capable of synthesizing bespoke glycans. Here, we outline our recently developed screening methodologies for rapidly detecting glycosynthase activity using a model fucosynthase enzyme engineered to be active on fucosyl azide as donor sugar...