Site Saturation Mutagenesis Applications onCandida methylicaFormate Dehydrogenase

In NADH regeneration,Candida methylicaformate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependentcmFDH from NAD+to NADP+and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+to5.6×104-fold and5×104-fold value, respectively. To increase the thermostability ofcmFDH, the conserved residue at position 1 in the catalytic domain ofcmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties ofcmFDH.

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Alteration of the coenzyme specificity of formate dehydrogenase from Candida methylica by site saturation mutagenesis

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2011.05.252 ◽

2011 ◽

Vol 22 ◽

pp. S83-S84

Author(s):

Gülşah Özgün ◽

Emel Ordu ◽

Emrah Yelboğa ◽

Nevin Gül Karagüler

Keyword(s):

Formate Dehydrogenase ◽

Saturation Mutagenesis ◽

Coenzyme Specificity ◽

Site Saturation

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Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties

Biomolecules ◽

10.3390/biom10060838 ◽

2020 ◽

Vol 10 (6) ◽

pp. 838 ◽

Cited By ~ 1

Author(s):

Raluca Bianca Tomoiagă ◽

Souad Diana Tork ◽

Ilka Horváth ◽

Alina Filip ◽

Levente Csaba Nagy ◽

...

Keyword(s):

Rational Design ◽

Stereoselective Synthesis ◽

Catalytic Properties ◽

Catalytic Efficiency ◽

High Specificity ◽

Saturation Mutagenesis ◽

Petroselinum Crispum ◽

Natural Substrate ◽

Whole Cells ◽

Pal Activity

Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of Petroselinum crispum PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify PcPAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V PcPAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, p-MeO-phenylalanine. Among the hits, besides the known I460V PcPAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-PcPAL in terms of catalytic efficiency within the reaction of p-MeO-Phe. Moreover, I460T PcPAL maintained the high specificity constant of the wild-type enzyme for the natural substrate, l-Phe. Molecular docking supported the favorable substrate orientation of p-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V PcPAL (PDB ID: 6RGS).

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An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase

Enzyme Research ◽

10.4061/2011/595793 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Joanna Griffin ◽

Paul C. Engel

Keyword(s):

Positive Charge ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Coenzyme Specificity ◽

E Coli ◽

Coenzyme Binding ◽

The Core ◽

Clostridium Symbiosum ◽

Key Residues

Sequence and structure comparisons of various glutamate dehydrogenases (GDH) and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination. We have mutated key residues in Clostridium symbiosum NAD+-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH. Comparisons with E. coli NADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purified and assessed at pH 6.0, 7.0, and 8.0. They showed markedly increased catalytic efficiency with NADPH, especially at pH 8.0 (∼170-fold for P262S and F238S/P262S with relatively small changes for NADH). A positive charge introduced through the D263K mutation also greatly increased catalytic efficiency with NADPH (over 100-fold at pH 8) and slightly decreased activity with NADH. At position 242, “P6” of the “core fingerprint,” where NAD+- and NADP+-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity.

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Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

Bioresources and Bioprocessing ◽

10.1186/s40643-019-0267-3 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Hui-Hui Su ◽

Fei Peng ◽

Pei Xu ◽

Xiao-Ling Wu ◽

Min-Hua Zong ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Rational Design ◽

Glucuronic Acid ◽

Specific Activity ◽

Value Added ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Glucaric Acid ◽

Triple Mutant ◽

Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

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Optimization and Engineering of a Self-Sufficient CYP102 Enzyme from Bacillus amyloliquefaciens towards Synthesis of In-Chain Hydroxy Fatty Acids

Catalysts ◽

10.3390/catal11060665 ◽

2021 ◽

Vol 11 (6) ◽

pp. 665

Author(s):

Li Zong ◽

Yan Zhang ◽

Zhengkang Shao ◽

Yingwu Wang ◽

Zheng Guo ◽

...

Keyword(s):

Fatty Acids ◽

Palmitic Acid ◽

Bacillus Amyloliquefaciens ◽

Catalytic Domain ◽

Hydroxy Fatty Acids ◽

Saturation Mutagenesis ◽

Long Chain Fatty Acids ◽

Medical Field ◽

Biotransformation Process ◽

Total Product

Cytochrome P450 (CYP) mediated enzymatic hydroxylation of fatty acids present a green alternative to chemical synthesis of hydroxy fatty acids (HFAs), which are high-value oleochemicals with various uses in materials industry and medical field. Although many CYPs require the presence of additional reductase proteins for catalytic activity, self-sufficient CYPs have their reductase partner naturally fused into their catalytic domain, leading to a greatly simplified biotransformation process. A recently discovered self-sufficient CYP, BAMF2522 from Bacillus amyloliquefaciens DSM 7, exhibits novel regioselectivity by hydroxylating in-chain positions of palmitic acid generating ω-1 to ω-7 HFAs, a rare regiodiversity profile among CYPs. Besides, F89I mutant of BAMF2522 expanded hydroxylation up to ω-9 position of palmitic acid. Here, we further characterize this enzyme by determining optimum temperature and pH as well as thermal stability. Moreover, using extensive site-directed and site-saturation mutagenesis, we obtained BAMF2522 variants that demonstrate greatly increased regioselectivity for in-chain positions (ω-4 to ω-9) of various medium to long chain fatty acids. Remarkably, when a six-residue mutant was reacted with palmitic acid, 84% of total product content was the sum of ω-7, ω-8 and ω-9 HFA products, the highest in-chain selectivity observed to date with a self-sufficient CYP. In short, our study demonstrates the potential of a recently identified CYP and its mutants for green and sustainable production of a variety of in-chain hydroxy enriched HFAs.

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Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+

FEBS Journal ◽

10.1111/j.1742-4658.2008.06533.x ◽

2008 ◽

Vol 275 (15) ◽

pp. 3859-3869 ◽

Cited By ~ 54

Author(s):

Aggeliki Andreadeli ◽

Dimitris Platis ◽

Vladimir Tishkov ◽

Vladimir Popov ◽

Nikolaos E. Labrou

Keyword(s):

Formate Dehydrogenase ◽

Saturation Mutagenesis ◽

Efficient Utilization ◽

Coenzyme Specificity

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Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library

Bioscience Biotechnology and Biochemistry ◽

10.1080/09168451.2018.1495550 ◽

2018 ◽

Vol 82 (10) ◽

pp. 1715-1723 ◽

Cited By ~ 3

Author(s):

Kota Nakatani ◽

Yuta Katano ◽

Kenji Kojima ◽

Teisuke Takita ◽

Rie Yatsunami ◽

...

Keyword(s):

Saturation Mutagenesis ◽

Bacillus Sp ◽

Site Saturation ◽

A Site

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Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

Biomolecules ◽

10.3390/biom11121882 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1882

Author(s):

Wei Xia ◽

Yingguo Bai ◽

Pengjun Shi

Keyword(s):

Hydrogen Bonding ◽

Rational Design ◽

Substrate Binding ◽

Enzymatic Saccharification ◽

Catalytic Efficiency ◽

Binding Pocket ◽

Glycoside Hydrolases ◽

Cellulosic Biomass ◽

Substrate Affinity ◽

Hydrogen Bonding Networks

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

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Biosynthesis, purification, characterization and immobilization of laccase from Lithothelium sp.

Chemija ◽

10.6001/chemija.v31i3.4292 ◽

2020 ◽

Vol 31 (3) ◽

Author(s):

Ingrida Radveikienė ◽

Ingrida Pilotaitė ◽

Rimgailė Dainytė ◽

Regina Vidžiūnaitė

Keyword(s):

Residual Activity ◽

Catalytic Efficiency ◽

Hydrophobic Interaction Chromatography ◽

Metal Salts ◽

Biotechnological Applications ◽

Affinity Constants ◽

Sds Page ◽

Wide Range ◽

Optimum Ph ◽

Sulphate Salts

Novel fungal laccase isoenzymes (namely L95-1 and L95-2) produced by the Ascomycete Lithothelium sp. isolated from the forest soil were purified. However, only one of them was characterized, because the other isoenzyme lost its activity during purification. Extracellular L95-1 laccase was purified 30-fold using ion-exchange and hydrophobic interaction chromatography, with an overall yield of 88%. The molecular mass of purified L95-1 was estimated to be 85 kDa by SDS-PAGE analysis. L95-1 laccase was stable at temperature 4–22°C and pH 6.0–6.5. The substrate specificity of L95-1 laccase was examined with various compounds. Determined affinity constants (KM) varied in a wide range of 3.7–2020.0 µM, whereas catalytic efficiency constants (kcat/KM) covered a range of 0.008–1.9 µM–1 s–1. The optimum pH for most substrates varied in a range from pH 5.0 to 6.0. Sodium azide and fluoride strongly inhibited L95-1 activity, whereas sulphate salts inhibited weakly. The laccase was immobilized on the Fe3O4 nanoparticles and characterized. Residual activity remained at 20% after ten cycles of ABTS oxidation reaction. The immobilized laccase showed higher tolerance to various metal salts. The properties of L95-1 laccase make it potentially useful in the biotechnological applications.

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