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

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Xiao-Ling Tang ◽  
Jian-Qiang Jin ◽  
Zhe-Ming Wu ◽  
Li-Qun Jin ◽  
Ren-Chao Zheng ◽  
...  
Keyword(s):  
Structure Function ◽  
Pyridine Ring ◽  
Double Mutant ◽  
Specific Activity ◽  
Function Analysis ◽  
Catalytic Efficiency ◽  
Saturation Mutagenesis ◽  
Nucleophilic Attack ◽  
High Catalytic Activity ◽  
Chlorine Substitution

ABSTRACT 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. In this study, an amidase from a Pantoea sp. (Pa-Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with kcat/Km values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 gproduct liter−1 day−1. These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships. IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa-Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa-Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa-Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid.

scholarly journals Active-site engineering of ω-transaminase from Ochrobactrum anthropi for preparation of L-2-aminobutyric acid

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhiwei Zhang ◽  
Yang Liu ◽  
Jing Zhao ◽  
Wenqiang Li ◽  
Ruiwen Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reaction System ◽  
Catalytic Efficiency ◽  
Unnatural Amino Acid ◽  
Aminobutyric Acid ◽  
Saturation Mutagenesis ◽  
High Catalytic Activity ◽  
Pharmaceutical Drugs ◽  
Ochrobactrum Anthropi ◽  
Threonine Deaminase

Abstract Background The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering. Results Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g. Conclusion This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.

scholarly journals Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

2021 ◽  
Vol 1 ◽  
Author(s):  
Eun Young Hong ◽  
Sun-Gu Lee ◽  
Hyungdon Yun ◽  
Byung-Gee Kim
Keyword(s):  
Disulfide Bond ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Arginine Decarboxylase ◽  
Saturation Mutagenesis ◽  
Alkaline Ph ◽  
Wild Type ◽  
Active Site Residues ◽  
Cellular Mechanisms

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5  h).

scholarly journals Exploration of strategies to altering thermal properties of industrial enzymes

2014 ◽  
Vol 70 (a1) ◽  
pp. C435-C435
Author(s):  
Gediminas Baltulionis ◽  
Maeve O'Neill ◽  
Denise Gallagher ◽  
Andrew Ellis ◽  
Dimitrios Charalampapolous ◽  
...  
Keyword(s):  
Thermal Properties ◽  
Elevated Temperatures ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Reaction Rates ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Salt Bridges ◽  
Cold Active Enzymes ◽  
Cold Active

Endoproteases and exopeptidases occupy a pivotal position with respect to their commercial applications in food (e.g. as additives in whey protein processing) and, as additives in detergent, textile and a number of other industries. Food processing at low temperatures by cold-active enzymes has many advantages as it minimises undesirable chemical reactions as well as the risk of microbial contamination. Cold-active enzymes were found to display higher specific activity and catalytic efficiency resulting in lower quantities of enzyme required and significantly shortened processing times. On the other hand, industrial hydrolysis typically occur at elevated temperatures due to the faster reaction rates, increased substrate solubility and thermophilic biocatalysts are required to maintain reactions at very high temperatures. The aim of our work is to exploit structure-function relationships of extremophilic enzymes that give rise to novel industrially useful proteases. We are using the high-throughput capability of the Oxford Protein Purification Facility (OPPF) to study a number of structural modifications leading to protein extremophilic functional behaviour. Several strategies to effectively alter the thermal properties of commercial serine endoproteases and aminopeptidases are being tested including; i) site directed mutagenesis targeted to reduce quantity of prolines, salt bridges, S-S bridges, and hydrophobic clusters, and ii) iterative saturation mutagenesis relying on residues with low B-factors (local rigidity) according to available 3D structures are currently being implemented. Our recent results reveal the potential for an emerging universal mechanism to modify the thermostability of any given enzyme.

scholarly journals Elucidating the degradation pattern of a new cold-tolerant pectate lyase used for efficient preparation of pectin oligosaccharides

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ling Zheng ◽  
Zilong Guo ◽  
Shengsheng Cao ◽  
Benwei Zhu
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
High Specific Activity ◽  
Pectate Lyase ◽  
Catalytic Efficiency ◽  
High Catalytic Activity ◽  
Ionization Mass ◽  
Biotechnological Applications ◽  
Product Analysis ◽  
Cold Tolerant

AbstractThe cold-active pectate lyases have drawn increasing attention in food and biotechnological applications due to their ability to retain high catalytic efficiency under lower temperatures, which could be helpful for energy saving, cost reduction and flavor preservation. Herein, a new cold-tolerant pectate lyase (ErPelPL1) gene from Echinicola rosea was cloned and heterologously expressed in Escherichia coli. Interestingly, ErPelPL1 retained high catalytic activity even at a low temperature (4 °C). ErPelPL1 exhibited optimal activity at 35 ℃, pH 8.0 with 1 mM of Ca2+. It showed high specific activity towards polygalacturonic acid (34.7 U/mg) and sodium polygalacturonate (59.3 U/mg). The combined thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC) and electrospray ionization mass spectrometry (ESI-MS) results indicated that ErPelPL1 endolytically degraded pectic substances into the oligosaccharides with degrees of depolymerization (Dps) of 1–6. In conclusion, this study mainly conducted biochemical characterization and product analysis of a cold-tolerant pectate lyase. Therefore, it provides a promising enzyme candidate for food and biotechnological applications. Graphical Abstract

Sign in / Sign up

Export Citation Format

Share Document