Enzyme engineering improves catalytic efficiency and enantioselectivity of hydroxynitrile lyase for promiscuous retro-nitroaldolase activity

2022 ◽  
pp. 105594
Author(s):  
Badipatla Vishnu Priya ◽  
D.H. Sreenivasa Rao ◽  
Rubina Gilani ◽  
Surabhi Lata ◽  
Nivedita Rai ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Enzyme Engineering ◽  
Hydroxynitrile Lyase

scholarly journals Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient l-homophenylalanine synthesis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Tao Wu ◽  
Xiaoqing Mu ◽  
Yuyan Xue ◽  
Yan Xu ◽  
Yao Nie
Keyword(s):  
Steric Hindrance ◽  
Degrees Of Freedom ◽  
Glucose Dehydrogenase ◽  
Reaction System ◽  
Docking Simulation ◽  
Catalytic Efficiency ◽  
Space Time ◽  
Enzyme Engineering ◽  
Phenylalanine Dehydrogenase ◽  
Phenylbutyric Acid

Abstract Background Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block l-homophenylalanine (l-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading. Results In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient l-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher kcat value, and accordingly a 12.9-fold higher kcat/Km value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to l-HPA in 210 min, and the specific space–time conversion was 30.9 mmol g−1 L−1 h−1. The substrate loading and specific space–time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones. Conclusions Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of l-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

scholarly journals The heterologous expression and characterization of a novel chitinase with dual catalytic domains discovered from a wetland soil metagenome

2020 ◽  
Author(s):  
Yumei Dai ◽  
Feng Yang ◽  
Xiao Liu ◽  
Hongling Wang ◽  
Zhiying Yan
Keyword(s):  
Target Genes ◽  
Catalytic Domain ◽  
Catalytic Efficiency ◽  
Enzyme Engineering ◽  
Wetland Soil ◽  
Substrate Affinity ◽  
The Novel ◽  
Catalytic Domains ◽  
Hydrolytic Activities ◽  
Metagenomic Approach

Abstract Background: Microbial chitinases have attracted a lot of attention because of their great potential in many applications. Metagenome-based approach obtains the target genes from the environment directly without culturing the microbes and is becoming a powerful tool to discover the novel chitinases.Results: Gene P1724 was found in a chitin-enriched microbial community from a wetland on the Qinghai-Tibetan plateau using the metagenomic approach. The translated protein sequence of P1724 was predicted to have two GH18 catalytic domains and showed very low similarities with any known and hypothetical chitinases. The gene sequence of P1724 , its N-terminal GH18 domain ( P1724nGH18 ), or C-terminal GH18 domain ( P1724cGH18 ) was cloned and expressed in Escherichia coli BL21 (DE3). Using colloid chitin as substrate, these purified recombinant chitinases showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive since they were still active at 4 °C; the activities of three chitinases were decreased with the presence of Cu 2+ and EDTA, but increased with Ba 2+ and Ca 3+ ; all three chitinases showed both chitotiosidase and endochitinase activities, and could hydrolyze chitosan as well. Other than these common characteristics, P1724 and P1724nGH18 shared more similarity in temperature and pH stabilities, NaCl tolerance and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. k cat / K m value (catalytic efficiency) of P1724 was significantly higher than the sum values of P1724nGH18 and P1724cGH18, which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin.Conclusion: Compared to most microbial chitinases that contain only one catalytic domain, P1724 contains two and was firstly discovered by the metagenomic approach. P1724, its N-terminal and C-terminal catalytic domains were heterologously expressed and characterized. These three recombinant chitinases are phylogenetically distant to any chitinases studied so far, have unique hydrolytic mode, high catalytic efficiency and so new promising candidates for chitinases in applications. In addition, this study broadens the knowledge of unknown chitinases in nature and shows a natural strategy for enzyme engineering by adding other catalytic domain(s).

scholarly journals Structure-Guided Steric Hindrance Engineering of Bacillus Badies Phenylalanine Dehydrogenase for Efficient L-Homophenylalanine Synthesis

2021 ◽  
Author(s):  
Tao Wu ◽  
Xiaoqing Mu ◽  
Yuyan Xue ◽  
Yan Xu ◽  
Yao Nie
Keyword(s):  
Steric Hindrance ◽  
Degrees Of Freedom ◽  
Glucose Dehydrogenase ◽  
Reaction System ◽  
Docking Simulation ◽  
Catalytic Efficiency ◽  
Space Time ◽  
Enzyme Engineering ◽  
Phenylalanine Dehydrogenase ◽  
Phenylbutyric Acid

Abstract Background: Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block L-homophenylalanine (L-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading.Results: In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badies to create an enhanced biocatalyst for efficient L-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic activity. Seven superior mutants were acquired, and the optimal triple-site mutant V309G/L306V/V144G showed a 12.9-fold higher kcat/Km value than wild-type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to L-HPA in 210 min, and the specific space-time conversion was 30.9 mmoL·g−1·L−1·h−1. The substrate loading and specific space-time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones.Conclusions: Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of L-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

scholarly journals Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

2019 ◽  
Vol 9 (2) ◽  
pp. 20180069 ◽  
Author(s):  
Mounir Benkoulouche ◽  
Régis Fauré ◽  
Magali Remaud-Siméon ◽  
Claire Moulis ◽  
Isabelle André
Keyword(s):  
Chemical Properties ◽  
Catalytic Efficiency ◽  
Enzyme Engineering ◽  
Catalytic Reactions ◽  
Glycoside Hydrolases ◽  
Controlled Synthesis ◽  
Substrate Specificities ◽  
Physico Chemical ◽  
Synthetic Routes ◽  
Remarkable Progress

Combined with chemical synthesis, the use of glycoenzyme biocatalysts has shown great synthetic potential over recent decades owing to their remarkable versatility in terms of substrates and regio- and stereoselectivity that allow structurally controlled synthesis of carbohydrates and glycoconjugates. Nonetheless, the lack of appropriate enzymatic tools with requisite properties in the natural diversity has hampered extensive exploration of enzyme-based synthetic routes to access relevant bioactive oligosaccharides, such as cell-surface glycans or prebiotics. With the remarkable progress in enzyme engineering, it has become possible to improve catalytic efficiency and physico-chemical properties of enzymes but also considerably extend the repertoire of accessible catalytic reactions and tailor novel substrate specificities. In this review, we intend to give a brief overview of the advantageous use of engineered glycoenzymes, sometimes in combination with chemical steps, for the synthesis of natural bioactive oligosaccharides or their precursors. The focus will be on examples resulting from the three main classes of glycoenzymes specialized in carbohydrate synthesis: glycosyltransferases, glycoside hydrolases and glycoside phosphorylases.

scholarly journals Catalytically Enhanced Cas9 through Directed Protein Evolution

2020 ◽  
Author(s):  
Travis H. Hand ◽  
Mitchell O. Roth ◽  
Chardasia L. Smith ◽  
Emily Shiel ◽  
Kyle N. Klein ◽  
...  
Keyword(s):  
Protein Evolution ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Human Colon ◽  
Enzyme Engineering ◽  
Human Colon Cancer ◽  
Homology Directed Repair ◽  
Short Palindromic Repeat ◽  
Low Efficiency

AbstractThe Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 system has found widespread applications in genome manipulations due to its simplicity and effectiveness. Significant efforts in enzyme engineering have been made to improve the CRISPR-Cas9 systems beyond their natural power with additional functionalities such as DNA modification, transcriptional regulation, and high target selectivity1–10. Relatively less attention, however, has been paid to improving the catalytic efficiency of CRISPR-Cas9. Increased catalytic efficiency may be desired in applications where the currently available CRISPR-Cas9 tools are either ineffective4, 11–14 or of low efficiency such as with type II-C Cas915–18 or in non-mammals19, 20. We describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9). We demonstrate the effectiveness of this method with a previously characterized Type IIC Cas9 from Acidothermus cellulolyticus (AceCas9) with up to 4-fold improvement of in vitro catalytic efficiency, as well as the widely used Streptococcus pyogenes Cas9 (SpyCas9), which showed a 2-fold increase in homology directed repair (HDR)-based gene insertion in human colon cancer cells.

scholarly journals Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Guang Yang ◽  
Hua Yao ◽  
Matteo Mozzicafreddo ◽  
Patrizia Ballarini ◽  
Sandra Pucciarelli ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Content Type ◽  
Cold Adapted ◽  
Wide Range ◽  
Cold Active ◽  
Surface Loops ◽  
The Antarctic

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

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