scholarly journals Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Min Li ◽  
Zhi-Jun Zhang ◽  
Xu-Dong Kong ◽  
Hui-Lei Yu ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Engineering ◽  
Directed Evolution ◽  
Specific Activity ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Carbonyl Reductase ◽  
Triple Mutant ◽  
Engineering Strategy ◽  
Substrate Tolerance

ABSTRACT Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T 50 15, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C 50 15, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter−1 day−1. These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor. IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

scholarly journals Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

2013 ◽  
Vol 79 (20) ◽  
pp. 6429-6438 ◽  
Author(s):  
Haiquan Yang ◽  
Long Liu ◽  
Hyun-dong Shin ◽  
Rachel R. Chen ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Active Site ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
C Terminus ◽  
N Terminus ◽  
Engineering Strategy ◽  
Specific Activities ◽  
Novel Protein

ABSTRACTIn this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) fromAlkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. Thekcat/Kmwas increased from 1.0 × 105liters · mol−1· s−1for AmyK to 30.6 × and 23.2 × 105liters · mol−1· s−1for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.

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

2019 ◽  
Vol 6 (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.

Stereoselective Synthesis of Chiral δ-Lactones via an Engineered Carbonyl Reductase

2021 ◽  
Author(s):  
Tao Wang ◽  
Xiaoyan Zhang ◽  
Yucong Zheng ◽  
Yunpeng Bai
Keyword(s):  
Serratia Marcescens ◽  
Directed Evolution ◽  
Stereoselective Synthesis ◽  
Specific Activity ◽  
Carbonyl Reductase

A carbonyl reductase variant, SmCRM5, from Serratia marcescens was obtained through structure-guided directed evolution. The variant showed improved specific activity (U mg-1) toward most of 16 tested substrates and gave...

scholarly journals Improvements of the productivity and saccharification efficiency of the cellulolytic β-glucosidase D2-BGL in Pichia pastoris via directed evolution

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Mu-Rong Kao ◽  
Su-May Yu ◽  
Tuan-H ua David Ho
Keyword(s):  
Pichia Pastoris ◽  
Directed Evolution ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Single Amino Acid ◽  
Cellulolytic Enzyme ◽  
Glycoside Hydrolase Family ◽  
Wild Type ◽  
Wild Type Enzyme

Abstract Background β-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient β-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass. Results We have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2. Conclusions D2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

scholarly journals Recapitulation of stability diversity of microbial α-amylases

Amylase ◽  
2020 ◽  
Vol 4 (1) ◽  
pp. 11-23
Author(s):  
Dhanya Gangadharan ◽  
Anu Jose ◽  
K. Madhavan Nampoothiri
Keyword(s):  
Genetic Engineering ◽  
Protein Engineering ◽  
Directed Evolution ◽  
High Stability ◽  
Catalytic Efficiency ◽  
Industrial Processes ◽  
Chemical Modifications ◽  
Huge Number ◽  
Standard Methods ◽  
Industrial Demand

Abstractα-Amylases from a huge number of sources have been isolated and characterised but very few of them meet the demands of the industries. The industrial processes take place under conditions hostile to biocatalysts thus increasing the industrial demand for a highly stable enzyme in good titre level. Improved understanding of biomolecular aspects of α-amylases has led to the advanced understanding of their catalytic nature. Enzymes with high stability are obtained from extremophiles. Extensive studies have demonstrated the importance of regulating expression and catalytic efficiency of nonextremophiles through genetic engineering, directed evolution and chemical modifications. The inability to culture most microorganisms in the environment by standard methods has also led to the focus on the development of metagenomics for getting improved biocatalytic functions. The present review aims to compile the studies reported by researchers in manipulating nonextremophiles and improving stability through directed evolution, metagenomics and protein engineering.

scholarly journals Tunnel engineering to accelerate product release for better biomass-degrading abilities in lignocellulolytic enzymes

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhenghui Lu ◽  
Xinzhi Li ◽  
Rui Zhang ◽  
Li Yi ◽  
Yanhe Ma ◽  
...  
Keyword(s):  
Active Sites ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Functional Verification ◽  
Lignocellulolytic Enzymes ◽  
Glycosyl Hydrolases ◽  
Tunnel Engineering ◽  
Deconvolution Analysis ◽  
Engineering Strategy ◽  
Ligands Exchange

Abstract Background For enzymes with buried active sites, transporting substrates/products ligands between active sites and bulk solvent via access tunnels is a key step in the catalytic cycle of these enzymes. Thus, tunnel engineering is becoming a powerful strategy to refine the catalytic properties of these enzymes. The tunnel-like structures have been described in enzymes catalyzing bulky substrates like glycosyl hydrolases, while it is still uncertain whether these structures involved in ligands exchange. Till so far, no studies have been reported on the application of tunnel engineering strategy for optimizing properties of enzymes catalyzing biopolymers. Results In this study, xylanase S7-xyl (PDB: 2UWF) with a deep active cleft was chosen as a study model to evaluate the functionalities of tunnel-like structures on the properties of biopolymer-degrading enzymes. Three tunnel-like structures in S7-xyl were identified and simultaneously reshaped through multi-sites saturated mutagenesis; the most advantageous mutant 254RL1 (V207N/Q238S/W241R) exhibited 340% increase in specific activity compared to S7-xyl. Deconvolution analysis revealed that all three mutations contributed synergistically to the improved activity of 254RL1. Enzymatic characterization showed that larger end products were released in 254RL1, while substrate binding and structural stability were not changed. Dissection of the structural alterations revealed that both the tun_1 and tun_2 in 254RL1 have larger bottleneck radius and shorter length than those of S7-xyl, suggesting that these tunnel-like structures may function as products transportation pathways. Attributed to the improved catalytic efficiency, 254RL1 represents a superior accessory enzyme to enhance the hydrolysis efficiency of cellulase towards different pretreated lignocellulose materials. In addition, tunnel engineering strategy was also successfully applied to improve the catalytic activities of three other xylanases including xylanase NG27-xyl from Bacillus sp. strain NG-27, TSAA1-xyl from Geobacillus sp. TSAA1 and N165-xyl from Bacillus sp. N16-5, with 80%, 20% and 170% increase in specific activity, respectively. Conclusions This study represents a pilot study of engineering and functional verification of tunnel-like structures in enzymes catalyzing biopolymer. The specific activities of four xylanases with buried active sites were successfully improved by tunnel engineering. It is highly likely that tunnel reshaping can be used to engineer better biomass-degrading abilities in other lignocellulolytic enzymes with buried active sites.

Characterization of Alanine Dehydrogenase and Its Effect on Streptomyces coelicolorA3(2) Development in Liquid Culture

2019 ◽  
Vol 29 (1-6) ◽  
pp. 57-65
Author(s):  
Arie Van Wieren ◽  
Ryan Cook ◽  
Sudipta Majumdar
Keyword(s):  
Enzyme Activity ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Lag Phase ◽  
Phase Growth ◽  
Carbon And Nitrogen ◽  
Alanine Dehydrogenase ◽  
Essential Enzyme ◽  
Deamination Reaction

<i>Streptomyces</i>, the most important group of industrial microorganisms, is harvested in liquid cultures for the production of two-thirds of all clinically relevant secondary metabolites. It is demonstrated here that the growth of <i>Streptomyces coelicolor</i> A3(2) is impacted by the deletion of the alanine dehydrogenase (ALD), an essential enzyme that plays a central role in the carbon and nitrogen metabolism. A long lag-phase growth followed by a slow exponential growth of <i>S. coelicolor</i> due to ALD gene deletion was observed in liquid yeast extract mineral salt culture. The slow lag-phase growth was replaced by the normal wild-type like growth by ALD complementation engineering. The ALD enzyme from <i>S. coelicolor</i> was also heterologously cloned and expressed in <i>Escherichia coli</i> for characterization. The optimum enzyme activity for the oxidative deamination reaction was found at 30°C, pH 9.5 with a catalytic efficiency, k<sub>cat</sub>/K<sub>M</sub>, of 2.0 ± 0.1 mM<sup>–1</sup> s<sup>–1</sup>. The optimum enzyme activity for the reductive amination reaction was found at 30°C, pH 9.0 with a catalytic efficiency, k<sub>cat</sub>/K<sub>M</sub>, of 1.9 ± 0.1 mM<sup>–1</sup> s<sup>–1</sup>.

scholarly journals „Schiffe Versenken“: Optimierung einer hochaktiven Saccharose-Isomerase

BIOspektrum ◽  
2021 ◽  
Vol 27 (6) ◽  
pp. 654-656
Author(s):  
Patrick Pilak ◽  
Arne Skerra
Keyword(s):  
Active Site ◽  
Food Industry ◽  
Catalytic Efficiency ◽  
Artificial Sweetener ◽  
Board Game ◽  
Triple Mutant ◽  
Serratia Plymuthica ◽  
Product Profile ◽  
Engineering Strategy ◽  
Sucrose Isomerase

AbstractThe sucrose isomerase SmuA from Serratia plymuthica catalyses the production of isomaltulose, an artificial sweetener used in the food industry. To suppress the formation of the hygroscopic by-product trehalulose we applied a semirational protein engineering strategy inspired by the “battleship” board game. After seven cycles of introducing amino acid exchanges around the active site and investigating their influence on the enzymatic product profile we obtained a triple mutant that showed 2.3 times less trehalulose formation but had retained high catalytic efficiency.

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