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

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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Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00973-08 ◽

2008 ◽

Vol 75 (3) ◽

pp. 589-595 ◽

Cited By ~ 150

Author(s):

Tae Seok Moon ◽

Sang-Hwal Yoon ◽

Amanda M. Lanza ◽

Joseph D. Roy-Mayhew ◽

Kristala L. Jones Prather

Keyword(s):

Escherichia Coli ◽

Pseudomonas Syringae ◽

Glucuronic Acid ◽

Value Added ◽

Culture Broth ◽

Glucaric Acid ◽

Synthetic Pathway ◽

Genes Encoding ◽

Microbial System ◽

Rate Limiting

ABSTRACT A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass.

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Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

Applied and Environmental Microbiology ◽

10.1128/aem.07012-11 ◽

2012 ◽

Vol 78 (11) ◽

pp. 3880-3884 ◽

Cited By ~ 17

Author(s):

Yu-Ri Lim ◽

Soo-Jin Yeom ◽

Deok-Kun Oh

Keyword(s):

Specific Activity ◽

Thermus Thermophilus ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Geobacillus Thermodenitrificans ◽

Wild Type Enzyme ◽

Content Type ◽

Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

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Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

10.22541/au.161875932.27635268/v1 ◽

2021 ◽

Author(s):

Kaifang Liu ◽

Xiulai Chen ◽

Yunlu Zhong ◽

Jia Liu ◽

Guipeng Hu ◽

...

Keyword(s):

Catalytic System ◽

Rational Design ◽

Specific Activity ◽

Catalytic Efficiency ◽

Disodium Salt ◽

In Vivo Testing ◽

Dynamics Simulations ◽

Rate Limiting

The compound 3′-phosphoadenosine-5′-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds, such as glycosaminoglycan and oxamniquine. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of ATP (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5′-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided “ADP expulsion” strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme’s flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor. The achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. The proposed strategy will facilitate industrial production of PAPS as well as the engineering of similar enzymes.

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Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications

Biotechnology for Biofuels ◽

10.1186/s13068-021-02052-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Lige Tong ◽

Jie Zheng ◽

Xiao Wang ◽

Xiaolu Wang ◽

Huoqing Huang ◽

...

Keyword(s):

Disulfide Bonds ◽

Specific Activity ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Dynamics Simulation ◽

Optimal Temperature ◽

Correlation Matrices ◽

Catalytic Center ◽

Starch Industry ◽

Temperature Melting

Abstract Background Glucoamylase is an important industrial enzyme in the saccharification of starch into glucose. However, its poor thermostability and low catalytic efficiency limit its industrial saccharification applications. Therefore, improving these properties of glucoamylase is of great significance for saccharification in the starch industry. Results In this study, a novel glucoamylase-encoding gene TlGa15B from the thermophilic fungus Talaromyces leycettanus JCM12802 was cloned and expressed in Pichia pastoris. The optimal temperature and pH of recombinant TlGa15B were 65 ℃ and 4.5, respectively. TlGa15B exhibited excellent thermostability at 60 ℃. To further improve thermostability without losing catalytic efficiency, TlGa15B-GA1 and TlGa15B-GA2 were designed by introducing disulfide bonds and optimizing residual charge–charge interactions in a region distant from the catalytic center. Compared with TlGa15B, mutants showed improved optimal temperature, melting temperature, specific activity, and catalytic efficiency. The mechanism underlying these improvements was elucidated through molecular dynamics simulation and dynamics cross-correlation matrices analysis. Besides, the performance of TlGa15B-GA2 was the same as that of the commercial glucoamylase during saccharification. Conclusions We provide an effective strategy to simultaneously improve both thermostability and catalytic efficiency of glucoamylase. The excellent thermostability and high catalytic efficiency of TlGa15B-GA2 make it a good candidate for industrial saccharification applications.

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Exploration of strategies to altering thermal properties of industrial enzymes

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095643 ◽

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.

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Enzymatic and Mutational Analysis of the PruA Pteridine Reductase Required for Pterin-Dependent Control of Biofilm Formation in Agrobacterium tumefaciens

Journal of Bacteriology ◽

10.1128/jb.00098-20 ◽

2020 ◽

Vol 202 (16) ◽

Author(s):

Monica Labine ◽

Lisa DePledge ◽

Nathan Feirer ◽

Jennifer Greenwich ◽

Clay Fuqua ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Signaling Pathway ◽

Plant Pathogen ◽

Biofilm Formation ◽

Mutational Analysis ◽

Reductase Activity ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Control Surface ◽

Content Type

ABSTRACT Pterins are ubiquitous biomolecules with diverse functions, including roles as cofactors, pigments, and redox mediators. Recently, a novel pterin-dependent signaling pathway that controls biofilm formation was identified in the plant pathogen Agrobacterium tumefaciens. A key player in this pathway is a pteridine reductase, termed PruA, where its enzymatic activity has been shown to control surface attachment and limit biofilm formation. Here, we biochemically characterized PruA to investigate the catalytic properties and the substrate specificity of this pteridine reductase. PruA demonstrated maximal catalytic efficiency with dihydrobiopterin and comparable activities with the stereoisomers dihydromonapterin and dihydroneopterin. Since A. tumefaciens does not synthesize or utilize biopterins, the likely physiological substrate is dihydromonapterin or dihydroneopterin or both. Notably, PruA did not exhibit pteridine reductase activity with dihydrofolate or fully oxidized pterins. Site-directed mutagenesis studies of a conserved tyrosine residue, the key component of a putative catalytic triad, indicated that this tyrosine is not directly involved in PruA catalysis but may be important for substrate or cofactor binding. Additionally, mutagenesis of the arginine residue in the N-terminal TGX3RXG motif significantly reduced the catalytic efficiency of PruA, supporting its proposed role in pterin binding and catalysis. Finally, we report on the enzymatic characterization of PruA homologs from Pseudomonas aeruginosa and Brucella abortus, thus expanding the roles and potential significance of pteridine reductases in diverse bacteria. IMPORTANCE Biofilms are complex multicellular communities that are formed by diverse bacteria. In the plant pathogen Agrobacterium tumefaciens, the transition from a free-living motile state to a nonmotile biofilm state is governed by a novel signaling pathway involving small molecules called pterins. The involvement of pterins in biofilm formation is unexpected and prompts many questions about the molecular details of this pathway. This work biochemically characterized the PruA pteridine reductase involved in the signaling pathway to reveal its enzymatic properties and substrate preference, thus providing important insight into pterin biosynthesis and its role in A. tumefaciens biofilm control. Additionally, the enzymatic characteristics of related pteridine reductases from mammalian pathogens were examined to uncover potential roles of these enzymes in other bacteria.

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Characterization of an Agrobacterium tumefaciensd-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

Applied and Environmental Microbiology ◽

10.1128/aem.72.2.981-985.2006 ◽

2006 ◽

Vol 72 (2) ◽

pp. 981-985 ◽

Cited By ~ 106

Author(s):

Hye-Jung Kim ◽

Eun-Kyung Hyun ◽

Yeong-Su Kim ◽

Yong-Joo Lee ◽

Deok-Kun Oh

Keyword(s):

Escherichia Coli ◽

Agrobacterium Tumefaciens ◽

Molecular Mass ◽

Specific Activity ◽

Catalytic Efficiency ◽

Maximal Activity ◽

Turnover Number ◽

Conversion Yield ◽

Equilibrium Ratio

ABSTRACT The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (k cat) and catalytic efficiency (k cat/Km ) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%.

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Enhancement of a protocol purifying T1 lipase through molecular approach

PeerJ ◽

10.7717/peerj.5833 ◽

2018 ◽

Vol 6 ◽

pp. e5833

Author(s):

Che Haznie Ayu Che Hussian ◽

Raja Noor Zaliha Raja Abd. Rahman ◽

Adam Leow Thean Chor ◽

Abu Bakar Salleh ◽

Mohd Shukuri Mohamad Ali

Keyword(s):

Rational Design ◽

Double Point ◽

Specific Activity ◽

Point Mutations ◽

Fold Increase ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Site Directed Mutagenesis ◽

Glutathione S Transferase ◽

Direct Separation

T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.

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Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

Journal of Bacteriology ◽

10.1128/jb.00586-08 ◽

2008 ◽

Vol 191 (5) ◽

pp. 1565-1573 ◽

Cited By ~ 38

Author(s):

Sang-Hwal Yoon ◽

Tae Seok Moon ◽

Pooya Iranpour ◽

Amanda M. Lanza ◽

Kristala Jones Prather

Keyword(s):

Agrobacterium Tumefaciens ◽

Pseudomonas Syringae ◽

Glucuronic Acid ◽

Glucaric Acid ◽

Michaelis Constant ◽

Turnover Number ◽

Reading Frame ◽

E Coli ◽

Uronate Dehydrogenase

ABSTRACT Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K m ) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k cat = 1.9 × 102 s−1 on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.

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Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

Applied and Environmental Microbiology ◽

10.1128/aem.00603-17 ◽

2017 ◽

Vol 83 (12) ◽

Cited By ~ 11

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.

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