DOCSLIB.ORG

Enhancing the Thermostability and Activity of Uronate Dehydrogenase from Agrobacterium Tumefaciens LBA4404 by Semi-Rational Engi

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Enhancing the Thermostability and Activity of Uronate Dehydrogenase from Agrobacterium Tumefaciens LBA4404 by Semi-Rational Engi Su et al. Bioresour. Bioprocess. (2019) 6:36 https://doi.org/10.1186/s40643-019-0267-3 RESEARCH Open Access Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering Hui‑Hui Su1†, Fei Peng1†, Pei Xu1, Xiao‑Ling Wu1, Min‑Hua Zong1, Ji‑Guo Yang2 and Wen‑Yong Lou1* 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, efcient and economical than chemical synthesis. Uronate dehydro‑ genases (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 diferent 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. tume- 1 1 faciens (At) LBA4404 had a better catalytic efciency than the other two UDHs (800 vs 600 and 530 s− mM− ). To fur‑ ther 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 10 5 °C improvement in T50 value and a 2.5‑fold improvement in specifc 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. Keywords: Uronate dehydrogenase, Semi‑rational engineering, Biocatalysis, Glucuronic acid, Glucaric acid Background and Hollmann 1976). Subsequently, UDHs were also Uronate dehydrogenase (EC 1.1.1.203, UDH), an cloned and characterized from other organisms, includ- NAD+-like oxidoreductase, can convert uronic acids ing Pseudomonas putida KT2440, Fulvimarina pelagi [e.g., glucuronic acid (GlcA)] into aldaric acids [e.g., glu- HTCC2506, Oceanicola granulosus DSM15982, Strep- caric acid (GA)] (Fig. 1). UDH was frst found in Agrobac- tomyces viridochromeogenes DSM40736, Polaromonas terium tumefaciens strain C58 and Pseudomonas syringae naphthalenivoransCJ2 and Termo bispora DSM43833 pv. tomato strain DC3000 (Zajic et al. 1959; Wagner (Moon et al. 2009; Pick et al. 2015; Wagschal et al. 2015; Li et al. 2018). *Correspondence: [email protected] Recently, UDH has attracted considerable attention as †Hui‑Hui Su and Fei Peng contributed equally to this work the key enzyme for GA and galactarate production from 1 Laboratory of Applied Biocatalysis, School of Food Science uronic acids. GA was identifed as a “top value-added and Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China chemical from biomass” by the US Department of Energy Full list of author information is available at the end of the article in 2004 (Werpy et al. 2004) and shows considerable © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Su et al. Bioresour. Bioprocess. (2019) 6:36 Page 2 of 9 potential in the food and pharmaceutical industries, with Materials and methods applications as a food additive, dietary supplement, ther- Bacterial strains and plasmids apeutic medicine, agent builder and polyamide derivative Tree A. tumefaciens strains, A. tumefaciens LBA4404 (Dwivedi et al. 1990; Bespalov and Aleksandrov 2012; (AtLBA4404), A. tumefaciens GV3101 (AtGV3101) Walaszek 1990; Zółtaszek et al. 2008; Morton and Kiely and A. tumefaciens EHA105 (AtEHA105), were used as 2000). Of all the characterized UDHs, the enzyme from genomic templates. Te above strains were donated by A. tumefaciens strain C58 shows the highest catalytic ef- Prof. Li from Guangdong Academy of Agricultural Sci- ciency (829 s−1 mM−1) with GlcA as substrate. However, ences (Guangdong, China). Te engineered Escherichia T50 its 50 value is only 37 °C (Pick et al. 2015). Subsequently, coli strains containing the target genes were cultured Roth et al. achieved a more thermostable triple variant in Luria–Bertani (LB) broth with antibiotics (50 μg/ T15 with a 50 value of 62 °C and a ∆∆GU of 2.3 kJ/mol com- ml kanamycin) at 37 °C. E. coli DH5α (Trans Gen Bio- pared to wild-type (Roth et al. 2017). However, this vari- tech, China) and E. coli BL21 (DE3) (Trans Gen Biotech, ant still does not meet the requirements of industry. China) were used as the cloning host and expression In line with the hypothesis that conformational host, respectively. Te plasmid pET-28a(+) (Jierui, changes in the catalytic center during heat treatment China) was employed as the template for overexpression afect enzyme activity, researchers have attempted to of the UDHs. improve the thermostability of various enzymes by increasing the stability of the catalytic center using a Expression and purifcation of recombinant proteins structure-guided enzyme engineering approach (Tanner Te preparation of genomic DNA and general cloning et al. 1996; Blair et al. 2007; Etzl et al. 2018; Zhang et al. steps were performed as previously described (Zhu et al. 2016). Tus, the thermostability of α-keto acid decarbox- 2014). We confrmed that the candidate genes of the ylase, measured as the temperature at which 50% activity three A. tumefaciens strains contained conserved UDH T1h remains after a 1-h incubation, 50 , improved by 3.3 °C domain sequences by PCR and sequencing. Te prim- compared to wild-type after structure-guided engineer- ers used to amplify the conserved domain sequences ing of the catalytic center (Sutiono et al. 2018). Surface from their genomic DNA are shown in Additional fle 1: engineering can also increase enzyme thermostability by Table S1. Te three target UDH genes were digested with altering hydrogen bonds, salt bridges and hydrophobic restriction enzymes HindIII and XhoI and then ligated interactions (Alponti et al. 2016; Eijsink et al. 2004; Yang into the corresponding restriction sites of pET-28a(+). et al. 2007). For example, the half-life and Tm of alkaline Te resulting constructs (pLBA-UDH, pGV-UDH and α-amylase from Alkalimonas amylolytica were raised by pEHA-UDH) were transformed into E. coli DH5α and 6.4-fold and 5.4 °C, respectively, after introducing multi- positive cells were confrmed by gene sequencing. ple arginines on the protein surface (Deng et al. 2014). Escherichia coli BL21 (DE3) carrying recombinant In this work, the enzyme characteristics of three UDHs plasmids was frst incubated overnight in LB medium from three A. tumefaciens strains were investigated. We containing kanamycin (50 μg/ml) and shaken at 37 °C attempted to enhance the thermostability and activity of and 200 rpm. Ten, 1% (v/v) of these cultures were used one particular example (AtLBA4404 UDH) by generating to seed fresh LB broth medium with kanamycin (50 μg/ small and smart enzyme libraries through semi-rational ml) and cultured at 37 °C and 200 rpm. When the OD600 engineering. Analysis of the structure of the most prom- reached 0.6–0.8, isopropyl β-d-1-thiogalactopyranoside ising variants showed that the mutated positions were (IPTG, fnal concentration 0.2 mM) was added to trig- mainly located in the fexible loop near the catalytic ger the expression of the target genes. After expression at center. 20 °C for 20–24 h, the cells were harvested at 8000×g for Fig. 1 Uronate dehydrogenase catalyzes a dehydrogenation reaction at C1, 6 position of glucuronic acid Su et al. Bioresour. Bioprocess. (2019) 6:36 Page 3 of 9 Site‑directed mutagenesis of A. tumefaciens LBA4404 10 min and resuspended in lysis bufer (20 mM Na2HPO4 (pH 7.4), 500 mM NaCl and 20 mM imidazole). Next, Te UDH from AtLBA4404 was selected for further cells were sonicated and the cell homogenates were molecular modifcation because it showed the highest −1 −1 centrifuged. Te supernatants were fltered and loaded kcat/Km (800 s mM ) of the three UDHs. A model of onto a nickel column (Ni–NTA, Bio-Rad laboratories, the UDH from AtLBA4404 was constructed by homology Hercules, CA) using the manufacturer’s instructions for modeling using Swiss-model (http://swiss model .expas purifcation. y.org/) (Biasini et al. 2014) with the crystal structure of UDH from AtC58 (PDB code 3RFT) as the template Enzyme activity assay (Parkkinen et al. 2011). Deep View (http://spdbv .wital -it. UDH activity was measured by determining NADH ch/) was used to evaluate which of the mutated amino generation at 340 nm and 30 °C in a potassium phos- acid positions were located on the protein surface and a phate bufer (KPi) reaction system (50 mM, pH 8.0) default value of 30% solvent exposure was chosen. containing GlcA (5 mM), galacturonic acid (5 mM) and To generate a mutant library, we performed the NAD+(2 mM). One unit of UDH activity was defned as polymerase chain reaction (PCR) using pET28a-AtL- the amount of UDH which generated 1 μmol NADH in BA4404UDH as the template and the degenerate codon 1 min. Protein concentration was measured using the NNK (Additional fle 1: Table S1).
Load more

Recommended publications
  • Global Regulation of Differential Gene Expression by C-Abl/Arg Oncogenic
    LAB/IN VITRO RESEARCH e-ISSN 1643-3750 © Med Sci Monit, 2017; 23: 2625-2635 DOI: 10.12659/MSM.904888 Received: 2017.04.16 Accepted: 2017.05.08 Global Regulation of Differential Gene Published: 2017.05.30 Expression by c-Abl/Arg Oncogenic Kinases Authors’ Contribution: ABCE 1 Qincai Dong* 1 Laboratory of Genetic Engineering, Beijing Institute of Biotechnology, Beijing, Study Design A BC 2 Chenggong Li* P.R. China Data Collection B 2 Institute of Health Sciences, Anhui University, Hefei, Anhui, P.R. China Statistical Analysis C BC 3 Xiuhua Qu 3 Center of Basic Medical Sciences, Navy General Hospital of PLA, Beijing, Data Interpretation D AFG 1 Cheng Cao P.R. China Manuscript Preparation E AEG 1 Xuan Liu Literature Search F Funds Collection G * These 2 authors contributed equally to this work Corresponding Authors: Xuan Liu, e-mail: [email protected], Cheng Cao, e-mail: [email protected] Source of support: This work was supported by the National Natural Science Foundation of China [31070674] Background: Studies have found that c-Abl oncogenic kinases may regulate gene transcription by RNA polymerase II phos- phorylation or by direct regulation of specific transcription factors or coactivators. However, the global regu- lation of differential gene expression by c-Abl/Arg is largely unknown. In this study, differentially expressed genes (DEGs) regulated by c-Abl/Arg were identified, and related cellular functions and associated pathways were investigated. Material/Methods: RNA obtained from wild-type and c-Abl/Arg gene-silenced MCF-7 cells was analyzed by RNA-Seq. DEGs were identified using edgeR software and partially validated by qRT-PCR.
    [Show full text]
  • Functional and Physiological Discovery in the Mannonate Dehydratase Subgroup of the Enolase Superfamily
    FUNCTIONAL AND PHYSIOLOGICAL DISCOVERY IN THE MANNONATE DEHYDRATASE SUBGROUP OF THE ENOLASE SUPERFAMILY BY DANIEL JOSEPH WICHELECKI DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Professor John Gerlt, Chair Professor John Cronan Professor Scott Silverman Professor Wilfred van der Donk ABSTRACT In the current post-genomic world, the exponential amassing of protein sequences is overwhelming the scientific community’s ability to experimentally assign each protein’s function. The use of automated, homology-based annotations has allowed a reprieve from this efflux of data, but has led to widespread misannotation and nonannotation in protein sequence databases. This dissertation details the functional and physiological characterization of the mannonate dehydratase subgroup (ManD) of the enolase superfamily (ENS). The outcome affirms the dangers of homology-based annotations while discovering novel metabolic pathways. Furthermore, the experimental verification of these pathways ( in vitro and in vivo ) has provided a platform to test the general strategies for improved functional and metabolic characterization being developed by the Enzyme Function Initiative (EFI). Prior to this study, one member of the ManD subgroup had been characterized and was shown to dehydrate D-mannonate to 2-keto-3-deoxy-D-gluconate. Forty-two additional members of the ManD, selected from across the sequence space of the subgroup, were screened for activity and kinetic constants were determined. The members of the once isofunctional subgroup were found to differ in both catalytic efficiency and substrate specificity: 1) high 3 4 -1 -1 efficiency (k cat /K M = 10 to 10 M s ) dehydration of D-mannonate, 2) low efficiency (k cat /K M = 10 1 to 10 2 M-1s-1) dehydration of D-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested).
    [Show full text]
  • The Regulation of Human Carbonyl Reductase 3 (CBR3) in Epithelial Cell Lines
    Charles university in Prague Faculty of pharmacy in Hradec Kralov´ e´ Department of biochemical sciences Diploma thesis The regulation of human carbonyl reductase 3 (CBR3) in epithelial cell lines Regulace lidske´ karbonylreduktasy 3 v buneˇcnˇ ych´ lini´ıch epithelu Petra Malatkov´ a´ 2009 Supervisors: Prof. Dr. Edmund Maser Dr. Bettina Ebert Christian Albrechts University in Kiel Faculty of Medicine Department of Toxicology and Pharmacology for Natural Scientists Prof. Ing. Vladim´ır Wsol,´ Ph.D. Charles University in Prague Faculty of Pharmacy in Hradec Kralov´ e´ Department of Biochemical Sciences Declaration I declare that this thesis is my original author’s work which I have worked out independently. All literature and other sources, from which I gather during elaboration, are listed in the bibli- ography and properly cited in the work. 4th May 2009 2 Acknowledgements First, I would like to express sincere thanks to Dr. Bettina Ebert for teaching me new techniques and performing cell culture experiments. I thank you for lending your time and providing de- tailed comments. Also, I would like to thank to Prof. Dr. Edmund Maser and Prof. Ing. Vladim´ır Wsol,´ Ph.D. for offering useful advice and, in particular, giving me chance to do the diploma thesis at Department of Toxicology and Pharmacology in Kiel. Next, I would like to thank to Yasser El-Hawari, who helps me with LaTex and contributes to improving my German skills. I would also like to thank to Michael Kisiela for his helpful assistance with any occurred problems. Finally, I would like to extend a big thank to my parents for their love and support.
    [Show full text]
  • Phenotypic and Proteomic Analysis of 5-Fluorouracil T Reated Normal And
    Phenotypic and Proteomic Analysis of 5-Fluorouracil T reated Normal and Carcinoma Cells A thesis submitted for the degree of Ph.D. Dublin City University By William Bryan, B.Sc. The research work described in this thesis was performed under the supervision of Prof. Martin Clynes National Institute for Cellular Biotechnology Dublin City University 2006 I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph-.O.-... (insert title of degree for which registered) is entirely my own work and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed (Candidate) ID No.: ffg5~72o3X Date:? )-/< - O b _________________ Acknowledgements Firstly, I’d like to thank Prof. Martin Clynes and Dr. Paula Meleady for the opportunity to pursue this Ph.D. in the National Institute for Cellular Biotechnology at Dublin City University and for their mentoring over the years - it’s very much appreciated. Also I’d like to thank the proteomics crew, Andrew, Paul, Jon and Mick for all the technical discussions on casting the perfect 2D gels and mastering of the mass spectrum. Also I’d like to thank the proteomics/toxicology crew, Lisa and Joanne, for the useful discussions over the years. Also I have to thank Lisa for the friendly ‘slagging’ towards the end of our Ph.D.’s, I guess you won the Ph.D. race, barely, by just 30 minutes. Cheers to Bella and Eadaoin for making those otherwise painful weekends pleasant.
    [Show full text]
  • Directed Mutagenesis Sites of Agrobacterium Tumefaciens Uronate Dehydrogenase
    Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 22, No. (1) : 2020 : 140-148 © Global Science Publications ISSN-0972-3005 EVOLUTION-GUIDED OPTIMIZATION FOR SELECTING SITE- DIRECTED MUTAGENESIS SITES OF AGROBACTERIUM TUMEFACIENS URONATE DEHYDROGENASE MURUGAN R., PRATHIVIRAJ R., DIPTI MOTHAY AND CHELLAPANDI P.* Molecular Systems Engineering Lab, Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India (Received 19 October, 2019; accepted 26 November, 2019) Key words : Uronate dehydrogenase, Agrobacterium tumefaciens, Site-directed mutagenesis, Coevolution, Mutant stability, Substrate specificity, Molecular evolution Abstract – Uronate dehydrogenase (EC:1.1.1.203) belongs to the NAD-dependent epimerase/dehydratase (NDE/D) subfamily, which converts D-galacturonic acid and D-glucuronic acid into D-galactaric acid and D-glucaric acid, respectively. Uronate dehydrogenase-catalyzed reaction is reversible and no substrate- specific activity in nature. Therefore, evolution-guided optimization approach was employed for screening, selection, and evaluation of its mutants to increase the substrate specificity with NAD+ and D-glucuronic acid. The phylogenetic analysis described that uronate dehydrogenase from A. tumefaciens evolved from the UDP-glucose-4-epimerase subfamily members and not related to closely related soil bacteria. Molecular conservation of its sequence-structure-function integrity was retained in this organism by imposing purifying selection and amino acid substitution patterns. A single amino acid substitution in its proton relay system or substrate-binding site found to bring several changes in the local structural environments. It hasenforced to optimize the substrate-binding site that recognizes the D-galacturonic acid or D-glucuronic acid. Hence, site-directed mutagenesis targets detectedinthis study would be useful for engineering uronate dehydrogenase subjected to be used in the biotransformation process of D-glucaric acid production.
    [Show full text]
  • V13a192-Bando Pgmkr
    Molecular Vision 2007; 13:1722-9 <http://www.molvis.org/molvis/v13/a192/> ©2007 Molecular Vision Received 14 February 2007 | Accepted 12 September 2007 | Published 18 September 2007 NADH photo-oxidation is enhanced by a partially purified λ-crystallin fraction from rabbit lens Masayasu Bando,1 Mikako Oka,2 Kenji Kawai,1 Hajime Obazawa,3 Makoto Takehana2 1Department of Ophthalmology, Tokai University School of Medicine, Isehara, Japan; 2Department of Molecular Function and Physiology, Kyoritsu University of Pharmacy, Tokyo, Japan; 3Eye Research Institute of Cataract Foundation, Tokyo, Japan Purpose: In the rabbit lens, high levels of reduced nicotinamide adenine dinucleotide (NADH) can function as a near- ultraviolet light (near-UV) filter, an effect apparently achieved by specific nucleotide binding to λ-crystallin. The present investigation asks whether λ-crystallin enhances NADH photo-oxidation by superoxide radicals produced via a photosen- sitization reaction of near-UV with NADH. Methods: λ-Crystallin was partially purified from rabbit lens soluble fraction by a two-step gel filtration and affinity column chromatography procedure. NADH solutions with or without partially purified λ-crystallin were subjected to near-UV irradiation or exposed to superoxide generated enzymatically by the xanthine/xanthine oxidase system. NADH oxidation was determined by assaying the decrease of absorbance at 340 nm. Results: When irradiated with near-UV, free NADH was oxidized very little in the absence of λ-crystallin. In contrast, NADH photo-oxidation was rapidly initiated in the presence of partially purified λ-crystallin. This λ-crystallin-enhanced NADH photo-oxidation was totally inhibited by adding superoxide dismutase. We also found that λ-crystallin largely increased NADH oxidation by a superoxide that is generated enzymatically.
    [Show full text]
  • All Enzymes in BRENDA™ the Comprehensive Enzyme Information System
    All enzymes in BRENDA™ The Comprehensive Enzyme Information System http://www.brenda-enzymes.org/index.php4?page=information/all_enzymes.php4 1.1.1.1 alcohol dehydrogenase 1.1.1.B1 D-arabitol-phosphate dehydrogenase 1.1.1.2 alcohol dehydrogenase (NADP+) 1.1.1.B3 (S)-specific secondary alcohol dehydrogenase 1.1.1.3 homoserine dehydrogenase 1.1.1.B4 (R)-specific secondary alcohol dehydrogenase 1.1.1.4 (R,R)-butanediol dehydrogenase 1.1.1.5 acetoin dehydrogenase 1.1.1.B5 NADP-retinol dehydrogenase 1.1.1.6 glycerol dehydrogenase 1.1.1.7 propanediol-phosphate dehydrogenase 1.1.1.8 glycerol-3-phosphate dehydrogenase (NAD+) 1.1.1.9 D-xylulose reductase 1.1.1.10 L-xylulose reductase 1.1.1.11 D-arabinitol 4-dehydrogenase 1.1.1.12 L-arabinitol 4-dehydrogenase 1.1.1.13 L-arabinitol 2-dehydrogenase 1.1.1.14 L-iditol 2-dehydrogenase 1.1.1.15 D-iditol 2-dehydrogenase 1.1.1.16 galactitol 2-dehydrogenase 1.1.1.17 mannitol-1-phosphate 5-dehydrogenase 1.1.1.18 inositol 2-dehydrogenase 1.1.1.19 glucuronate reductase 1.1.1.20 glucuronolactone reductase 1.1.1.21 aldehyde reductase 1.1.1.22 UDP-glucose 6-dehydrogenase 1.1.1.23 histidinol dehydrogenase 1.1.1.24 quinate dehydrogenase 1.1.1.25 shikimate dehydrogenase 1.1.1.26 glyoxylate reductase 1.1.1.27 L-lactate dehydrogenase 1.1.1.28 D-lactate dehydrogenase 1.1.1.29 glycerate dehydrogenase 1.1.1.30 3-hydroxybutyrate dehydrogenase 1.1.1.31 3-hydroxyisobutyrate dehydrogenase 1.1.1.32 mevaldate reductase 1.1.1.33 mevaldate reductase (NADPH) 1.1.1.34 hydroxymethylglutaryl-CoA reductase (NADPH) 1.1.1.35 3-hydroxyacyl-CoA
    [Show full text]
  • Cloning and Characterization of Uronate Dehydrogenases from Two
    JOURNAL OF BACTERIOLOGY, Mar. 2009, p. 1565–1573 Vol. 191, No. 5 0021-9193/09/$08.00ϩ0 doi:10.1128/JB.00586-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58ᰔ‡ Sang-Hwal Yoon, Tae Seok Moon, Pooya Iranpour,† Amanda M. Lanza, and Kristala Jones Prather* Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 Received 28 April 2008/Accepted 30 November 2008 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 comple- mentation 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 (kcat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (Km) for galacturonate was lower than that for glucuronate.
    [Show full text]
  • Synthetic Scaffold Systems for Increasing the Efficiency Of
    biology Review Synthetic Scaffold Systems for Increasing the Efficiency of Metabolic Pathways in Microorganisms Almando Geraldi 1,2,* , Fatiha Khairunnisa 2,3, Nadya Farah 4, Le Minh Bui 5 and Ziaur Rahman 6 1 Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya 60115, Indonesia 2 Research Center for Bio-Molecule Engineering, Universitas Airlangga, Surabaya 60115, Indonesia; [email protected] 3 Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya 60115, Indonesia 4 Department of Biology, Faculty of Mathematics and Life Sciences, Indonesia Defense University, Bogor 16810, Indonesia; [email protected] 5 NTT Hi-Tech Institute, Nguyen Tat Thanh University (NTTU), Ho Chi Minh City 700000, Vietnam; [email protected] 6 Department of Microbiology, Abdul Wali Khan University Mardan, Mardan, Khyber Pakhtunkhwa 23200, Pakistan; [email protected] * Correspondence: [email protected]; Tel.: +62-31-5936501 Simple Summary: Biotechnology involves the use of living organisms to create high-value products. Bacteria and yeast, in particular, are widely applied for such processes, but they may not naturally produce certain products, such as amino acids, organic acids, and alcohols in large amounts, if at all. Hence, the field of metabolic engineering has emerged for “tweaking” the biosynthetic pathways of these cells to encourage the high production of desired products. However, the complexity of the many metabolic pathways in natural cells makes it difficult to ensure that only the molecular components and pathways related to the desired product are enhanced. Very often, competing Citation: Geraldi, A.; Khairunnisa, F.; metabolic pathways and toxic intermediates will lower the production efficiency.
    [Show full text]
  • Uncorrected Proof
    Process Biochemistry xxx (xxxx) xxx-xxx Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com Chromohalobacter salixigens uronate dehydrogenase: Directed evolution for improved thermal stability and mutant CsUDH-inc X-ray crystal structure Kurt Wagschal a⁠ ,⁠ ⁎, Victor J. Chan a⁠ , Jose H. Pereira b⁠ , Peter H. Zwart c⁠ , Banumathi Sankaran d⁠ ,⁠ ⁎ a USDA Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, USA b Molecular Biophysics and Integrated Bioimaging, Joint BioEnergy Institute, Emeryville, CA, 94608, USA c Molecular Biophysics and Integrated Bioimaging & Center for Advanced Mathematics for Energy Research Applications, Lawrence Berkeley National Laboratories,1 Cyclotron Road, Berkeley, CA, 94703, USA d Molecular Biophysics and Integrated Bioimaging, Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley,PROOFCalifornia, 94720, USA ARTICLE INFO ABSTRACT Keywords: Chromohalobacter salixigens contains a uronate dehydrogenase termed CsUDH that can convert uronic acids to Uronate dehydrogenase their corresponding C1,C6-dicarboxy aldaric acids, an important enzyme reaction applicable for biotechnological Directed evolution use of sugar acids. To increase the thermal stability of this enzyme for biotechnological processes, directed evolu- Pectin tion using gene family shuffling was applied, and the hits selected from 2-tier screening of a shuffled gene family Alginate library contained in total 16 mutations, only some of which when examined individually appreciably increased Gene shuffling thermal stability. Most mutations, while having minimal or no effect on thermal stability when tested in isola- Protein thermal stability 0.5⁠ tion, were found to exhibit synergy when combined; CsUDH-inc containing all 16 mutations had ΔK t⁠ +18 °C, such that kcat⁠ was unaffected by incubation for 1 h at ∼70 °C.
    [Show full text]
  • Bioassay Technology
    BIOASSAY TECHNOLOGY - CHINA SR#: CAT#: PRODUCT NAME SIZE 1 E0000Hu Human transthyretin,TTR ELISA Kit 96 Tests 2 E0001Hu Human True insulin,TI ELISA Kit 96 Tests 3 E0002Hu Human Islet amyloid polypeptide,IAPP ELISA Kit 96 Tests 4 E0003Hu Human advanced glycation end products,AGEs ELISA Kit 96 Tests 5 E0004Hu Human Pentosidine ELISA Kit 96 Tests 6 E0005Hu Human ReverseTri-iodothyronine,rT3 ELISA Kit 96 Tests 7 E0006Hu Human C-Polypeptide,CP ELISA Kit 96 Tests 8 E0007Hu Human beta cellulin,BTC ELISA Kit 96 Tests 9 E0008Hu Human Thyroglobulin,TG ELISA Kit 96 Tests 10 E0009Hu Human C-Peptide ELISA Kit 96 Tests 11 E0010Hu Human Insulin,INS ELISA Kit 96 Tests 12 E0011Hu Human Neonatal Thyroxine,NN-T4 ELISA Kit 96 Tests 13 E0012Hu Human Free Tri-iodothyronine Indes,Free-T3 ELISA Kit 96 Tests 14 E0013Hu Human Free Thyroxine,FT4 ELISA Kit 96 Tests 15 E0014Hu Human trypsinogen activation peptide,TAP ELISA Kit 96 Tests 16 E0015Hu Human Ultrasensitivity Tri-iodothyronine,u-T3 ELISA Kit 96 Tests 17 E0016Hu Human Amylin ELISA Kit 96 Tests Human Glucose dependent insulin releasing polypeptide,GIP 18 E0017Hu 96 Tests ELISA Kit 19 E0018Hu Human Proinsulin,PI ELISA Kit 96 Tests 20 E0019Hu Human beta,beta-carotene 9',10'-oxygenase,BCO2 ELISA Kit 96 Tests 21 E0020Hu Human Tri-iodothyronine,T3 ELISA Kit 96 Tests 22 E0021Hu Human thyroxine,T4 ELISA Kit 96 Tests 23 E0022Hu Human Glucagon-like peptide 1,GLP-1 ELISA Kit 96 Tests 24 E0023Hu Human Glycated hemoglobin A1c,GHbA1c ELISA Kit 96 Tests 25 E0024Hu Human Insulin Receptor β,ISR-β ELISA KIT 96 Tests 26 E0025Hu
    [Show full text]
  • Thermostabilization of the Uronate Dehydrogenase From
    Roth et al. AMB Expr (2017) 7:103 DOI 10.1186/s13568-017-0405-2 ORIGINAL ARTICLE Open Access Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi‑rational design Teresa Roth1,2, Barbara Beer1, André Pick1 and Volker Sieber1,3,4* Abstract Aldaric acids represent biobased ‘top value-added chemicals’ that have the potential to substitute petroleum-derived chemicals. Until today they are mostly produced from corresponding aldoses using strong chemical oxidizing agents. An environmentally friendly and more selective process could be achieved by using natural resources such as sea- weed or pectin as raw material. These contain large amounts of uronic acids as major constituents such as glucuronic acid and galacturonic acid which can be converted into the corresponding aldaric acids via an enzyme-based oxida- tion using uronate dehydrogenase (Udh). The Udh from Agrobacterium tumefaciens (UdhAt) features the highest cata- 1 1 lytic efciency of all characterized Udhs using glucuronic acid as substrate (829 s− mM− ). Unfortunately, it sufers from poor thermostability. To overcome this limitation, we created more thermostable variants using semi-rational design. The amino acids for substitution were chosen according to the B factor in combination with four additional knowledge-based criteria. The triple variant A41P/H101Y/H236K showed higher kinetic and thermodynamic stability 15 with a T50 value of 62.2 °C (3.2 °C improvement) and a ∆∆GU of 2.3 kJ/mol compared to wild type. Interestingly, it was only obtained when including a neutral mutation in the combination. Keywords: Uronate dehydrogenase, Glucuronic acid, Agrobacterium tumefaciens, Thermostability, B factor, Neutral drift Introduction not selective.
    [Show full text]