双头菌WH-1环氧乙烷二酸水解酶的克隆和酶学性质的研究

Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1 # # 双头菌WH-1环氧乙烷二酸水解酶的克隆和酶学性质的研究 1 2 3 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:luozisheng@zju.edu.cn †E-mail:luozisheng@zju.edu.cn luozisheng@zju.edu.cn 2 3 2 3 2 3 2 3 2 3 College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China Zhejiang Provincial Key Laboratory for Chemical and Biological Processing Technology of Farm Products, Hangzhou 310023, China Zhejiang Provincial Key Laboratory for Chemical and Biological Processing Technology of Farm Products, Hangzhou 310023, China Objective: 2 This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1. Methods: 2 Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed. Results: 2 The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (p I ) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min ( T 50 15 ) of 61 °C and the half-life at 50 °C ( t 1/2, 50 °C ) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu 2+ , Ag + , and anionic surfactants. Its kinetic parameters ( K m , k cat , and k cat / K m ) were 18.7 mmol/L, 222.3 s −1 , and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain. Conclusions: 2 The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of L (+)-2,3-dihydrobutanedioic acid. 目的 2 克隆双头菌WH-1环氧乙烷二酸水解酶(ORCH)的基因并研究其酶学性质. 创新点 2 首次获得双头菌ORCH的基因,且该酶催化效率高,热稳定性好. 方法 2 柱层析纯化ORCH后,进行酶学性质研究;通过蛋白末端测序和PCR获得其基因序列;通过二级结构预测和多序列比对进行ORCH序列分析. 结论 2 来源于双头菌WH-1的ORCH是迄今报道的催化效率和热稳定性最好的ORCH,为 L (+)-2,3-二羟基丁二酸的生产提供了新的催化剂. Oxiranedicarboxylate hydrolase (ORCH) L (+)-2,3-Dihydrobutanedioic acid Characterization Cloning 环氧乙烷二酸水解酶 L (+)-2,3-二羟基丁二酸 酶学性质 克隆 Introduction 1 Oxiranedicarboxylate hydrolase (ORCH) belongs to the epoxide hydrolase (EC 3.3.2.3) family and can asymmetrically hydrolyze oxiranedicarboxylate to 2,3-dihydroxybutanedioate. The 2,3-dihydroxybutanedioate product has three enantiomers ( L (+)-configuration, D (−)-configuration, and meso -configuration), formation of which is determined by the properties of the ORCH. Many species have been reported to produce L (+)-dihydroxybutanedioate, including Pseudomonas , Rhizobium , Corynebacterium , Acetobacter , Labrys , Rhodococcus , Nocardia , and Klebsiella (Liu et al., 2007a 2007a ; Cheng et al., 2014a 2014a ; Bao et al., 2015 2015 ). The ORCHs from the latter three species have been characterized and their genes have been cloned (Liu et al., 2007b 2007b ; Wang et al., 2013 2013 ; Cheng et al., 2014b 2014b ). They proceed through a two-step mechanism using a nucleophile-histidine-acid catalytic triad, involving the formation of a covalent intermediate between enzyme and substrate (Pan et al., 2011 2011 ; Wang et al., 2013 2013 ; Cheng et al., 2014b 2014b ). A few genera have been reported to produce D (−)-dihydroxybutanedioate, including Pseudomonas , Alcaligenes , and Bordetella (Pan et al., 2008 2008 ). The amino acid sequences of the ORCHs from the latter two have been determined (Pan et al., 2010 2010 ). The crystal structure of ORCH from Bordetella and its possible catalytic mechanism have been reported (Bao et al., 2014 2014 ; Dong et al., 2018 2018 ). The only genus known to produce meso- dihydroxybutanedioate is Flavobacterium (Martin and Foster, 1955 1955 ), but there have been no further reports on the gene sequence, enzyme properties, or catalytic mechanism of the enzyme(s) involved. L (+)-Dihydrobutanedioic acid is the most abundantly produced dextrorotatory isomer of 2,3-dihydrobutanedioic acid. It is widely used in the food, textile, chemical, pharmaceutical, and building industries. Biocatalytic preparation by ORCH is a trend in L (+)-dihydrobutanedioic acid production. However, poor stability of reported ORCHs restricts the efficiency of L (+)-dihydrobutanedioic acid production. Labrys sp. WH-1 has been isolated by our laboratory, which produces L (+)-dihydroxybutanedioate and shows good production stability with high enantioselectivity. In this study, the ORCH of Labrys sp. WH-1 was purified, characterized, cloned, and expressed in Escherichia coli . Materials and methods 1 Purification of ORCH from Labrys sp. WH-1 2 Labrys sp. WH-1 was stored by our laboratory and it was incubated aerobically at 30 °C for 24 h in a fermentation medium (1 g/L K 2 HPO 4 ·3H 2 O, 5 mL/L corn steep liquor, 5 g/L saccharose, 0.5 g/L MgSO 4 · 7H 2 O, 2.7 g/L NH 4 Cl, and 10 g/L L (+)-tartrate, pH 8.0). Purification of ORCH from Labrys sp. WH-1 was performed by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose chromatography, phenyl-Sepharose chromatography, and Sephadex G100 chromatography at 4 °C, according to the method of Cheng et al. ( 2014a 2014a ), except that the (NH 4 ) 2 SO 4 precipitation ranged from 40% to 60% saturation. The content of dihydrobutanedioic was determined by high-performance liquid chromatography (HPLC) and the ORCH activity was assayed according to the method of Cheng et al. ( 2014a 2014a ). The yield of each step was calculated, and the purified samples were analyzed by 12% (0.12 g/mL) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Molecular weight analysis 2 Purified ORCH was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS; Sangon Biotech Co., Ltd., Shanghai, China) to determine the accurate molecular weight (Zhu et al., 2018 2018 ). Purified ORCH was loaded on a Biosep SEC-s3000 column (300.0 mm×7.8 mm) using an ÄKTA purification system (GE Healthcare, Uppsala, Sweden) at a flow rate of 1 mL/min and 4 °C to calculate the molecular mass of the nature protein in solution by size exclusion chromatography. Thyroglobulin (669.0 kDa), immunoglobulin A (380.0 kDa), immunoglobulin G (150.0 kDa), transferrin (80.0 kDa), ovalbumin (45.0 kDa), β-lactoglobulin (35.0 kDa), chymotrypsinogen (25.0 kDa), and ribonuclease A (13.7 kDa) were used as molecular mass standards. Determination of isoelectric point 2 Capillary isoelectric focusing (CIEF) was used for determination of the isoelectric point (p I ) of ORCH loaded on a Beckman Coulter P/ACE MDQ capillary electrophoresis system (Fullerton, CA, USA) according to the method by Salmanowicz et al. ( 2014 2014 ). The apparent p I was calculated via linear regression analysis of marker p I values (p I values of 4.1, 5.5, 7.0, and 9.5; Beckman Coulter) versus migration time. Characterization of ORCH 2 The effects of temperature, pH, metal ions, and other reagents on ORCH activity were determined. Kinetic parameters ( K m and k cat ) were determined at substrate concentrations from 10 to 200 mmol/L (Yuan et al., 2018 2018 ). Circular dichroism (CD) spectra were measured on a Jasco-815 spectropolarimeter (Jasco, Japan), and the content of secondary structural elements was predicted using the program K2D according to the method of Bao et al. ( 2014 2014 ). Protein terminal sequencing 2 The purified ORCH was electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The transferred protein band was subjected to N-terminal and C-terminal sequence analysis on an ABI Procise N sequencer (Applied Biosystems, CA, USA). Cloning and expression of ORCH in E. coli 2 Genomic DNA of Labrys sp. WH-1 was extracted using an Ezup spin column bacterial genomic DNA isolation kit (Sangon Biothech, Shanghai, China). ORCH was amplified with degenerate primers P1 and P2 (Table S1 S1 ), which were designed based on the sequences of the N-terminus and C-terminus of ORCH . The PCR products were purified and ligated with pUCm-T by using the T/A cloning procedure, and the recombinant plasmid was transformed into E. coli DH5α cells. A positive clone was sequenced. Primers P3 and P4 (Table S1 S1 ) were designed to amplify ORCH and cloned into vector pTrc99A with Nco I and Bam HI. ORCH was over-expressed in E. coli JM109 cells with addition of isopropyl thiogalactoside (IPTG) at 30 °C for 18 h. Nucleotide sequence accession number 2 The nucleotide sequence of ORCH from Labrys sp. WH-1 was submitted to the GenBank nucleotide sequence database (https:// www.ncbi.nlm.nih.gov/ www.ncbi.nlm.nih.gov/ genbank) under accession No. MK994516. Results 1 Purification and analysis of ORCH 2 The ORCH from Labrys sp. WH-1 was purified with 12.7% yield by (NH 4 ) 2 SO 4 precipitation and three chromatographic steps (Table 1 1 ). The specific activity increased from 8.9 to 231.7 U/mg, indicating a 26-fold purification of the enzyme. SDS-PAGE (Fig. 1a 1a ) revealed that the ORCH was essentially pure with only very faint bands of contaminating protein being visible, and the molecular mass of the ORCH was between 25 and 35 kDa. MALDI-TOF-MS analysis (Fig. 1b 1b ) showed that the accurate molecular weight was 30.2 kDa. (a) Purification of the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1. Samples were taken at different stages of ORCH purification (Table 1 1 ) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Lanes: M, protein molecular weight standards; 1, crude enzyme; 2, 40%–60% saturated (NH 4 ) 2 SO 4 precipitate; 3, pooled fractions after diethylaminoethyl (DEAE)-Sepharose chromatography; 4, pooled fractions after phenyl-Sepharose chromatography; 5, pooled fractions after Sephadex G100 chromatography. (b) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of the ORCH from Labrys sp. WH-1. (c) Size exclusion chromatography profile. The peak at 10.05 min corresponds to a molecular mass of 30 kDa. Inset, the standard curve (see Section 2.1). (d) Capillary isoelectric focusing profile of ORCH from Labrys sp. WH-1 and standard p I markers  Step Activity (U) Protein (mg) Specific activity (U/mg) Recovery (%) Purification (fold) Crude enzyme 15 903 1796.0 8.9 100.0 1.0 (NH 4 ) 2 SO 4 10 982 896.0 12.3 69.1 1.4 Diethylaminoethyl (DEAE)-Sepharose 7681 188.0 40.9 48.3 4.6 Phenyl-Sepharose 5662 28.9 195.9 35.6 22.0 Sephadex G100 2016 8.7 231.7 12.7 26.0 In size-exclusion chromatography (Fig. 1c 1c ), the purified ORCH from Labrys sp. WH-1 had one main peak with an elution time of 10.05 min, corresponding to an apparent molecular mass of 30 kDa. Given that the ORCH monomeric unit has a molecular mass of about 30 kDa (Figs. 1a and 1b 1a and 1b ); this suggests that ORCH from Labrys sp. WH-1 is a monomer in solution. CIEF is a special mode of capillary electrophoresis that is used for the separation and charge-based characterization of protein and for the determination of p I . The mobilization stage from 15–38 min shows data for p I peptide markers and ORCH (Fig. 1d 1d ). The apparent p I of ORCH was calculated from a curve of the linear relationship between the detection time and the theoretical p I values of the peptide markers. The experimental p I value of the ORCH was determined to be 8.57 with retention time of 19.29 min. Characterization of ORCH 2 As Fig. 2a 2a shows, the ORCH from Labrys sp. WH-1 was most active between 50 and 60 °C with an optimum temperature for activity of 55 °C, and the enzyme was stable at up to 55 °C for 30 min. The temperature at which the activity of the enzyme decreased by 50% in 15 min ( T 50 15 ) and the half-life at 50 °C ( t 1/2, 50 °C ) of ORCH were about 61 °C (Fig. 2b 2b ) and 51 min (Fig. 2c 2c ), respectively. As shown in Fig. 2d 2d , the ORCH showed high activity between pH 7.5 and 9.5 with optimal pH for activity of 8.5. The ORCH showed high stability in a broad pH range, from 4.0 to 10.0 (Fig. 2d 2d ). (a) The optimum temperature for enzyme activity and the thermal stability of purified oxiranedicarboxylate hydrolase (ORCH). The optimum temperature was determined by varying the temperature at pH 8.0. Thermal stability was determined after preincubation of purified ORCH at different temperatures for 30 min at pH 8.0. (b) The temperature at which the activity of the enzyme decreased by 50% in 15 min ( T 50 15 ) was determined as follows: purified ORCH was heated for 15 min at different temperatures in thin-walled tubes, cooled immediately on ice, and the residual activity was measured. (c) The half-life at 50 °C ( t 1/2, 50 °C ) was determined by incubating purified ORCH in a water bath at 50 °C for various time, cooling immediately in an ice-water bath, and measuring the residual activity. (d) The optimum pH for enzyme activity and the pH stability of purified ORCH. The optimum pH was determined by assay at various pH values at 37 °C. pH stability was investigated by preincubation of ORCH in different buffers for 30 min at 25 °C. (e) Effects of metal ions (1 mmol/L) and chemicals (0.1% (1 g/L), except for 20 mmol/L ethylenediaminetetraacetic acid (EDTA)-Na 2 ) on the activity of purified ORCH. The activity of the ORCH from Labrys sp. WH-1 in standard reaction conditions (37 °C, pH 8.0) was (231±10.3) U/mg, and the residual activity was taken as 100%. Each value is expressed as mean±standard deviation (SD), n =3. SDS: sodium dodecyl sulfate-polyacrylamide; DTT: dithiothreitol As Fig. 2e 2e shows, the ORCH from Labrys sp. WH-1 retained high relative activity in the presence of a metal chelating agent (20 mmol/L ethylenediaminetetraacetic acid (EDTA)-Na 2 ), indicating that it is a metal-independent enzyme. It was strongly inhibited by Cu 2+ and Ag + . The ORCH was not stable in the presence of anionic surfactants (SDS and sodium deoxycholate), while it was only slightly affected by zwitterionic surfactants (Triton X-100 and Tween 80), suggesting that zwitterionic surfactants may be more suitable for increasing cell permeability during catalytic conversion by free cells. Dithiothreitol (DTT) is a powerful reducing agent, which can protect the reducing groups (such as –SH) of enzymes and block the formation of disulfide bonds. DTT only slightly affected the activity of the ORCH, suggesting that the enzyme may not contain disulfide bonds. Other metal ions (Fe 2+ , Ca 2+ , Ni 2+ , Mg 2+ , Al 3+ , Mn 2+ , Co 2+ , Ba 2+ , and Zn 2+ ) and ethanol could weakly affect the activity of the enzyme. Kinetic parameters of the ORCH for disodium cis -2,3-oxiranedicarboxylate were determined in the optimal reaction conditions (55 °C, pH 8.5). The values of K m , k cat , and k cat / K m were calculated to be 18.7 mmol/L, 222.3 s −1 , and 11.9 mmol/(L·s), respectively, which showed stronger affinity toward cis- 2,3-oxiranedicarboxylate and higher catalytic efficiency. Ultraviolet (UV) CD may be used to investigate the secondary structure of proteins. CD spectra of the ORCH are shown in Fig. S1 S1 , and the secondary structural content of the protein was predicted using the software K2D. The predicted contents of helix and sheet in ORCH were 40.3% and 12.5%, respectively. Protein terminal sequencing 2 Protein terminal sequence analysis was determined at the N-terminal and C-terminal ends of the ORCH, separately. The N-terminal amino acid sequence was MKFSGASLFA, and the C-terminal was FTELADRLGA. Cloning and expression of ORCH 2 Based on the result of the N-terminal and C-terminal sequence analysis, degenerate primers P1 and P2 (Table S1 S1 ) were designed to amplify the ORCH gene, and a sequence of about 0.8 kb was achieved. The sequence analysis using DNAMAN Version 6.0 (LynnonBiosoft, CA, USA) indicated that the fragment contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues (GenBank accession No. MK994516). The deduced molecular mass and p I of ORCH from Labrys sp. WH-1 were 30.2 kDa and 8.43, respectively, which were consistent with the actual measured results (Figs. 1c and 1d 1c and 1d ). ORCH was over expressed in E. coli JM109 and the recombinant bacteria showed the high-level expression of recombinant protein induced by IPTG (Fig. S2 S2 ). The enzyme activity of engineering bacteria could achieve (128 200±7400) U/g biomass, which was about 33 times as much as that of the original strain ((3800±360) U/g biomass). This established a beneficial foundation for industrial production of L (+)-dihydrobutanedioic acid. Discussion 1 The ORCH from Labrys sp. WH-1 was purified and some characteristics were determined in the study. The ORCHs from Rhodococcus sp. ML-0004 (Liu et al., 2007a 2007a ) and Klebsiella sp. BK-58 (Cheng et al., 2014a 2014a ) were reported to produce L (+)-dihydrobutanedioic acid, and are commonly used strains in industrial production. The properties of ORCHs from these different sources (Liu et al., 2007a 2007a ; Pan et al., 2011 2011 ; Cheng et al., 2014a 2014a , 2014b 2014b ; Qiao et al., 2015 2015 ) are compared and summarized in Table 2 2 . The ORCH from Labrys sp. WH-1 is a monomer in solution and is a metal-independent enzyme, similar to the other two ORCHs. The p I of ORCH from Labrys sp. WH-1 is higher than those from Rhodococcus and Klebsiella , which were predicted from their amino acid sequences using Compute pI/Mw software (https://web.expasy.org/compute_pi). T 50 15 : temperature at which the activity of the enzyme decreased by 50% in 15 min; t 1/2, 50 °C : half-life at 50 °C; K m , k cat : kinetic parameters; EE: enantiomeric excess. Isoelectric point (p I ) was predicted from the amino acid sequence using Compute pI/Mw software Species Molecular weight (kDa) Subunit Optimum temperature (°C) Thermal stability (°C) 2 T 50 15 (°C) 2 t 1/2, 50 °C (min) 2 Optimal pH Range of pH stability Rhodococcus 28.1 Monomer 37 <35 2 44 2 8.5 2 7.5 7–8 Klebsiella 30.1 50 <50 2 2 2 8.5 5–10 Labrys 30.2 Monomer 55 <55 2 61 2 51.0 2 8.5 4–10 12 12 Species Metal-dependence K m (mmol/L) k cat (s −1 ) k cat / K m (mmol/(L·s)) p I 2 Conversion rate (%) 2 EE value (%) 2 Reference 12 Rhodococcus No 43.3 290.1 6.7 4.75 a a 2 >99.0 2 >99.5 2 Liu et al., 2007a 2007a ; Pan et al., 2011 2011 ; Qiao et al., 2015 2015 Klebsiella No 19.6 217.7 11.1 7.78 a a 2 >99.0 2 >99.5 2 Cheng et al., 2014a 2014a , 2014b 2014b Labrys No 18.7 222.3 11.9 8.57 2 >99.0 2 >99.5 2 This study ORCH from Labrys sp. WH-1 was more thermostable than the ORCHs from Rhodococcus and Klebsiella in terms of optimum temperature, T 50 15 , and t 1/2, 50 °C (Qiao et al., 2015 2015 ). The activity range of ORCH from Labrys sp. WH-1 was wider than that of the ORCH from Rhodococcus and similar to that of the ORCH from Klebsiella . The optimum pH for each of these L (+)-dihydroxybutanedioate-producing ORCHs was moderately alkaline. The mechanism of the ORCHs from Rhodococcus (Pan et al., 2011 2011 ) and Klebsiella (Cheng et al., 2014b 2014b ) was proposed to involve a two-step reaction involving the formation of a covalent intermediate between enzyme and substrate, which was easier to generate in weakly alkaline conditions. Although the ORCH from Rhodococcus had a higher k cat , it had a lower affinity for substrate ( K m ), resulting in a lower enzyme efficiency ( k cat / K m ) (Liu et al., 2007b 2007b ). The k cat / K m of the ORCH from Labrys sp. WH-1 was the best among the reported L (+)-dihydroxybutanedioate-producing ORCHs. Taken together, although the ORCH from Rhodococcus had the highest enzyme activity, its thermal and pH stability were the poorest. The properties of the ORCH from Klebsiella are intermediate between those of the enzymes from Labrys and Rhodococcus . The ORCH from Labrys sp. WH-1 showed the best catalytic efficiency and stability (thermal and pH stability), and is less sensitive to most metal ions and chemicals, indicating that it could be a good industrial biocatalyst for production of L (+)-dihydrobutanedioic acid. ORCH from Labrys sp. WH-1 was further cloned and its amino acid sequence was aligned with those from Rhodococcus and Klebsiella using ClustalW2 program (Fig. 3 3 ). It showed 37% and 47% identity with the ORCHs from Rhodococcus and Klebsiella , respectively. The amino acid composition of these three ORCHs is shown in Table S2 S2 . The ORCH from Labrys has higher basic amino acid content (Arg, Lys, and His) than the other two, consistent with its higher isoelectric point (Fig. 1d 1d ). The ORCH from Labrys sp. WH-1 has the highest hydrophobic amino acid content (Ala, Val, Ile, Leu, Phe, and Trp) and the lowest polar amino acid content (Cys, Ser, Thr, Tyr, Asn, and Gln) of these three enzymes, indicating that it has the best stability, which was confirmed by our experiments (the hydrophobic amino acid content is related to protein stability). The secondary structures of ORCHs were predicted by PredictProtein ( http://www.predictprotein.org http://www.predictprotein.org ), and it gave similar results to those from the amino acid sequence alignment data (Fig. 3 3 ), except for N-terminal sequences. UV CD was used to investigate the secondary structure, and the contents of helix and sheet in ORCH from Labrys sp. WH-1 were 40.3% and 12.5%, respectively, which is consistent with software predictions. The nine residues for ORCH from Labrys sp. WH-1 (D48, T52, R85, N165, K195, Y201, A219, H221, and D224) are completely conserved with the important catalytic residues for ORCHs from Rhodococcus (D18, T22, R55, N134, K164, Y170, A188, H190, and D193) and Klebsiella (D48, T52, R85, N165, K195, Y201, A219, H221, and D224), suggesting that they may adopt the same catalytic mechanism (Pan et al., 2011 2011 ; Cheng et al., 2014b 2014b ). The sequences of oxiranedicarboxylate hydrolases (ORCHs) from Labrys sp. WH-1 (this study), Klebsiella sp. BK-58 (GenBank accession No. KF977193) and Rhodococcus sp. ML-0004 (GenBank accession No. DQ471957) were aligned with ClustalW2. Identical amino acids are marked with an asterisk, conserved substitution residues with a colon, semiconserved substitution residues with a period. The predicted secondary structure elements of ORCHs by PredictProtein are shown. α-Helices are shaded black and β-sheets are shaded gray. The indispensable catalytic amino acids for Klebsiella and Rhodococcus are shown in the box Conclusions 1 The ORCH from Labrys sp. WH-1 was cloned and some characteristics were determined and compared with those of other reported ORCHs. Its catalytic efficiency and thermal stability were the best among the reported L (+)-dihydroxybutanedioate-producing ORCHs. Sequence alignments showed that the residues involved in catalysis were conserved. As these L (+)-dihydroxybutanedioate-producing ORCHs have the same or similar functions, we can in principle engineer a novel ORCH enzyme with high catalytic activity and good stability by comparison between ORCH sequences and molecular structure modification, combining the favorable characteristics of the ORCH from each source. Achieving this will require knowledge of their 3D structures, which may be the top priority in future research into ORCH enzymes. References WN Bao , HF Pan , ZH Zhang , 等 . 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Proteomic analysis of human umbilical vein endothelial cells exposed to PM2.5 Compliance with ethics guidelines Wen-na BAO, Zi-sheng LUO, Shi-wang LIU, Yuan-feng WU, Pei-lian WEI, Gong-nian XIAO, and Yong LIU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1900392 Project supported by the Zhejiang Provincial Natural Science Foundation of China (No. LQ19C200001), the Education Department of Zhejiang Province Scientific Research Project (No. Y201737925), the Zhejiang Provincial Key Laboratory for Chemical and Biological Processing Technology of Farm Products (No. 2016KF0048), and the Key Laboratory of Bioorganic Synthesis of Zhejiang Province (No. 20170110), China 2019-07-08 2019-09-05 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 20