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Mining of aminotransferase gene ota3 from Bacillus pumilus W3 via genome analysis, gene cloning and expressing for compound bioamination
Gene 686 (2019) 21–28
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Mining of aminotransferase gene ota3 from Bacillus pumilus W3 via genome analysis, gene cloning and expressing for compound bioamination
T
Lixin Zhaia, Shaolan Yanga, Yingjie Laib, Di Menga, Qiaopeng Tiana, Zhengbing Guana, ⁎ ⁎ Yujie Caia, , Xiangru Liaoa, a b
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China Chem-Stone (Guangzhou) Co. Ltd., Scientific and Technological Enterprise Accelerator, 11 Kaiyuan Avenue, Guangzhou 510530, China
ARTICLE INFO
ABSTRACT
Keywords: Aminotransferase Genome analysis Bioinformatics Chiral amine Bacillus pumilus W3
Aminotransferases are widely employed as biocatalysts to produce chiral amines and biologically active pharmaceuticals via asymmetric synthesis. In this study, transaminase genes in the Bacillus pumilus W3 genome were analysed, and gene ota3 encoding a putative (R)-selective transaminase was identified. The sequence of ota3 shares highest sequence identity (24.7%) with the first (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Amino acid sequence and conserved domains analyses indicated that ω-BPAT encoded by ota3 belonged to the pyridoxal 5′-phosphate-dependent class IV (PLPDE_IV) superfamily. Both native and codon-optimised ωBPAT genes were recombinantly expressed, and the purified proteins had a molecular mass of ~33.4 kDa. Furthermore, enantioselectivity tests with (S)- and (R)-α-phenethylamine revealed its (R)-selectivity. The optimal conditions for catalytic reaction were 45 °C and pH 7.0, and ω-BPAT retained stability at 20 °C and pH 7.0. Thus, ω-BPAT is a novel (R)-selective aminotransferase with great potential as a universal biocatalyst.
1. Introduction Chiral amines are important pharmaceutical intermediates because > 70% of drugs currently on the market are chiral amines or their derivatives (Ward & Wohlgemuth, 2010; Calcaterra, 2018). However, the present industrial production of chiral amines is largely achieved through chemical synthesis (Tan et al., 2018), which has disadvantages including high cost and environmental pollution (Bagal & Bhanage, 2015; Fleury-Brégeot et al., 2010; Wang & Xiao, 2014; Grogan, 2018). In recent years, aminotransferases that are active under mild reaction conditions, exhibit rigorous stereoselectivity, are simple to prepare, and can achieve total substrate conversion in environmentally friendly reaction processes have been reported, and these novel biocatalysts are expected to replace poor traditional transition metal catalysts in the process of amine production (Guo & Berglund, 2017; Ghislieri & Turner, 2014; Kohls et al., 2014; Fuchs et al., 2015; Simon et al., 2014; Malik et al., 2012; Mathew et al., 2013; Gao et al., 2017; Cerioli et al., 2015). Through the synergistic action of aminotransferases and their pyridoxal 5′-phosphate (PLP) coenzyme, amino groups on amino donors can be quickly transferred to prochiral acceptor ketones to produce chiral
amines, and ketones or α-keto acids as by-products (Schmidt et al., 2015; Wu et al., 2014) (Fig. 1). Thus, in biotechnological application fields, aminotransferase is an irreplaceable member of enzyme family originating from its extraordinary potential. According to multiple sequence alignment using the PFAM database (http://www.sanger.ac.uk/Software/Pfam/), aminotransferases can be divided into five categories, its representative enzymes have aspartate aminotransferase (AspATs), aromatic transaminase (AroATs), ω-transaminase (ω-ATs), branched-chain transaminase (BCATs) and serine aminotransferase (SerATs), respectively (Hwang et al., 2005). Among these classes, ω-transaminases are used for asymmetric synthesis of pharmaceutical intermediates in varying degree due to their wide substrate specificity compared with other aminotransferases, and they are key enzymes for the synthesis of chiral compounds (Park et al., 2014; Park et al., 2013a; Park et al., 2013b). Furthermore, enzymes can be engineered to increase stability and alter substrate specificity, and much effort has been expended on molecular modelling and structureactivity relationships (Weiß et al., 2017; Deszcz et al., 2015; Skalden et al., 2015; Chen et al., 2015; Nobili et al., 2015), especially for glutamate decarboxylase and α-L-rhamnosidase (Fan et al., 2018; Ge et al.,
Abbreviations: PLPDE_IV, pyridoxal 5′-phosphate dependent enzyme class IV; CDD, conservation domain database; PLP, pyridoxal 5′-phosphate; ω-BPAT, Bacillus pumilus W3 ω-transaminase; ALDH-SF, aldehyde dehydrogenase superfamily of NAD(P)+-dependent enzymes; AAT, aspartate aminotransferase ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Cai), [email protected] (X. Liao). https://doi.org/10.1016/j.gene.2018.10.082 Received 6 July 2018; Received in revised form 16 October 2018; Accepted 28 October 2018 Available online 05 November 2018 0378-1119/ © 2018 Published by Elsevier B.V.
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Fig. 1. Asymmetric synthesis by amine transaminase enzymes.
2018). For example, Savile et al. (2010)) reported the successful synthesis of Sitagliptin from a large substrate precursor by engineering ATA-117, an (R)-selective variant of a ω-transaminase from Arthrobacter sp. KNK 168. Many novel applicable ω-ATs have been found and further applications for these enzymes have been explored (Shi et al., 2017). The complete Bacillus pumilus W3 genome is available from NCBI (National Center for Biotechnology Information) (Guan et al., 2015). Then, the BLASTP (Basic Local Alignment Search Tool Protein sequences) system basing on the amino acid sequence of the (R)-selective ω-aminotransferases was executed from Arthrobacter sp. KNK 168 as the template. In this article, one hypothetical protein ω-BPTA as pyridoxal 5′-phosphate dependent enzymes class IV (PLPDE_IV; aminotransferase class IV) was chosen from Bacillus pumilus W3 aiming to more detailed analytical investigation and identification. Gene ota3 encoding ω-BPTA was cloned, overexpressed, and the enzyme was purified to homogeneity. The activity and enantioselectivity of ω-aminotransferases were verified via the related experimental techniques. Moreover, the relationships between comprehensive performance of ω-BPTA (activity and stability) and reaction conditions (pH and temperature) were investigated by carrying out a single-variable test with phenethylamine and pyruvate as donor and acceptor respectively.
branched-chain amino acid aminotransferase family a member of the PLPDE_IV superfamily. Then the amino acid sequence of ω-BPTA was compared with the ω-aminotransferase from Arthrobacter sp. KNK 168 for ω-aminotransferase activity prediction. 2.3. Molecular biology tools, enzymes, bacteria and chemicals The MiniBEST Bacterial Genomic DNA Extraction Kit (Version 3.0) and all other kits were purchased from TaKaRa (Otsu, Japan). PrimeSTAR® HS DNA polymerase, 5× PrimeSTAR® buffer (Mg2+ plus), protein markers, and all other molecular biology reagents including isopropyl-β-D-thiogalactopyranoside (IPTG) were also purchased from TaKaRa. PCR primers for cloning and gene sequencing were manufactured by Hongxun (Suzhou, China). The pColdII vector was purchased from TaKaRa (Dalian, China). Ampicillin sodium salt was purchased from Molekula Ltd. (Gillingham, UK). All analytical grade chemical reagents, unless stated otherwise, were purchased from Sigma-Aldrich (Shanghai, China). 2.4. Codon optimisation of ota3 and construction of the expression vector The ota3 gene was obtained by PCR amplification from B. pumilus W3 genomic DNA using PrimeSTAR® HS DNA polymerase and oligonucleotide primers FWD-ota (5′-GCCGCTCGAGAAGGAACAGTGGATC TTTTTAAACG-3′) and REW-ota (5′-GCCGTGCAGTGCTTGCGTGAATGT TCTCATCGGTA-3′), incorporating XhoI and PstI restriction sites, respectively (underlined). PCR was performed following the polymerase manufacturer's protocol in 50 μL reactions containing 0.5 μL enzyme, 2 ng/μL template (B. pumilus W3 genomic DNA), 0.5 μM of each primer. Thermal cycling included 30 cycles of denaturation at 98 °C for 10 s, annealing at 53 °C for 15 s, and extension 72 °C for 1 min. According to the manufacturer's guidelines, gel-purified PCR products were digested with XhoI and PstI for 16 h, and cloned into digested pColdII plasmid. The recombinant plasmid was verified by sequencing and named pColdII-ota3. The gene sequence of ota3 annotated from NCBI was codon-optimised using the online program Optimizer (http://sg.idtdna.com/ CodonOpt) based on the codon preference of Escherichia coli BL21 (DE3). The codon-optimised ota3 gene was synthesized by Hongxun (Suzhou, China) and cloned into the pColdII expression vector. The recombinant plasmid was verified by sequencing and named pCold IIotas3.
2. Materials and methods 2.1. Analysis of putative aminotransferase genes in the Bacillus pumilus W3 genome The annotated genome of Bacillus pumilus W3 (GenBank No: CP011150.1), isolated from raw gallnut honey in Nandan County, Guangxi Province, China, was obtained from the NCBI database, and sequences of aminotransferase genes were analysed using MEGA version 7.0 (Kumar et al., 2016). Enzymes encoded by these genes were subjected to preliminary domain identification. A putative aminotransferase gene (ota3) consistent with the structural characteristics of an ω-aminotransferase from Arthrobacter sp. KNK 168 was further identified. 2.2. Bioinformatic analysis of ota3 nucleotide and ω-BPTA amino acid sequences Gene ota3 (GenBank No: MH196528) was identified in the genome of B. pumilus W3. The NCBI conservation domain database (CDD) (http://www.ncbi.nlm.nih.gov/cdd/) was employed to analyse the amino acid sequence in ω-BPTA conserved domain. Some specific sites were identified with the D-AAT_like (D-alanine aminotransferase),
2.5. Recombinant expression of ota3 and otas3 in E. coli The pColdII-ota3 and pColdII-otas3 plasmids were separately 22
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Table 1 Bioinformatics information for all putative aminotransferases in the Bacillus pumilus W3 genome. Gene name
Gene_ID
Length
Location
CDD
Ota1 Ota2 Ota3
fig|6,666,666.73146.peg.1094 fig|6,666,666.73146.peg.120 fig|6,666,666.73146.peg.1413
1338 1377 915
NODE_1_length_1095241_cov_117.046_ID_1_1042308_1040971 NODE_1_length_1095241_cov_117.046_ID_1_132475_131099 NODE_2_length_808211_cov_119.051_ID_3_254110_255024
Ota4 Ota5 Ota6 Ota7 Ota8
fig|6,666,666.73146.peg.1433 fig|6,666,666.73146.peg.1720 fig|6,666,666.73146.peg.1794 fig|6,666,666.73146.peg.1916 fig|6,666,666.73146.peg.2289
1293 1098 1308 1098 1092
NODE_2_length_808211_cov_119.051_ID_3_278194_279486 NODE_2_length_808211_cov_119.051_ID_3_528624_529721 NODE_2_length_808211_cov_119.051_ID_3_597590_598897 NODE_2_length_808211_cov_119.051_ID_3_702604_703701 NODE_3_length_576529_cov_129.492_ID_5_261854_260763
Ota9 Ota10 Ota11 Ota12 Ota13 Ota14
fig|6,666,666.73146.peg.2356 fig|6,666,666.73146.peg.2365 fig|6,666,666.73146.peg.2632 fig|6,666,666.73146.peg.2841 fig|6,666,666.73146.peg.2874 fig|6,666,666.73146.peg.2877
1179 1197 1803 1338 1425 1395
NODE_3_length_576529_cov_129.492_ID_5_322557_323735 NODE_3_length_576529_cov_129.492_ID_5_332595_331399 NODE_4_length_423185_cov_130.202_ID_9_20422_22224 NODE_4_length_423185_cov_130.202_ID_9_256275_257612 NODE_4_length_423185_cov_130.202_ID_9_288425_287001 NODE_4_length_423185_cov_130.202_ID_9_291235_292629
Ota15 Ota16 Ota17 Ota18 Ota19 Ota20 Ota21 Ota22 Ota23 Ota24 Ota25 Ota26 Ota27 Ota28
fig|6,666,666.73146.peg.3101 fig|6,666,666.73146.peg.3105 fig|6,666,666.73146.peg.3198 fig|6,666,666.73146.peg.3345 fig|6,666,666.73146.peg.3356 fig|6,666,666.73146.peg.3499 fig|6,666,666.73146.peg.3507 fig|6,666,666.73146.peg.3709 fig|6,666,666.73146.peg.3855 fig|6,666,666.73146.peg.608 fig|6,666,666.73146.peg.649 fig|6,666,666.73146.peg.948 fig|6,666,666.73146.peg.958 fig|6,666,666.73146.peg.991
1194 1176 1227 1194 1164 1431 1365 1296 1479 1173 1191 1347 1869 1440
NODE_5_length_400015_cov_125.854_ID_7_68796_67603 NODE_5_length_400015_cov_125.854_ID_7_70641_71816 NODE_5_length_400015_cov_125.854_ID_7_159314_158088 NODE_5_length_400015_cov_125.854_ID_7_301264_300071 NODE_5_length_400015_cov_125.854_ID_7_313246_312083 NODE_6_length_268780_cov_123.362_ID_11_56611_58041 NODE_6_length_268780_cov_123.362_ID_11_65607_66971 NODE_6_length_268780_cov_123.362_ID_11_265690_264395 NODE_9_length_18562_cov_138.564_ID_17_5857_4379 NODE_1_length_1095241_cov_117.046_ID_1_592196_593368 NODE_1_length_1095241_cov_117.046_ID_1_634491_633301 NODE_1_length_1095241_cov_117.046_ID_1_899405_898059 NODE_1_length_1095241_cov_117.046_ID_1_909849_907981 NODE_1_length_1095241_cov_117.046_ID_1_941309_942748
Aspartate aminotransferase (AAT)superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Pyridoxine 5′-phosphate-dependent enzyme class IV (PLPDE_IV) Aspartate aminotransferase (AAT) superfamily (type I) Aminomethyltransferase folate binding domain Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Pyridoxine 5′-phosphate-dependent enzyme class IV (PLPDE_IV) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Glutamine transaminase II (GATase) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aldehyde dehydrogenase superfamily of NAD(P) + −dependent enzymes (ALDH-SF) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Glutamine transaminase II (GATase) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Glutamine transaminase II (GATase) Aspartate aminotransferase (AAT) superfamily (type I)
transformed into E. coli BL21 (DE3) competent cells and expressed in Luria-Bertani (LB) broth. A 1 mL sample of overnight seed culture was inoculated into 50 mL LB broth containing 100 μg/mL ampicillin and grown at 37 °C with shaking at 200 rpm to an OD600 of ~0.6. Protein expression was then induced with an 0.4 mM IPTG and culturing was continued at 15 °C with shaking at 200 rpm for 24 h. Cells were harvested by centrifugation (8000 ×g, 4 °C, 10 min), and the cell pellet was weighed, frozen and stored at −20 °C. The pellet was dissolved at a final concentration of 5.0 mL/g in lysis buffer containing sodium phosphate buffer pH 7.0 (20 mM) and PLP (0.1 mM). Cells were disrupted by ultrasonication in an ice bath using a SonicsVCX130 instrument with 12 cycles of 3 s sonication and 2 s cooling. After centrifugation (10,000 ×g, 4 °C, 10 min), debris and unbroken cells were removed and the supernatant was collected as crude enzyme. Crude enzyme activity was measured by high-performance liquid chromatography (HPLC) and the protein was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE; see below).
and −20 °C for long periods. Enzymatic activity was measured by HPLC assay, and purified enzymes were analysed by SDS-PAGE (see below) and named ω-BPAT (B. pumilus W3 ω-transaminase). 2.7. Enzyme activity assay and SDS-PAGE analysis Measurement of the aminotransferase activity of ω-BPAT was performed using the method described below. A 20 mM sample of (R)-αphenethylamine or (S)-α-phenethylamine was reacted with 20 mM sodium pyruvate in the presence of 0.1 mM PLP and an appropriate amount of purified enzyme (or crude enzyme) at 45 °C and pH 7.0. The reaction was terminated after 15 min by adding 500 μL ethyl acetate. The reaction mixture was centrifuged (12,000 ×g, 1 min), and the organic phase was analysed by HPLC using an Agilent C18 column (250 × 4.6 mm; Agilent, USA) at a UV absorbance of 254 nm, with a mobile phase of acetonitrile/ultrapure water (50/50, v/v) and a flow rate of 0.6 mL/min. Enzyme activity was estimated based on the yield of acetophenone, and all experiments were conducted with three replicates. The relative molecular weight of pure enzyme was determined by employing 12% SDS-PAGE (Laemmli, 1970), staining with Coomassie Brilliant Blue R250, and comparing with broad-range protein markers (14.3–97.2 kDa).
2.6. Purification of recombinant ω-BPAT enzymes The protein products of recombinant ota3 and otas3 genes were purified by chromatography using an Avant purifier (GE Healthcare, Little Chalfont, UK). Briefly, the cell supernatant containing the target protein was passed through a 0.22 μm cellulose filter and loaded onto a 1 mL Ni-HisTrap column (GE Healthcare, Shanghai, China). The column was washed at a flow rate of 1 mL/min with at least 10 column volumes of binding buffer (20 mM sodium phosphate, 500 mM NaCl, 0.1 mM PLP, 5 mM imidazole, pH 7.4), and the enzyme was eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCl, 0.1 mM PLP, 500 mM imidazole, pH 7.0). Enzyme-containing fractions were desalted using a HiTrap TM Desalting column (5 mL) and concentrated by centrifugal ultrafiltration. Purified enzymes were stored at 4 °C for a short period,
2.8. Effect of pH and temperature on activity and stability of ω-BPAT To determine the optimal temperature for pure enzyme, (R)-αphenethylamine as amino donor was reacted with sodium pyruvate as amino acceptor at temperatures ranging from 30 °C to 70 °C and at pH 7.0. The optimal pH of purified enzyme was determined at optimum temperature in different pH buffers from 5.0 to 13.0 (pH range from 5.0 to 8.0 using sodium phosphate buffer, from 9.0 to 11.0 using glycineNaOH buffer, and from 12.0 to 13.0 using Tris-NaOH buffer). 23
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Fig. 2. Phylogenetic analysis of all putative aminotransferases in the Bacillus pumilus W3 genome and (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Enzymes in red belong to the pyridoxine 5′-phosphate (PLP)-dependent enzyme class IV (PLPDE_IV). Enzymes in blue belong to the glutamine transaminase II (GATase) class. Enzymes in yellow belong to the aminomethyltransferase folate binding domain class. The enzyme in magenta belongs to the aldehyde dehydrogenase superfamily of NAD(P)+-dependent enzymes (ALDH-SF). Enzymes in black belong to the aspartate aminotransferase (AAT) superfamily (type I). Phylogenetic trees were built using the neighbour-joining algorithm within the Molecular Evolutionary Genetics Analysis (MEGA 7.0.) program. The reliability of each branch was evaluated with 1000 bootstrap replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Under standard reaction conditions employing the above method, residual enzyme activity was measured after incubating the enzyme for 30 min at temperatures ranging from 25 to 65 °C to evaluate the thermostability of ω-BPAT. Moreover, under the standard reaction conditions mentioned above, the pH stability of ω-BPAT was evaluated by measuring residual activity after incubating the enzyme for 30 min in different pH buffers ranging from 5.0 to 13.0. Relative activities (%) were calculated in comparison with maximal activity (100%), and all experiments were conducted with three replicates.
3. Results and discussion 3.1. Analysis of putative aminotransferase genes in the Bacillus pumilus W3 genome The B. pumilus W3 (GenBank No: CP011150.1) genome was obtained from the NCBI database. And new aminotransferase genes were fish out by making use of the microbial genome database as a source of new biocatalysts, which was called genome mining. Through analysis of gene sequences and conserved domains, 28 aminotransferase genes (ota1–28) were found in the genome of Bacillus pumilus W3, in which 24
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Fig. 3. Amino acid sequence alignment of ω-BPTA and the (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Identical amino acid residues in the two enzymes are highlighted by a red background. Black boxes represent conserved domains of the two enzymes, and contain a catalytic site, a substrate-cofactor binding pocket, and a PLP binding site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
consistent with (R)-selective aminotransferase from Arthrobacter sp. KNK 168 (Fig. 3). It is concluded that the ω-BPTA may have the activity of the ω-aminotransferase. Further analysis found that the ω-BPTA as PLPDE_IV (aminotransferase class IV) was presumed to have the properties of branched chain amino acid aminotransferase (BCAT), and Dalanine aminotransferase (D-AAT_like), respectively (Fig. S1). In view of this, ω-BPTA was further studied as a potential novel ω-transaminase.
the enzymes encoded by the genes ota3 and ota8 belong to PLPDE_IV, the enzymes encoded by the genes ota11, ota20 and ota27 belong to glutamine transaminase II (GATase), the enzyme encoded by ota5 belongs to aminomethyltransferase folate binding domain, the enzyme encoded by ota14 belongs to aldehyde dehydrogenase superfamily of NAD(P)+-dependent enzymes (ALDH-SF), and the enzymes encoded by the remaining 21 aminotransferase genes belong to aspartate aminotransferase (AAT) superfamily (type I) (Table 1). In order to screen ω-aminotransferases, the gene sequence and deduced amino acid sequence of an (R)-selective aminotransferase from Arthrobacter sp. KNK 168 was utilised as a template for BLASTP searches of the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with default settings. Considering this, genes ota3 (GenBank No. MH196528) and ota8, annotated as PLPDE_IV family members, were selected for further study. Although the enzyme encoded by ota8 belongs to the PLPDE_IV family, pyridoxal 5′-phosphate binding site like typical (R)-selective aminotransferase does not exist. The sequence of ota3 shares highest sequence identity (24.7%) with the first (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Therefore, ota3 may be used as a novel gene encoding ω-aminotransferase, and was chosen for further study (Fig. 2). In recent years, classification via genomic mining (Zhang et al., 2018) and in silico property prediction (Tikariha et al., 2016) have proven popular, but these techniques have not been applied to transaminases from B. pumilus.
3.3. Codon optimisation of the ota3 gene Gene ota3 has been cloned from Bacillus pumilus W3 by PCR amplification and expressed in host cell E. coli BL21 (DE3). However, the extremely low expression level of the gene ota3 results in ω-BPTA being not easily purified. In order to increase the expression level of ota3 in E. coli, the ota3 gene was codon-optimised based on the ota3 gene sequence by making use of E. coli codon preference principle (Fig. 4). 187 bases were substituted and 183 codons were modified in the gene ota3 by codon optimization. However, amino acid sequences were absolutely identical. The modified ota3 gene was not only codon-optimised but the GC content was also appropriately increased from 43.06% to 46.34%. In this study, we found that the optimised ota3 gene had a significantly increased expression level in E. coli than the native ota3 via employing E. coli preferred codons. One of the reasons may be that codon optimization can significantly eliminate rare codons of ota3 gene. Another reason may be that change in the GC content of ota3 gene might enhance the stability of ota3 mRNA secondary structure, particularly RNA stem structure (data not shown). A moderate amount of GC content is helpful for protein translation. The results demonstrated that codon optimization is effective in increasing the expression level of genes and helps to reduce the cost of large-scale production of enzymes.
3.2. Bioinformatics analysis of ω-BPTA Through bioinformatics analysis of ω-BPTA amino acid sequences, residue F10 Phenylalanine-F283 Phenylalanine constitutes a conserved domain database (CDD). The K155 Lysine residue considered to be responsible for the aminotransferase catalytic activity. The residue in ω-BPTA catalytic site is the same as (R)-selective aminotransferase from Arthrobacter sp. KNK 168. The ω-BPTA substrate-cofactor binding pocket is composed of nine residues: Y32 Tyrosine, F37 Phenylalanine, R56 Arginine, K155 Lysine, Y159 Tyrosine, E188 Glutamic acid, I215 Isoleucine, T216 Threonine, and T252 Threonine. Of these, five residues, e.g. F37 Phenylalanine, R56 Arginine, K155 Lysine, T216 Threonine, and T252 Threonine also constitute pyridoxal 5′-phosphate binding site. Moreover, Y32 Tyrosine, F37 Phenylalanine and Y159 Tyrosine in these nine residues are not identical to the same function residues of T62 Threonine, Y67 Tyrosine, and W192 tryptophan in (R)selective aminotransferase from Arthrobacter sp. KNK 168. This shown that the amino acid residues constituting the pyridoxal 5′-phosphate binding site and the substrate-cofactor binding pocket are also basically
3.4. Purification of ω-BPTA and molecular mass determination The gene ota3 and otas3 were expressed mainly as soluble proteins in LB broth with IPTG, and the His-tagged proteins were both subsequently purified by immobilized metal affinity chromatography (IMAC). The purified enzymes of the codon optimised enzyme or native enzyme were obtained (Fig. 5). Nonetheless, the codon optimised protein was expressed higher concentration than native protein. The codon optimised enzyme was named with the acronym ω-BPTA (Bacillus pumilus W3 ω-aminotransferase). The molecular mass of ω-BPTA was estimated to be 33.4 kDa by SDS-PAGE which showed a single band and it was in accord with the theoretical value. SDS-PAGE of B. pumilus 25
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Fig. 4. DNA and amino acid sequence alignment of the native ota3 gene and its protein product, and the codon-optimised variant designed for optimal expression in the E. coli BL21 (DE3) host.
aminotransferase purification fractions. The purified ω-BPTA showed much higher activity toward (R)phenethylamine than the (S)-phenethylamine and it has almost no activity on (S)-phenethylamine, clearly indicating its (R)-selectivity (Table 2).
the pH was in the range of 7.0 to 13.0, the enzyme activity was gradually decreased with increasing pH, and its activity decreased sharply at pH 8.0. The enzyme in this study showed the optimal activity at neutral pH, indicating that neutral condition would promote the enzymatic reaction process. The results displayed that ω-BPTA was sensitive to pH and preferred neutral condition at pH 7.0. The effect of temperature on ω-BPTA activity was reported under different conditions of a temperature range from 30 °C to 70 °C and at pH 7.0 (Fig. 6B). The figure showed that the enzyme exhibited the highest activity at 45 °C, whereas the enzyme activity was weak at temperatures below 45 °C. The activity of ω-BPTA was almost completely lost at temperatures above 45 °C. The results demonstrated that although ω-BPTA was highly activity at above room temperature, it was unstable in the condition of higher than 45 °C. To sum up, ω-BPTA is a neutral enzyme, but it prefers higher temperatures.
3.5. Effects of temperature and pH on ω-BPAT activity The effect of pH on ω-BPTA activity is reported (Fig. 6A). The activity of ω-BPTA was investigated under various conditions of a pH range from 5.0 to 13.0 at 45 °C toward (R)-phenethylamine and sodium pyruvate. As can be seen from the figure, ω-BPTA exhibited maximum activity at pH 7.0. In the pH range from 5.0 to 7.0, the enzyme activity was gradually enhanced with the increase of pH, and only approximate 30% of its maximum activity was exhibited at pH 6.0. However, when
26
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ranging from 5.0 to 13.0 and stored at 4 °C for 30 min. After incubation the residual activity was determined with the HPLC assay carried out at each test conditions. The ω-BPTA activity reached a maximum at pH 7.0, and the enzyme activity decreased rapidly when the pH was < 7.0 or > 7.0. It was indicated that ω-BPTA was sensitive to pH and only stored in a neutral environment. The effect of temperature on ω-BPTA stability is displayed (Fig. 7B). Thermal stability was tested after incubation of the enzyme at variable temperatures ranging from 20 °C to 70 °C and stored in phosphate buffer 50 mM pH 7.0 for 30 mins. The highest activity was reached when the ω-BPTA was stored at 20 °C, and the enzyme activity was decreased as the storage temperature was increased. The enzyme was loss almost all its activity at 60 °C–70 °C. It was showed that the stability of ω-BPTA is related to the temperature, and the low temperature is conducive to the retention of enzyme activity. 4. Conclusions In the present study, all transaminase genes in the B. pumilus W3 genome were subjected to preliminary bioinformatics analysis and categorisation. This provided a theoretical basis for future investigation of transaminase function. Gene ota3 encoded transaminase ω-BPTA that belonged to the PLPDE_IV superfamily. The conserved domains of ωBPTA were analysed in detail, along with the catalytic site, substratecofactor binding pocket, and pyridoxal 5′-phosphate binding site, further implicating ω-aminotransferase activity. This method provided an approach for the rapid screening of transaminases, and a reference for future rational engineering of ω-BPTA to improve substrate specificity and conversion efficiency. We prepared a codon-optimised version of the ota3 gene, which increased expression levels in the E. coli host, facilitating easier purification of recombinant ω-BPTA enzyme. ω-BPTA preferentially utilised (R)-phenethylamine as an amino donor, and displayed almost no activity with (S)- phenethylamine, indicating (R)-enantioselectivity. ωBPTA activity was optimal at neutral pH and higher temperatures (< 45 °C). These findings could assist the large-scale production of ωBPTA, an (R)-selective ω-transaminase. Moreover, the enzyme could be further engineered to generate a more versatile biocatalyst for the synthesis of chiral amines such as chiral 5-membered ring amine intermediates of Sitafloxacin. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.10.082.
Fig. 5. SDS-PAGE analysis of ω-BPTA from Bacillus pumilus W3. Lane 1, protein markers (14.3–97.2 kDa); lane 2, crude fractions of the native enzyme; lane 3, crude fractions of the codon-optimised enzyme; lane 4, purification fractions of the codon-optimised enzyme; lane 5, purification fractions of the native enzyme. Table 2 ω-BPTA specificity toward (R)-phenethylamine and (S)-phenethylamine as amino donor. protein
(R)-phenethylamine Activity (U/mg)
Purified ω-BPTA
1.1760
a
(S)-phenethylamine Activity (U/mg)a 0.0005
a One unit of enzyme activity was defined as the amount of enzyme catalysing the formation of 1 μmol of ketone product from amine in 1 min under the reaction conditions described above.
3.6. Effects of temperature and pH on ω-BPAT stability The effect of pH on ω-BPTA stability is displayed (Fig. 7A). ω-BPTA preparation was dissolved in a universal buffer solution at various pH
Fig. 6. Effects of pH (A) and temperature (B) on enzyme activity. The highest activity in A (1.49 ± 0.18 U/mg) and B (1.35 ± 0.15 U/mg) was set as 100%, and relative activity was calculated by comparison with the maximal activity.
27
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Fig. 7. Effects of pH and temperature on enzyme stability. (A) Effect of pH on the stability of ω-BPTA. (B) The thermostability of ω-BPTA. The highest activity in A (1.62 ± 0.17 U/mg) and B (1.19 ± 0.18 U/mg) was set as 100%, and relative activity was calculated by comparison with the maximal activity.
Acknowledgments
analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227, 680–685. Malik, M.S., Park, E.S., Shin, J.S., 2012. Features and technical applications of ω-transaminases. Appl. Microbiol. Biotechnol. 94, 1163–1171. Mathew, S., Shin, G., Shon, M., Yun, H., 2013. High throughput screening methods for ωtransaminases. Biotechnol. Bioprocess Eng. 18, 1–7. Nobili, A., Steffen-Munsberg, F., Kohls, H., Trentin, I., Schulzke, C., Höhne, M., Bornscheuer, U.T., 2015. Engineering the active site of the amine transaminase from Vibrio fluvialis for the asymmetric synthesis of aryl-alkyl amines and amino alcohols. ChemCatChem 7, 757–760. Park, E.S., Dong, J.Y., Shin, J.S., 2013a. Biocatalytic asymmetric synthesis of unnatural amino acids through the cascade transfer of amino groups from primary amines onto keto acids. ChemCatChem 5, 3538–3542. Park, E.S., Dong, J.Y., Shin, J.S., 2013b. ω-Transaminase-catalyzed asymmetric synthesis of unnatural amino acids using isopropylamine as an amino donor. Org. Biomol. Chem. 11, 6929–6933. Park, E.S., Dong, J.Y., Shin, J.S., 2014. Active site model of (R)-selective ω-transaminase and its application to the production of D-amino acids. Appl. Microbiol. Biotechnol. 98, 651–660. Savile, C.K., Janey, J.M., Mundorff, E.C., Moore, J.C., Tam, S., Jarvis, W.R., Colbeck, J.C., Krebber, A., Fleitz, F.J., Brands, J., Devine, P.N., Huisman, G.W., Hughes, G.J., 2010. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309. Schmidt, N.G., Simon, R.C., Kroutil, W., 2015. Biocatalytic asymmetric synthesis of optically pure aromatic propargylic amines employing ω-transaminases. Adv. Synth. Catal. 357, 1815–1821. Shi, F., Si, H., Ni, Y., Zhang, L., Li, Y., 2017. Transaminase encoded by NCgl2515 gene of Corynebacterium glutamicum ATCC13032 is involved in γ-aminobutyric acid decomposition. Process Biochem. 55, 55–60. Simon, R.C., Richter, N., Busto, E., Kroutil, W., 2014. Recent developments of cascade reactions involving ω-transaminases. ACS Catal. 4, 129–143. Skalden, L., Thomsen, M., Höhne, M., Bornscheuer, U.T., Hinrichs, W., 2015. Structural and biochemical characterization of the dual substrate recognition of the (R)-selective amine transaminase from Aspergillus fumigatus. FEBS J. 282, 407–415. Tan, X., Gao, S., Zeng, W., Xin, S., Yin, Q., Zhang, X., 2018. Asymmetric synthesis of chiral primary amines by ruthenium-catalyzed direct reductive amination of alkyl aryl ketones with ammonium salts and molecular H2. J. Am. Chem. Soc. 140, 2024–2027. Tikariha, H., Pal, R.R., Qureshi, A., Kapley, A., Purohit, H.J., 2016. In silico analysis for prediction of degradative capacity of Pseudomonas putida SF1. Gene 591, 382–392. Wang, C., Xiao, J., 2014. Asymmetric reductive amination. In: Li, W., Zhang, X. (Eds.), Stereoselective Formation of Amines. Springer, Berlin, pp. 261–282. Ward, J., Wohlgemuth, R., 2010. High-yield biocatalytic amination reactions in organic synthesis. Curr. Org. Chem. 14, 1914–1927. Weiß, M.S., Pavlidis, L.V., Spurr, P., Hanlon, S.P., Wirz, B., Iding, H., Bornscheuer, U.T., 2017. Amine transaminase engineering for spatially bulky substrate acceptance. Chembiochem 18, 1022–1026. Wu, X., Fei, M., Chen, Y., Wang, Z., Chen, Y., 2014. Enzymatic synthesis of L-norephedrine by coupling recombinant pyruvate decarboxylase and ω-transaminase. Appl. Microbiol. Biotechnol. 98, 7399–7408. Zhang, H., Jin, J., Jin, L., Li, Z., Xu, G., Wang, R., Zhang, J., Zhai, N., Chen, Q., Liu, P., Chen, X., Zheng, Q., Zhou, H., 2018. Identification and analysis of the chloride channel gene family members in tobacco (Nicotiana tabacum). Gene. https://doi.org/ 10.1016/j.gene.2018.06.073.
This work was financially supported by the Collaborative Innovation Involving Production, Teaching & Research Funds of Jiangsu Province (BY2014023-28) and the Agricultural Support Project, Wuxi Science & Technology Development (CLE01N1310). We thank Chem-Stone (Guangzhou) Co., Ltd., (Project No. P314) for financial support and collaboration. References Bagal, D.B., Bhanage, B.M., 2015. Recent advances in transition metal-catalyzed hydrogenation of nitriles. Adv. Synth. Catal. 357, 883–900. Calcaterra, A., D'Acquarica, I., 2018. The market of chiral drugs: chiral switches versus de novo enantiomerically pure compounds. J. Pharm. Biomed. Anal. 147, 323–340. Cerioli, L., Planchestainer, M., Cassidy, J., Tessaro, D., Paradisi, F., 2015. Characterization of a novel amine transaminase from Halomonas elongata. J. Mol. Catal. B Enzym. 120, 141–150. Chen, Y., Yi, D., Jiang, S., Wei, D., 2015. Identification of novel thermostable taurinepyruvate transaminase from Geobacillus thermodenitrificans for chiral amine synthesis. Appl. Microbiol. Biotechnol. 100, 3101–3111. Deszcz, D., Affaticati, P., Ladkau, N., Gegel, A., Ward, J.M., Hailes, N.C., Dalby, P.A., 2015. Single active-site mutants are sufficient to enhance serine: pyruvate α-transaminase activity in an ω-transaminase. FEBS J. 282, 2512–2526. Fan, L.Q., Li, M.W., Qiu, Y.J., Chen, Q.M., Jiang, S.J., Shang, Y.J., Zhao, L.M., 2018. Increasing thermal stability of glutamate decarboxylase from Escherichia coli by sitedirected saturation mutagenesis and its application in GABA production. J. Biotechnol. 278, 1–9. Fleury-Brégeot, N., Fuente, V., Castillón, S., Claver, C., 2010. Highlights of transition metal-catalyzed asymmetric hydrogenation of imines. ChemCatChem 2, 1346–1371. Fuchs, M., Farnberger, J.E., Kroutil, W., 2015. The industrial age of biocatalytic transamination. Eur. J. Org. Chem. 32, 6965–6982. Gao, S., Su, Y., Zhao, L., Li, G., Zheng, G., 2017. Characterization of a (R)-selective amine transaminase from Fusarium oxysporum. Process Biochem. 63, 130–136. Ge, L., Li, D., Wu, T., Zhao, L., Ding, G., Wang, Z., Xiao, W., 2018. B-factor-saturation mutagenesis as a strategy to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus. J. Biotechnol. 275, 17–23. Ghislieri, D., Turner, N.J., 2014. Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines. Top. Catal. 57, 284–300. Grogan, G., 2018. Synthesis of chiral amines using redox biocatalysis. Curr. Opin. Chem. Biol. 43, 15–22. Guan, Z.B., Cai, Y.J., Zhang, Y.Z., Zhao, H., Liao, X.R., 2015. Complete genome sequence of Bacillus pumilus W3: a strain exhibiting high laccase activity. J. Biotechnol. 207, 8–9. Guo, F., Berglund, P., 2017. Transaminase biocatalysis: optimization and application. Green Chem. 19, 333–360. Hwang, B.Y., Cho, B.K., Yun, H., Koteshwar, K., Kim, B.G., 2005. Revisit of aminotransferase in the genomic era and its application to biocatalysis. J. Mol. Catal. B Enzym. 37, 47–55. Kohls, H., Steffen-Munsberg, F., Höhne, M., 2014. Recent achievements in developing the biocatalytic toolbox for chiral amine synthesis. Curr. Opin. Chem. Biol. 19, 180–192. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics
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Gene journal homepage: www.elsevier.com/locate/gene
Mining of aminotransferase gene ota3 from Bacillus pumilus W3 via genome analysis, gene cloning and expressing for compound bioamination
T
Lixin Zhaia, Shaolan Yanga, Yingjie Laib, Di Menga, Qiaopeng Tiana, Zhengbing Guana, ⁎ ⁎ Yujie Caia, , Xiangru Liaoa, a b
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China Chem-Stone (Guangzhou) Co. Ltd., Scientific and Technological Enterprise Accelerator, 11 Kaiyuan Avenue, Guangzhou 510530, China
ARTICLE INFO
ABSTRACT
Keywords: Aminotransferase Genome analysis Bioinformatics Chiral amine Bacillus pumilus W3
Aminotransferases are widely employed as biocatalysts to produce chiral amines and biologically active pharmaceuticals via asymmetric synthesis. In this study, transaminase genes in the Bacillus pumilus W3 genome were analysed, and gene ota3 encoding a putative (R)-selective transaminase was identified. The sequence of ota3 shares highest sequence identity (24.7%) with the first (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Amino acid sequence and conserved domains analyses indicated that ω-BPAT encoded by ota3 belonged to the pyridoxal 5′-phosphate-dependent class IV (PLPDE_IV) superfamily. Both native and codon-optimised ωBPAT genes were recombinantly expressed, and the purified proteins had a molecular mass of ~33.4 kDa. Furthermore, enantioselectivity tests with (S)- and (R)-α-phenethylamine revealed its (R)-selectivity. The optimal conditions for catalytic reaction were 45 °C and pH 7.0, and ω-BPAT retained stability at 20 °C and pH 7.0. Thus, ω-BPAT is a novel (R)-selective aminotransferase with great potential as a universal biocatalyst.
1. Introduction Chiral amines are important pharmaceutical intermediates because > 70% of drugs currently on the market are chiral amines or their derivatives (Ward & Wohlgemuth, 2010; Calcaterra, 2018). However, the present industrial production of chiral amines is largely achieved through chemical synthesis (Tan et al., 2018), which has disadvantages including high cost and environmental pollution (Bagal & Bhanage, 2015; Fleury-Brégeot et al., 2010; Wang & Xiao, 2014; Grogan, 2018). In recent years, aminotransferases that are active under mild reaction conditions, exhibit rigorous stereoselectivity, are simple to prepare, and can achieve total substrate conversion in environmentally friendly reaction processes have been reported, and these novel biocatalysts are expected to replace poor traditional transition metal catalysts in the process of amine production (Guo & Berglund, 2017; Ghislieri & Turner, 2014; Kohls et al., 2014; Fuchs et al., 2015; Simon et al., 2014; Malik et al., 2012; Mathew et al., 2013; Gao et al., 2017; Cerioli et al., 2015). Through the synergistic action of aminotransferases and their pyridoxal 5′-phosphate (PLP) coenzyme, amino groups on amino donors can be quickly transferred to prochiral acceptor ketones to produce chiral
amines, and ketones or α-keto acids as by-products (Schmidt et al., 2015; Wu et al., 2014) (Fig. 1). Thus, in biotechnological application fields, aminotransferase is an irreplaceable member of enzyme family originating from its extraordinary potential. According to multiple sequence alignment using the PFAM database (http://www.sanger.ac.uk/Software/Pfam/), aminotransferases can be divided into five categories, its representative enzymes have aspartate aminotransferase (AspATs), aromatic transaminase (AroATs), ω-transaminase (ω-ATs), branched-chain transaminase (BCATs) and serine aminotransferase (SerATs), respectively (Hwang et al., 2005). Among these classes, ω-transaminases are used for asymmetric synthesis of pharmaceutical intermediates in varying degree due to their wide substrate specificity compared with other aminotransferases, and they are key enzymes for the synthesis of chiral compounds (Park et al., 2014; Park et al., 2013a; Park et al., 2013b). Furthermore, enzymes can be engineered to increase stability and alter substrate specificity, and much effort has been expended on molecular modelling and structureactivity relationships (Weiß et al., 2017; Deszcz et al., 2015; Skalden et al., 2015; Chen et al., 2015; Nobili et al., 2015), especially for glutamate decarboxylase and α-L-rhamnosidase (Fan et al., 2018; Ge et al.,
Abbreviations: PLPDE_IV, pyridoxal 5′-phosphate dependent enzyme class IV; CDD, conservation domain database; PLP, pyridoxal 5′-phosphate; ω-BPAT, Bacillus pumilus W3 ω-transaminase; ALDH-SF, aldehyde dehydrogenase superfamily of NAD(P)+-dependent enzymes; AAT, aspartate aminotransferase ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Cai), [email protected] (X. Liao). https://doi.org/10.1016/j.gene.2018.10.082 Received 6 July 2018; Received in revised form 16 October 2018; Accepted 28 October 2018 Available online 05 November 2018 0378-1119/ © 2018 Published by Elsevier B.V.
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Fig. 1. Asymmetric synthesis by amine transaminase enzymes.
2018). For example, Savile et al. (2010)) reported the successful synthesis of Sitagliptin from a large substrate precursor by engineering ATA-117, an (R)-selective variant of a ω-transaminase from Arthrobacter sp. KNK 168. Many novel applicable ω-ATs have been found and further applications for these enzymes have been explored (Shi et al., 2017). The complete Bacillus pumilus W3 genome is available from NCBI (National Center for Biotechnology Information) (Guan et al., 2015). Then, the BLASTP (Basic Local Alignment Search Tool Protein sequences) system basing on the amino acid sequence of the (R)-selective ω-aminotransferases was executed from Arthrobacter sp. KNK 168 as the template. In this article, one hypothetical protein ω-BPTA as pyridoxal 5′-phosphate dependent enzymes class IV (PLPDE_IV; aminotransferase class IV) was chosen from Bacillus pumilus W3 aiming to more detailed analytical investigation and identification. Gene ota3 encoding ω-BPTA was cloned, overexpressed, and the enzyme was purified to homogeneity. The activity and enantioselectivity of ω-aminotransferases were verified via the related experimental techniques. Moreover, the relationships between comprehensive performance of ω-BPTA (activity and stability) and reaction conditions (pH and temperature) were investigated by carrying out a single-variable test with phenethylamine and pyruvate as donor and acceptor respectively.
branched-chain amino acid aminotransferase family a member of the PLPDE_IV superfamily. Then the amino acid sequence of ω-BPTA was compared with the ω-aminotransferase from Arthrobacter sp. KNK 168 for ω-aminotransferase activity prediction. 2.3. Molecular biology tools, enzymes, bacteria and chemicals The MiniBEST Bacterial Genomic DNA Extraction Kit (Version 3.0) and all other kits were purchased from TaKaRa (Otsu, Japan). PrimeSTAR® HS DNA polymerase, 5× PrimeSTAR® buffer (Mg2+ plus), protein markers, and all other molecular biology reagents including isopropyl-β-D-thiogalactopyranoside (IPTG) were also purchased from TaKaRa. PCR primers for cloning and gene sequencing were manufactured by Hongxun (Suzhou, China). The pColdII vector was purchased from TaKaRa (Dalian, China). Ampicillin sodium salt was purchased from Molekula Ltd. (Gillingham, UK). All analytical grade chemical reagents, unless stated otherwise, were purchased from Sigma-Aldrich (Shanghai, China). 2.4. Codon optimisation of ota3 and construction of the expression vector The ota3 gene was obtained by PCR amplification from B. pumilus W3 genomic DNA using PrimeSTAR® HS DNA polymerase and oligonucleotide primers FWD-ota (5′-GCCGCTCGAGAAGGAACAGTGGATC TTTTTAAACG-3′) and REW-ota (5′-GCCGTGCAGTGCTTGCGTGAATGT TCTCATCGGTA-3′), incorporating XhoI and PstI restriction sites, respectively (underlined). PCR was performed following the polymerase manufacturer's protocol in 50 μL reactions containing 0.5 μL enzyme, 2 ng/μL template (B. pumilus W3 genomic DNA), 0.5 μM of each primer. Thermal cycling included 30 cycles of denaturation at 98 °C for 10 s, annealing at 53 °C for 15 s, and extension 72 °C for 1 min. According to the manufacturer's guidelines, gel-purified PCR products were digested with XhoI and PstI for 16 h, and cloned into digested pColdII plasmid. The recombinant plasmid was verified by sequencing and named pColdII-ota3. The gene sequence of ota3 annotated from NCBI was codon-optimised using the online program Optimizer (http://sg.idtdna.com/ CodonOpt) based on the codon preference of Escherichia coli BL21 (DE3). The codon-optimised ota3 gene was synthesized by Hongxun (Suzhou, China) and cloned into the pColdII expression vector. The recombinant plasmid was verified by sequencing and named pCold IIotas3.
2. Materials and methods 2.1. Analysis of putative aminotransferase genes in the Bacillus pumilus W3 genome The annotated genome of Bacillus pumilus W3 (GenBank No: CP011150.1), isolated from raw gallnut honey in Nandan County, Guangxi Province, China, was obtained from the NCBI database, and sequences of aminotransferase genes were analysed using MEGA version 7.0 (Kumar et al., 2016). Enzymes encoded by these genes were subjected to preliminary domain identification. A putative aminotransferase gene (ota3) consistent with the structural characteristics of an ω-aminotransferase from Arthrobacter sp. KNK 168 was further identified. 2.2. Bioinformatic analysis of ota3 nucleotide and ω-BPTA amino acid sequences Gene ota3 (GenBank No: MH196528) was identified in the genome of B. pumilus W3. The NCBI conservation domain database (CDD) (http://www.ncbi.nlm.nih.gov/cdd/) was employed to analyse the amino acid sequence in ω-BPTA conserved domain. Some specific sites were identified with the D-AAT_like (D-alanine aminotransferase),
2.5. Recombinant expression of ota3 and otas3 in E. coli The pColdII-ota3 and pColdII-otas3 plasmids were separately 22
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Table 1 Bioinformatics information for all putative aminotransferases in the Bacillus pumilus W3 genome. Gene name
Gene_ID
Length
Location
CDD
Ota1 Ota2 Ota3
fig|6,666,666.73146.peg.1094 fig|6,666,666.73146.peg.120 fig|6,666,666.73146.peg.1413
1338 1377 915
NODE_1_length_1095241_cov_117.046_ID_1_1042308_1040971 NODE_1_length_1095241_cov_117.046_ID_1_132475_131099 NODE_2_length_808211_cov_119.051_ID_3_254110_255024
Ota4 Ota5 Ota6 Ota7 Ota8
fig|6,666,666.73146.peg.1433 fig|6,666,666.73146.peg.1720 fig|6,666,666.73146.peg.1794 fig|6,666,666.73146.peg.1916 fig|6,666,666.73146.peg.2289
1293 1098 1308 1098 1092
NODE_2_length_808211_cov_119.051_ID_3_278194_279486 NODE_2_length_808211_cov_119.051_ID_3_528624_529721 NODE_2_length_808211_cov_119.051_ID_3_597590_598897 NODE_2_length_808211_cov_119.051_ID_3_702604_703701 NODE_3_length_576529_cov_129.492_ID_5_261854_260763
Ota9 Ota10 Ota11 Ota12 Ota13 Ota14
fig|6,666,666.73146.peg.2356 fig|6,666,666.73146.peg.2365 fig|6,666,666.73146.peg.2632 fig|6,666,666.73146.peg.2841 fig|6,666,666.73146.peg.2874 fig|6,666,666.73146.peg.2877
1179 1197 1803 1338 1425 1395
NODE_3_length_576529_cov_129.492_ID_5_322557_323735 NODE_3_length_576529_cov_129.492_ID_5_332595_331399 NODE_4_length_423185_cov_130.202_ID_9_20422_22224 NODE_4_length_423185_cov_130.202_ID_9_256275_257612 NODE_4_length_423185_cov_130.202_ID_9_288425_287001 NODE_4_length_423185_cov_130.202_ID_9_291235_292629
Ota15 Ota16 Ota17 Ota18 Ota19 Ota20 Ota21 Ota22 Ota23 Ota24 Ota25 Ota26 Ota27 Ota28
fig|6,666,666.73146.peg.3101 fig|6,666,666.73146.peg.3105 fig|6,666,666.73146.peg.3198 fig|6,666,666.73146.peg.3345 fig|6,666,666.73146.peg.3356 fig|6,666,666.73146.peg.3499 fig|6,666,666.73146.peg.3507 fig|6,666,666.73146.peg.3709 fig|6,666,666.73146.peg.3855 fig|6,666,666.73146.peg.608 fig|6,666,666.73146.peg.649 fig|6,666,666.73146.peg.948 fig|6,666,666.73146.peg.958 fig|6,666,666.73146.peg.991
1194 1176 1227 1194 1164 1431 1365 1296 1479 1173 1191 1347 1869 1440
NODE_5_length_400015_cov_125.854_ID_7_68796_67603 NODE_5_length_400015_cov_125.854_ID_7_70641_71816 NODE_5_length_400015_cov_125.854_ID_7_159314_158088 NODE_5_length_400015_cov_125.854_ID_7_301264_300071 NODE_5_length_400015_cov_125.854_ID_7_313246_312083 NODE_6_length_268780_cov_123.362_ID_11_56611_58041 NODE_6_length_268780_cov_123.362_ID_11_65607_66971 NODE_6_length_268780_cov_123.362_ID_11_265690_264395 NODE_9_length_18562_cov_138.564_ID_17_5857_4379 NODE_1_length_1095241_cov_117.046_ID_1_592196_593368 NODE_1_length_1095241_cov_117.046_ID_1_634491_633301 NODE_1_length_1095241_cov_117.046_ID_1_899405_898059 NODE_1_length_1095241_cov_117.046_ID_1_909849_907981 NODE_1_length_1095241_cov_117.046_ID_1_941309_942748
Aspartate aminotransferase (AAT)superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Pyridoxine 5′-phosphate-dependent enzyme class IV (PLPDE_IV) Aspartate aminotransferase (AAT) superfamily (type I) Aminomethyltransferase folate binding domain Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Pyridoxine 5′-phosphate-dependent enzyme class IV (PLPDE_IV) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Glutamine transaminase II (GATase) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aldehyde dehydrogenase superfamily of NAD(P) + −dependent enzymes (ALDH-SF) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Glutamine transaminase II (GATase) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Aspartate aminotransferase (AAT) superfamily (type I) Glutamine transaminase II (GATase) Aspartate aminotransferase (AAT) superfamily (type I)
transformed into E. coli BL21 (DE3) competent cells and expressed in Luria-Bertani (LB) broth. A 1 mL sample of overnight seed culture was inoculated into 50 mL LB broth containing 100 μg/mL ampicillin and grown at 37 °C with shaking at 200 rpm to an OD600 of ~0.6. Protein expression was then induced with an 0.4 mM IPTG and culturing was continued at 15 °C with shaking at 200 rpm for 24 h. Cells were harvested by centrifugation (8000 ×g, 4 °C, 10 min), and the cell pellet was weighed, frozen and stored at −20 °C. The pellet was dissolved at a final concentration of 5.0 mL/g in lysis buffer containing sodium phosphate buffer pH 7.0 (20 mM) and PLP (0.1 mM). Cells were disrupted by ultrasonication in an ice bath using a SonicsVCX130 instrument with 12 cycles of 3 s sonication and 2 s cooling. After centrifugation (10,000 ×g, 4 °C, 10 min), debris and unbroken cells were removed and the supernatant was collected as crude enzyme. Crude enzyme activity was measured by high-performance liquid chromatography (HPLC) and the protein was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE; see below).
and −20 °C for long periods. Enzymatic activity was measured by HPLC assay, and purified enzymes were analysed by SDS-PAGE (see below) and named ω-BPAT (B. pumilus W3 ω-transaminase). 2.7. Enzyme activity assay and SDS-PAGE analysis Measurement of the aminotransferase activity of ω-BPAT was performed using the method described below. A 20 mM sample of (R)-αphenethylamine or (S)-α-phenethylamine was reacted with 20 mM sodium pyruvate in the presence of 0.1 mM PLP and an appropriate amount of purified enzyme (or crude enzyme) at 45 °C and pH 7.0. The reaction was terminated after 15 min by adding 500 μL ethyl acetate. The reaction mixture was centrifuged (12,000 ×g, 1 min), and the organic phase was analysed by HPLC using an Agilent C18 column (250 × 4.6 mm; Agilent, USA) at a UV absorbance of 254 nm, with a mobile phase of acetonitrile/ultrapure water (50/50, v/v) and a flow rate of 0.6 mL/min. Enzyme activity was estimated based on the yield of acetophenone, and all experiments were conducted with three replicates. The relative molecular weight of pure enzyme was determined by employing 12% SDS-PAGE (Laemmli, 1970), staining with Coomassie Brilliant Blue R250, and comparing with broad-range protein markers (14.3–97.2 kDa).
2.6. Purification of recombinant ω-BPAT enzymes The protein products of recombinant ota3 and otas3 genes were purified by chromatography using an Avant purifier (GE Healthcare, Little Chalfont, UK). Briefly, the cell supernatant containing the target protein was passed through a 0.22 μm cellulose filter and loaded onto a 1 mL Ni-HisTrap column (GE Healthcare, Shanghai, China). The column was washed at a flow rate of 1 mL/min with at least 10 column volumes of binding buffer (20 mM sodium phosphate, 500 mM NaCl, 0.1 mM PLP, 5 mM imidazole, pH 7.4), and the enzyme was eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCl, 0.1 mM PLP, 500 mM imidazole, pH 7.0). Enzyme-containing fractions were desalted using a HiTrap TM Desalting column (5 mL) and concentrated by centrifugal ultrafiltration. Purified enzymes were stored at 4 °C for a short period,
2.8. Effect of pH and temperature on activity and stability of ω-BPAT To determine the optimal temperature for pure enzyme, (R)-αphenethylamine as amino donor was reacted with sodium pyruvate as amino acceptor at temperatures ranging from 30 °C to 70 °C and at pH 7.0. The optimal pH of purified enzyme was determined at optimum temperature in different pH buffers from 5.0 to 13.0 (pH range from 5.0 to 8.0 using sodium phosphate buffer, from 9.0 to 11.0 using glycineNaOH buffer, and from 12.0 to 13.0 using Tris-NaOH buffer). 23
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Fig. 2. Phylogenetic analysis of all putative aminotransferases in the Bacillus pumilus W3 genome and (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Enzymes in red belong to the pyridoxine 5′-phosphate (PLP)-dependent enzyme class IV (PLPDE_IV). Enzymes in blue belong to the glutamine transaminase II (GATase) class. Enzymes in yellow belong to the aminomethyltransferase folate binding domain class. The enzyme in magenta belongs to the aldehyde dehydrogenase superfamily of NAD(P)+-dependent enzymes (ALDH-SF). Enzymes in black belong to the aspartate aminotransferase (AAT) superfamily (type I). Phylogenetic trees were built using the neighbour-joining algorithm within the Molecular Evolutionary Genetics Analysis (MEGA 7.0.) program. The reliability of each branch was evaluated with 1000 bootstrap replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Under standard reaction conditions employing the above method, residual enzyme activity was measured after incubating the enzyme for 30 min at temperatures ranging from 25 to 65 °C to evaluate the thermostability of ω-BPAT. Moreover, under the standard reaction conditions mentioned above, the pH stability of ω-BPAT was evaluated by measuring residual activity after incubating the enzyme for 30 min in different pH buffers ranging from 5.0 to 13.0. Relative activities (%) were calculated in comparison with maximal activity (100%), and all experiments were conducted with three replicates.
3. Results and discussion 3.1. Analysis of putative aminotransferase genes in the Bacillus pumilus W3 genome The B. pumilus W3 (GenBank No: CP011150.1) genome was obtained from the NCBI database. And new aminotransferase genes were fish out by making use of the microbial genome database as a source of new biocatalysts, which was called genome mining. Through analysis of gene sequences and conserved domains, 28 aminotransferase genes (ota1–28) were found in the genome of Bacillus pumilus W3, in which 24
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Fig. 3. Amino acid sequence alignment of ω-BPTA and the (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Identical amino acid residues in the two enzymes are highlighted by a red background. Black boxes represent conserved domains of the two enzymes, and contain a catalytic site, a substrate-cofactor binding pocket, and a PLP binding site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
consistent with (R)-selective aminotransferase from Arthrobacter sp. KNK 168 (Fig. 3). It is concluded that the ω-BPTA may have the activity of the ω-aminotransferase. Further analysis found that the ω-BPTA as PLPDE_IV (aminotransferase class IV) was presumed to have the properties of branched chain amino acid aminotransferase (BCAT), and Dalanine aminotransferase (D-AAT_like), respectively (Fig. S1). In view of this, ω-BPTA was further studied as a potential novel ω-transaminase.
the enzymes encoded by the genes ota3 and ota8 belong to PLPDE_IV, the enzymes encoded by the genes ota11, ota20 and ota27 belong to glutamine transaminase II (GATase), the enzyme encoded by ota5 belongs to aminomethyltransferase folate binding domain, the enzyme encoded by ota14 belongs to aldehyde dehydrogenase superfamily of NAD(P)+-dependent enzymes (ALDH-SF), and the enzymes encoded by the remaining 21 aminotransferase genes belong to aspartate aminotransferase (AAT) superfamily (type I) (Table 1). In order to screen ω-aminotransferases, the gene sequence and deduced amino acid sequence of an (R)-selective aminotransferase from Arthrobacter sp. KNK 168 was utilised as a template for BLASTP searches of the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with default settings. Considering this, genes ota3 (GenBank No. MH196528) and ota8, annotated as PLPDE_IV family members, were selected for further study. Although the enzyme encoded by ota8 belongs to the PLPDE_IV family, pyridoxal 5′-phosphate binding site like typical (R)-selective aminotransferase does not exist. The sequence of ota3 shares highest sequence identity (24.7%) with the first (R)-selective aminotransferase from Arthrobacter sp. KNK 168. Therefore, ota3 may be used as a novel gene encoding ω-aminotransferase, and was chosen for further study (Fig. 2). In recent years, classification via genomic mining (Zhang et al., 2018) and in silico property prediction (Tikariha et al., 2016) have proven popular, but these techniques have not been applied to transaminases from B. pumilus.
3.3. Codon optimisation of the ota3 gene Gene ota3 has been cloned from Bacillus pumilus W3 by PCR amplification and expressed in host cell E. coli BL21 (DE3). However, the extremely low expression level of the gene ota3 results in ω-BPTA being not easily purified. In order to increase the expression level of ota3 in E. coli, the ota3 gene was codon-optimised based on the ota3 gene sequence by making use of E. coli codon preference principle (Fig. 4). 187 bases were substituted and 183 codons were modified in the gene ota3 by codon optimization. However, amino acid sequences were absolutely identical. The modified ota3 gene was not only codon-optimised but the GC content was also appropriately increased from 43.06% to 46.34%. In this study, we found that the optimised ota3 gene had a significantly increased expression level in E. coli than the native ota3 via employing E. coli preferred codons. One of the reasons may be that codon optimization can significantly eliminate rare codons of ota3 gene. Another reason may be that change in the GC content of ota3 gene might enhance the stability of ota3 mRNA secondary structure, particularly RNA stem structure (data not shown). A moderate amount of GC content is helpful for protein translation. The results demonstrated that codon optimization is effective in increasing the expression level of genes and helps to reduce the cost of large-scale production of enzymes.
3.2. Bioinformatics analysis of ω-BPTA Through bioinformatics analysis of ω-BPTA amino acid sequences, residue F10 Phenylalanine-F283 Phenylalanine constitutes a conserved domain database (CDD). The K155 Lysine residue considered to be responsible for the aminotransferase catalytic activity. The residue in ω-BPTA catalytic site is the same as (R)-selective aminotransferase from Arthrobacter sp. KNK 168. The ω-BPTA substrate-cofactor binding pocket is composed of nine residues: Y32 Tyrosine, F37 Phenylalanine, R56 Arginine, K155 Lysine, Y159 Tyrosine, E188 Glutamic acid, I215 Isoleucine, T216 Threonine, and T252 Threonine. Of these, five residues, e.g. F37 Phenylalanine, R56 Arginine, K155 Lysine, T216 Threonine, and T252 Threonine also constitute pyridoxal 5′-phosphate binding site. Moreover, Y32 Tyrosine, F37 Phenylalanine and Y159 Tyrosine in these nine residues are not identical to the same function residues of T62 Threonine, Y67 Tyrosine, and W192 tryptophan in (R)selective aminotransferase from Arthrobacter sp. KNK 168. This shown that the amino acid residues constituting the pyridoxal 5′-phosphate binding site and the substrate-cofactor binding pocket are also basically
3.4. Purification of ω-BPTA and molecular mass determination The gene ota3 and otas3 were expressed mainly as soluble proteins in LB broth with IPTG, and the His-tagged proteins were both subsequently purified by immobilized metal affinity chromatography (IMAC). The purified enzymes of the codon optimised enzyme or native enzyme were obtained (Fig. 5). Nonetheless, the codon optimised protein was expressed higher concentration than native protein. The codon optimised enzyme was named with the acronym ω-BPTA (Bacillus pumilus W3 ω-aminotransferase). The molecular mass of ω-BPTA was estimated to be 33.4 kDa by SDS-PAGE which showed a single band and it was in accord with the theoretical value. SDS-PAGE of B. pumilus 25
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Fig. 4. DNA and amino acid sequence alignment of the native ota3 gene and its protein product, and the codon-optimised variant designed for optimal expression in the E. coli BL21 (DE3) host.
aminotransferase purification fractions. The purified ω-BPTA showed much higher activity toward (R)phenethylamine than the (S)-phenethylamine and it has almost no activity on (S)-phenethylamine, clearly indicating its (R)-selectivity (Table 2).
the pH was in the range of 7.0 to 13.0, the enzyme activity was gradually decreased with increasing pH, and its activity decreased sharply at pH 8.0. The enzyme in this study showed the optimal activity at neutral pH, indicating that neutral condition would promote the enzymatic reaction process. The results displayed that ω-BPTA was sensitive to pH and preferred neutral condition at pH 7.0. The effect of temperature on ω-BPTA activity was reported under different conditions of a temperature range from 30 °C to 70 °C and at pH 7.0 (Fig. 6B). The figure showed that the enzyme exhibited the highest activity at 45 °C, whereas the enzyme activity was weak at temperatures below 45 °C. The activity of ω-BPTA was almost completely lost at temperatures above 45 °C. The results demonstrated that although ω-BPTA was highly activity at above room temperature, it was unstable in the condition of higher than 45 °C. To sum up, ω-BPTA is a neutral enzyme, but it prefers higher temperatures.
3.5. Effects of temperature and pH on ω-BPAT activity The effect of pH on ω-BPTA activity is reported (Fig. 6A). The activity of ω-BPTA was investigated under various conditions of a pH range from 5.0 to 13.0 at 45 °C toward (R)-phenethylamine and sodium pyruvate. As can be seen from the figure, ω-BPTA exhibited maximum activity at pH 7.0. In the pH range from 5.0 to 7.0, the enzyme activity was gradually enhanced with the increase of pH, and only approximate 30% of its maximum activity was exhibited at pH 6.0. However, when
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ranging from 5.0 to 13.0 and stored at 4 °C for 30 min. After incubation the residual activity was determined with the HPLC assay carried out at each test conditions. The ω-BPTA activity reached a maximum at pH 7.0, and the enzyme activity decreased rapidly when the pH was < 7.0 or > 7.0. It was indicated that ω-BPTA was sensitive to pH and only stored in a neutral environment. The effect of temperature on ω-BPTA stability is displayed (Fig. 7B). Thermal stability was tested after incubation of the enzyme at variable temperatures ranging from 20 °C to 70 °C and stored in phosphate buffer 50 mM pH 7.0 for 30 mins. The highest activity was reached when the ω-BPTA was stored at 20 °C, and the enzyme activity was decreased as the storage temperature was increased. The enzyme was loss almost all its activity at 60 °C–70 °C. It was showed that the stability of ω-BPTA is related to the temperature, and the low temperature is conducive to the retention of enzyme activity. 4. Conclusions In the present study, all transaminase genes in the B. pumilus W3 genome were subjected to preliminary bioinformatics analysis and categorisation. This provided a theoretical basis for future investigation of transaminase function. Gene ota3 encoded transaminase ω-BPTA that belonged to the PLPDE_IV superfamily. The conserved domains of ωBPTA were analysed in detail, along with the catalytic site, substratecofactor binding pocket, and pyridoxal 5′-phosphate binding site, further implicating ω-aminotransferase activity. This method provided an approach for the rapid screening of transaminases, and a reference for future rational engineering of ω-BPTA to improve substrate specificity and conversion efficiency. We prepared a codon-optimised version of the ota3 gene, which increased expression levels in the E. coli host, facilitating easier purification of recombinant ω-BPTA enzyme. ω-BPTA preferentially utilised (R)-phenethylamine as an amino donor, and displayed almost no activity with (S)- phenethylamine, indicating (R)-enantioselectivity. ωBPTA activity was optimal at neutral pH and higher temperatures (< 45 °C). These findings could assist the large-scale production of ωBPTA, an (R)-selective ω-transaminase. Moreover, the enzyme could be further engineered to generate a more versatile biocatalyst for the synthesis of chiral amines such as chiral 5-membered ring amine intermediates of Sitafloxacin. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.10.082.
Fig. 5. SDS-PAGE analysis of ω-BPTA from Bacillus pumilus W3. Lane 1, protein markers (14.3–97.2 kDa); lane 2, crude fractions of the native enzyme; lane 3, crude fractions of the codon-optimised enzyme; lane 4, purification fractions of the codon-optimised enzyme; lane 5, purification fractions of the native enzyme. Table 2 ω-BPTA specificity toward (R)-phenethylamine and (S)-phenethylamine as amino donor. protein
(R)-phenethylamine Activity (U/mg)
Purified ω-BPTA
1.1760
a
(S)-phenethylamine Activity (U/mg)a 0.0005
a One unit of enzyme activity was defined as the amount of enzyme catalysing the formation of 1 μmol of ketone product from amine in 1 min under the reaction conditions described above.
3.6. Effects of temperature and pH on ω-BPAT stability The effect of pH on ω-BPTA stability is displayed (Fig. 7A). ω-BPTA preparation was dissolved in a universal buffer solution at various pH
Fig. 6. Effects of pH (A) and temperature (B) on enzyme activity. The highest activity in A (1.49 ± 0.18 U/mg) and B (1.35 ± 0.15 U/mg) was set as 100%, and relative activity was calculated by comparison with the maximal activity.
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Fig. 7. Effects of pH and temperature on enzyme stability. (A) Effect of pH on the stability of ω-BPTA. (B) The thermostability of ω-BPTA. The highest activity in A (1.62 ± 0.17 U/mg) and B (1.19 ± 0.18 U/mg) was set as 100%, and relative activity was calculated by comparison with the maximal activity.
Acknowledgments
analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227, 680–685. Malik, M.S., Park, E.S., Shin, J.S., 2012. Features and technical applications of ω-transaminases. Appl. Microbiol. Biotechnol. 94, 1163–1171. Mathew, S., Shin, G., Shon, M., Yun, H., 2013. High throughput screening methods for ωtransaminases. Biotechnol. Bioprocess Eng. 18, 1–7. Nobili, A., Steffen-Munsberg, F., Kohls, H., Trentin, I., Schulzke, C., Höhne, M., Bornscheuer, U.T., 2015. Engineering the active site of the amine transaminase from Vibrio fluvialis for the asymmetric synthesis of aryl-alkyl amines and amino alcohols. ChemCatChem 7, 757–760. Park, E.S., Dong, J.Y., Shin, J.S., 2013a. Biocatalytic asymmetric synthesis of unnatural amino acids through the cascade transfer of amino groups from primary amines onto keto acids. ChemCatChem 5, 3538–3542. Park, E.S., Dong, J.Y., Shin, J.S., 2013b. ω-Transaminase-catalyzed asymmetric synthesis of unnatural amino acids using isopropylamine as an amino donor. Org. Biomol. Chem. 11, 6929–6933. Park, E.S., Dong, J.Y., Shin, J.S., 2014. Active site model of (R)-selective ω-transaminase and its application to the production of D-amino acids. Appl. Microbiol. Biotechnol. 98, 651–660. Savile, C.K., Janey, J.M., Mundorff, E.C., Moore, J.C., Tam, S., Jarvis, W.R., Colbeck, J.C., Krebber, A., Fleitz, F.J., Brands, J., Devine, P.N., Huisman, G.W., Hughes, G.J., 2010. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309. Schmidt, N.G., Simon, R.C., Kroutil, W., 2015. Biocatalytic asymmetric synthesis of optically pure aromatic propargylic amines employing ω-transaminases. Adv. Synth. Catal. 357, 1815–1821. Shi, F., Si, H., Ni, Y., Zhang, L., Li, Y., 2017. Transaminase encoded by NCgl2515 gene of Corynebacterium glutamicum ATCC13032 is involved in γ-aminobutyric acid decomposition. Process Biochem. 55, 55–60. Simon, R.C., Richter, N., Busto, E., Kroutil, W., 2014. Recent developments of cascade reactions involving ω-transaminases. ACS Catal. 4, 129–143. Skalden, L., Thomsen, M., Höhne, M., Bornscheuer, U.T., Hinrichs, W., 2015. Structural and biochemical characterization of the dual substrate recognition of the (R)-selective amine transaminase from Aspergillus fumigatus. FEBS J. 282, 407–415. Tan, X., Gao, S., Zeng, W., Xin, S., Yin, Q., Zhang, X., 2018. Asymmetric synthesis of chiral primary amines by ruthenium-catalyzed direct reductive amination of alkyl aryl ketones with ammonium salts and molecular H2. J. Am. Chem. Soc. 140, 2024–2027. Tikariha, H., Pal, R.R., Qureshi, A., Kapley, A., Purohit, H.J., 2016. In silico analysis for prediction of degradative capacity of Pseudomonas putida SF1. Gene 591, 382–392. Wang, C., Xiao, J., 2014. Asymmetric reductive amination. In: Li, W., Zhang, X. (Eds.), Stereoselective Formation of Amines. Springer, Berlin, pp. 261–282. Ward, J., Wohlgemuth, R., 2010. High-yield biocatalytic amination reactions in organic synthesis. Curr. Org. Chem. 14, 1914–1927. Weiß, M.S., Pavlidis, L.V., Spurr, P., Hanlon, S.P., Wirz, B., Iding, H., Bornscheuer, U.T., 2017. Amine transaminase engineering for spatially bulky substrate acceptance. Chembiochem 18, 1022–1026. Wu, X., Fei, M., Chen, Y., Wang, Z., Chen, Y., 2014. Enzymatic synthesis of L-norephedrine by coupling recombinant pyruvate decarboxylase and ω-transaminase. Appl. Microbiol. Biotechnol. 98, 7399–7408. Zhang, H., Jin, J., Jin, L., Li, Z., Xu, G., Wang, R., Zhang, J., Zhai, N., Chen, Q., Liu, P., Chen, X., Zheng, Q., Zhou, H., 2018. Identification and analysis of the chloride channel gene family members in tobacco (Nicotiana tabacum). Gene. https://doi.org/ 10.1016/j.gene.2018.06.073.
This work was financially supported by the Collaborative Innovation Involving Production, Teaching & Research Funds of Jiangsu Province (BY2014023-28) and the Agricultural Support Project, Wuxi Science & Technology Development (CLE01N1310). We thank Chem-Stone (Guangzhou) Co., Ltd., (Project No. P314) for financial support and collaboration. References Bagal, D.B., Bhanage, B.M., 2015. Recent advances in transition metal-catalyzed hydrogenation of nitriles. Adv. Synth. Catal. 357, 883–900. Calcaterra, A., D'Acquarica, I., 2018. The market of chiral drugs: chiral switches versus de novo enantiomerically pure compounds. J. Pharm. Biomed. Anal. 147, 323–340. Cerioli, L., Planchestainer, M., Cassidy, J., Tessaro, D., Paradisi, F., 2015. Characterization of a novel amine transaminase from Halomonas elongata. J. Mol. Catal. B Enzym. 120, 141–150. Chen, Y., Yi, D., Jiang, S., Wei, D., 2015. Identification of novel thermostable taurinepyruvate transaminase from Geobacillus thermodenitrificans for chiral amine synthesis. Appl. Microbiol. Biotechnol. 100, 3101–3111. Deszcz, D., Affaticati, P., Ladkau, N., Gegel, A., Ward, J.M., Hailes, N.C., Dalby, P.A., 2015. Single active-site mutants are sufficient to enhance serine: pyruvate α-transaminase activity in an ω-transaminase. FEBS J. 282, 2512–2526. Fan, L.Q., Li, M.W., Qiu, Y.J., Chen, Q.M., Jiang, S.J., Shang, Y.J., Zhao, L.M., 2018. Increasing thermal stability of glutamate decarboxylase from Escherichia coli by sitedirected saturation mutagenesis and its application in GABA production. J. Biotechnol. 278, 1–9. Fleury-Brégeot, N., Fuente, V., Castillón, S., Claver, C., 2010. Highlights of transition metal-catalyzed asymmetric hydrogenation of imines. ChemCatChem 2, 1346–1371. Fuchs, M., Farnberger, J.E., Kroutil, W., 2015. The industrial age of biocatalytic transamination. Eur. J. Org. Chem. 32, 6965–6982. Gao, S., Su, Y., Zhao, L., Li, G., Zheng, G., 2017. Characterization of a (R)-selective amine transaminase from Fusarium oxysporum. Process Biochem. 63, 130–136. Ge, L., Li, D., Wu, T., Zhao, L., Ding, G., Wang, Z., Xiao, W., 2018. B-factor-saturation mutagenesis as a strategy to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus. J. Biotechnol. 275, 17–23. Ghislieri, D., Turner, N.J., 2014. Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines. Top. Catal. 57, 284–300. Grogan, G., 2018. Synthesis of chiral amines using redox biocatalysis. Curr. Opin. Chem. Biol. 43, 15–22. Guan, Z.B., Cai, Y.J., Zhang, Y.Z., Zhao, H., Liao, X.R., 2015. Complete genome sequence of Bacillus pumilus W3: a strain exhibiting high laccase activity. J. Biotechnol. 207, 8–9. Guo, F., Berglund, P., 2017. Transaminase biocatalysis: optimization and application. Green Chem. 19, 333–360. Hwang, B.Y., Cho, B.K., Yun, H., Koteshwar, K., Kim, B.G., 2005. Revisit of aminotransferase in the genomic era and its application to biocatalysis. J. Mol. Catal. B Enzym. 37, 47–55. Kohls, H., Steffen-Munsberg, F., Höhne, M., 2014. Recent achievements in developing the biocatalytic toolbox for chiral amine synthesis. Curr. Opin. Chem. Biol. 19, 180–192. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics
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