J. Microbiol. Biotechnol. (2015), 25(10), 1660–1669 http://dx.doi.org/10.4014/jmb.1502.02032 Research Article Review jmb Expression and Characterization of a Novel Nitrilase from Hyperthermophilic Bacterium Thermotoga maritima MSB8 Zhi Chen1, Huayou Chen1,2*, Zhong Ni1, Rui Tian1, Tianxi Zhang1, Jinru Jia1, and Shengli Yang1 1Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China 2National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China Received: February 12, 2015 Revised: June 8, 2015 The present study describes the gene cloning, overexpression and characterization of a novel Accepted: June 9, 2015 nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The nitrilase gene consisted of 804 base pairs, encoding a protein of 268 amino acid residues with a molecular mass of 30.07 kDa after SDS-PAGE analysis. The optimal temperature and pH of the purified First published online enzyme were 45°C and 7.5, respectively. The enzyme demonstrated good temperature June 9, 2015 tolerance, with 40% residual activity after 60 min of heat treatment at 75°C. The kinetic *Corresponding author constants Vmax and Km of this nitrilase toward 3-cyanopyridine were 3.12 µmol/min/mg and Phone: +86-13912800258; 7.63 mM, respectively. Furthermore, this novel nitrilase exhibited a broad spectrum toward Fax: +86-51188791702; the hydrolysis of the aliphatic nitriles among the tested substrates, and particularly was E-mail: [email protected] specific to aliphatic dinitriles like succinonitrile, which was distinguished from most nitrilases ever reported. The catalytic efficiency kcat/Km was 0.44 /mM/s toward succinonitrile. This distinct characteristic might enable this nitrilase to be a potential candidate for industrial applications for biosynthesis of carboxylic acid. pISSN 1017-7825, eISSN 1738-8872 Keywords: Nitrilase, hyperthermophilic bacterium, Thermotoga maritima, temperature tolerance, Copyright© 2015 by The Korean Society for Microbiology aliphatic dinitriles and Biotechnology Introduction tremendous recognition of their potential due to the possibility of performing such biotransformation under Biocatalysis, as the core of industrial biotechnology, has mild condition that would not alter other labile reactive been widely investigated to improve the sustainability and groups [2]. efficiency so as to prepare industrial fine chemicals, mainly Nitrilase (E.C. 3.5.5.1), as one kind of valuable biocatalyst, focusing on intermediates for pharmaceuticals, agrochemicals, was utilized for the enzymatic biocatalysis of nitrile materials, and food ingredients [23, 35]. Nitrile compounds, compounds directly to corresponding carboxylic acids, generally speaking, are synthetically more available for the liberating ammonia. It had drawn sustainable attention to production of high-value carboxylic acids and amides, chemical hydrolysis in the organic chemical industry. Over which are important intermediates in producing the fine the past few decades, a considerable amount of nitrilases, chemicals and pharmaceuticals [32]. Nitrilase-mediated mainly derived from bacteria, yeasts, fungi, and plants, biocatalysis reactions of nitrile compounds to their had been acquired and reviewed in details, some of which corresponding carboxylic acids provide an ecofriendly had already been applied into the production of carboxylic alternative allowing clean and mild synthesis combined acids in the chemical industry [8, 11]. Furthermore, in- with high yield and selectivity when compared with depth investigations on nitrilases had been widely dwelt conventional chemical approaches typically requiring harsh upon with respect to their natural sources function basic or acidic reaction conditions and usually producing mechanisms, enzyme structure, screening pathways, undesired byproducts [36]. This has increasingly aroused biocatalytic properties, immobilization, purification, cloning J. Microbiol. Biotechnol. A Novel Nitrilase from Hyperthermophilic Bacterium 1661 and modifications of the nitrilase gene [4, 8, 9, 14-16, 22, commercially available and of analytical grade. 24, 28, 30]. On the basis of the broad substrate spectrum, nitrilases were commonly classified into three major groups, Sequence Analysis which included aliphatic, aromatic, and arylacetonitrilases, Amplified DNA fragments were sequenced by Sangon Biotech making them useful for the hydrolysis of a large number of (Shanghai, China). Nucleotide and protein sequence homology analyses in the NCBI database (http://blast.ncbi.nlm.nih.gov/ nitriles [19]. For example, a new nitrilase from Fusarium Blast.cgi) were conducted using the BLAST algorithm. Multiple proliferatum AUF-2 was characterized to be specific towards sequence alignment was carried out using MEGA 6.06 software. aliphatic, heterocyclic, and aromatic nitriles, which exhibited good catalytical efficiency for detoxification of nitriles [29]. The Gene Cloning and Expression of Thermotoga maritima MSB8 nitrilase from Rhodobacter sphaeroides could enantioselectively Nitrilase hydrolyze aliphatic dinitriles to corresponding cyanocarboxylic The primers that were designed based on the reported amino acids, which demonstrated great potential for commercial acid sequences of nitrilase in NCBI for the amplification of genes production of various cyanocarboxylic acids by readily were as follows: Forward: 5’-CGCGGATCCTTGCGAGTGGCGGC available dinitriles [26]. The nitrilase from Pseudomonas putida AGTACAGAT-3’ (BamHI restriction site is underlined) and CGMCC3830 was indentified as an aromatic nitrilase and Reverse 5'-CCGCTCGAGTCATAACCTCCCCTTCTGAAGC-3’ harbored high conversion efficiency toward cyanopyridine (XholI restriction site is underlined), which incorporated BamHI [37]. In addition, some arylacetonitrilases from Burkholderia and XholI restriction sites, respectively. The amplified 804 bp DNA fragment digested with BamHI and XhoI was ligated into the cenocepacia J2315 and Alcaligenes sp. ECU0401 were expression vector pet-28a(+) digested with the same restriction reported to harbor the merits of high substrate tolerance, enzymes, and then transformed into the E. coli BL21 (DE3) cells by yield, and optical purity while converting mandelonitrile heat shock. The positive clones were identified by colony PCR and to (R)-(−)-mandelic acid [10, 25, 31, 33, 34]. Moreover, a few double digestion. Sequencing of the cloned nitrilase gene was fungal nitrilases from Gibberella intermedia and Aspergillus subsequently performed at Sangon Biotech. niger were reported to be highly specific toward 3- For the expression of the nitrilase, the resulting recombinant cyanopyridine and 2-cyanopridine, respectively [7, 12]. E. coli cells were cultivated in Luria–Bertani liquid medium Although nitrilases to some extent exhibited prominent containing 50 mg/ml kanamycin at 37°C on a rotary shaker at 220 ×g. potential in the chemical industry, some drawbacks A final concentration of 0.1 mM isopropyl-β-D-thiogalactoside including insufficient stability, narrow spectrum, low was added for the induction when the optical density at 600 nm of specific activity, and poor selectivity still exist at present the culture broth reached between 0.6 and 0.8. The cells were then and limit their applications [21]. Seeking for novel nitrilase further incubated at 28°C and 160 ×g for another 20 h. After centrifugation at 8,000 ×g for 20 min, the cells were harvested and resources and their potential applications would be a preserved at -20°C for further experiments. constant demand for researchers for a long period of time. In this paper, the gene encoding the nitrilase from Purification of Thermotoga maritima MSB8 Nitrilase Thermotoga maritima MSB8 was cloned and overexpressed Nickle affinity chromatography (Ni-NTA) was applied to purify in E. coli BL21 (DE3). The recombinant nitrilase was purified the recombinant nitrilase by exploiting the histidine tag. The and biochemically characterized in detail in order to gain a obtained cells were suspended and washed twice with 10 ml of deeper understanding about the properties and application phosphate-buffered saline (50 mM, pH 8.0). Then the cells were potential of this enzyme. resuspended in 20 ml of the same buffer and disrupted by sonication on ice at 200 W for 10 min. The soluble fractions of the Materials and Methods sonicated solution were obtained by centrifugation at 8,000 ×g for 20 min to remove the cell debris. The resulting supernatant was Material passed through a 0.22 µm filter, and then loaded onto a Ni-NTA The genomic DNA of Thermotoga maritima MSB8 was obtained column previously equilibrated with a binding buffer (50 mM NaH PO , 300 mM NaCl, pH 8.0). The column was subsequently from Professor Weilan Shao at Jiangsu University. The E. coli 2 4 washed with 10 ml of wash buffer (50 mM NaH PO , 300 mM strains DH5α and BL21 (DE3) (Gene Copoeia, USA) were used as 2 4 hosts for cloning and expression experiments of the nitrilase, NaCl, 10 mM imidazole, pH 8.0) to wipe out the unbound proteins and eluted with the elution buffer (50 mM NaH PO , respectively. The plasmid pET-28a (+) (Novagen, Shanghai, China), 2 4 carrying an N-terminal and a C-terminal His6-Tag sequence, was 300 mM NaCl, 250 mM imidazole, pH 8.0). The purified enzyme used for the cloning and expression of the nitrilase. The nitrile was further analyzed by 12% sodium dodecyl sulfate polyacrylamide substrates were purchased from Sinopharm Chemical Reagent gel electrophoresis