A hyperthermophilic hydantoinase from Methanococcus jannaschii with an optimum growth at 85°C was cloned and expressed in E. coli. The recombinant hydantoinase was purified by affinity and anion-exchange chromatography and determined to be homotetrameric protein by gel filtration chromatography. The best substrate for the hydantoinase was D,L-5-hydroxyhydantoin, which has the specific activity of 183.4 U/mg. The optimum pH and temperature for the hydantoinase activity was 8.0 and 80°C, respectively. The half-life of the hydantoinase was measured to be 100 min at 90°C in the buffer containing 500 mM KCl. Manganese ions were the most effective for the hydantoinase activity. Stereospecificity was determined to be L-specific for the 5-hydroxymethylhydantoin and 5-methylhydantoin by chiral TLC. The activity yields as well as the operational stabilities of the thermostable M. jannaschii hydantoinase could be significantly improved by immobilization method.
Introduction
Hydantoin-hydrolyzing enzymes (hydantoinases and hydantoin amidohydrolases) belong to the cyclic amidases (EC group 3.5.2), which include dihydropyrimidinase, dihydroorotase, and allantoinase [1]. Even though the substrates on which the various cyclic amidases act are slightly different, the enzymes probably have a similar action on the cyclic amide bonds because the basic aspects of their catalytic mechanisms are believed to be similar [2]. A wide range of 5-monosubstituted hydantoins is hydrolyzed stereospecifically into corresponding D-, L-N-carbamoyl amino acids by the hydantoinases [3], [4]. Hydantoinases are used for industrial production of optically pure amino acids from racemic D,L-5-monosubstituted hydantoin derivatives in combination with highly stereoselective N-carbamoylases and hydantoin racemase [5]. With growing interest in industrial production of optically pure D-amino acids for semi-synthetic antibiotics, many microbial hydantoinases with different substrate specificity and stereospecificity have been isolated and characterized [6].
Many studies reported thermostable D-hydanoinases from thermophiles [7], [8], [9]. A thermostable hydantoinase from a mesophilic Bacillus sp. AR9 has a half life of 80 min at 70°C and loses only 33% of its activity in 4 h at 60°C [10]. Kim et al. reported that a gene encoding thermostable D-hydantoinase of Bascillus stearothermophilus, which has the half-life of 25 min at 80°C [11].
Hyperthermophilic microorganism provided thermostable enzymes for application in industrial processes. M. jannaschii is a methanogen, which grows at between 51 and 94°C, with and optimum growth at 85°C, and its entire genome has been completely sequenced [12].
In this study a hyperthermophilic hydantoinase from M. jannaschii was cloned and its catalytic activity and thermostability were investigated.
Section snippets
Chemicals
Hydantoin, hydantoic acid, allantoin, 5,5-diphenylhydantoin, uracil, dihydrouracil, dihydrothymine, dihydroorotic acid, 5-oxo-proline, barbituric acid, creatinine, lysine-lactam, and p-dimethylaminobenzaldehyde were purchased from Sigma. 1-Methylhydantoin and 2-thiohydantoin were obtained from Aldrich. 5-Methylhydantoin, 5-benzylhydantoin, 5-hydroxymethylhydantoin, 5-phenylhydantoin, 5-hydroxyhydantoin, 5-(δ-hydroxybutyl)hydantoin, 5-(δ-bromobutyl) hydantoin, and
Cloning and purification of hydantoinase
We identified the ORF Mj0963 from M. jannaschii as a presumed hydantoinase showing sequence homology to hydantoinases of other organisms by the BLAST program at NCBI [14]. Using the SEQSEE program [20], the amino acid sequence of Mj0963 aligns with that of hydantoinase from Pseudomonas sp. NS671 [21] (Fig. 1). From the analysis of sequence alignment, several regions are highly conserved and these proteins have the conserved histidines. The amino acid sequence of Mj0963 also exhibited 20–50%
Discussion
We cloned a hyperthermophilic hydantoinase from Methanococcus jannaschii. The hydantoinase expressed in E. coli was purified and characterized. The amino acid sequence of the Mj0903 was aligned with the hydantoinase from Pseudomonas sp. and found to have the conserved histidines (Fig. 1). The role of four strictly conserved histidines has been investigated in mammalian dihydroorotase [29]. The conserved histidines possibly play an essential role in metal binding and participate in catalysis.
Acknowledgements
This research was supported by the Korea Institute of Science and Technology following the advice of Dr. Sung-Hou Kim.
The control of pH is effective for inhibiting methanogenesis in the chain elongation fermentation (CEF) system. However, obscure conclusions exist especially with regard to the underlying mechanism. This study comprehensively explored the responses of methanogenesis in granular sludge at various pH levels, ranging from 4.0 to 10.0, from multiple aspects including methane production, methanogenesis pathway, microbial community structure, energy metabolism and electron transport. Results demonstrated that compared with that at pH 7.0, pH at 4.0, 5.5, 8.5 and 10.0 triggered a 100%, 71.7%, 23.8% and 92.1% suppression on methanogenesis by the end of 3 cycles lasting 21 days. This might be explained by the remarkably inhibited metabolic pathways and intracellular regulations. To be more specific, extreme pH conditions decreased the abundance of the acetoclastic methanogens. However, obligate hydrogenotrophic and facultative acetolactic/hydrogenotrophic methanogens were significantly enriched by 16.9%-19.5 fold. pH stress reduced the gene abundance and/or activity of most enzymes involved in methanogenesis such as acetate kinase (by 81.1%–93.1%), formylmethanofuran dehydrogenase (by 10.9%–54.0%) and tetrahydromethanopterin S-methyltransferase (by 9.3%–41.5%). Additionally, pH stress suppressed electron transport via improper electron carriers and decreased electron amount as evidenced by 46.3%–70.4% reduced coenzyme F420 content and diminished abundance of CO dehydrogenase (by 15.5%–70.5%) and NADH:ubiquinone reductase (by 20.2%–94.5%). pH stress also regulated energy metabolism with inhibited ATP synthesis (e.g., ATP citrate synthase level reduced by 20.1%–95.3%). Interestingly, the protein and carbohydrate content secreted in EPS failed to show consistent responses to acidic and alkaline conditions. Specifically, when compared with pH 7.0, the acidic condition remarkably reduced the levels of total EPS and EPS protein while both levels were enhanced in the alkaline condition. However, the EPS carbohydrate content at pH 4.0 and 10.0 both decreased. This study is expected to promote the understanding of the pH control-induced methanogenesis inhibition in the CEF system.
Interestingly, supplementation of the buffer with 500 mM KCl reduced the thermostability of MCM B-887 hydantoinase as compared to the control. Chung et al. reported the reverse of this [39] wherein they reported an enhancement in the thermostability of hydantoinase from M. jannaschii by adding 500 mM KCl. Dihydrouracil, hydantoin, allantoin, DL-phenylhydantoin, and DL-methylhydantoin, were used as substrates to investigate the kinetic properties of the d-hydantoinase.
Overexpression of a novel hydantoinase (hyuH) from P. aeruginosa (MCM B-887) in E. coli yielded optically pure carbamoyl amino acids. The use of optically pure carbamoyl amino acids as substrates facilitates the synthesis of non-proteinogenic amino acids. The enzyme hyuH shared a maximum of 92 % homology with proven hydantoinase protein sequences from the GenBank database, highlighting its novelty. Expression of hydantoinase gene was improved by >150 % by overexpressing it as a fusion protein in specialized E. coli CODON + host cells, providing adequate machinery for effective translation of the GC-rich gene. The presence of distinct residues in the substrate binding and active site of MCM B-887 hydantoinase enzyme explained its unique and broad substrate profile desirable for industrial applications. The purified enzyme, with a specific activity of 53U/mg of protein, was optimally active at 42 °C and pH 9.0 with a requirement of 2 mM Mn2+ ions. Supplementation of 500 mM of Na-glutamate enhanced the thermostability of the enzyme by more than 200 %.
So, Mn2+, Zn2+ and Mg2+ were evaluated as stabilizers able to reduce the deleterious effects of sodium borohydride on the enzyme activity. These ions were chosen because it has been previously reported that they exert a positive effect on the activity and the stability of most DHTases, at least from microbial sources [67–72]. Arcuri et al. [4] reported that the requirements for optimal activity of DHTase from Vigna angularis are simpler than those required by bacterial DHTases, not requiring the addition of divalent cations, such as Fe2+, Mn2+ or Zn+2 for their activity.
This work reports the immobilization of a multimeric d-hydantoinase (DHTase) from Vigna angularis (E.C. 3.5.2.2.) on agarose beads activated with glyoxyl groups aiming to improve its stability via multipoint covalent attachment. The final reduction with sodium borohydride resulted in a drop in enzyme activity that could be decreased by adding Zn2+ or Mg2+. The optimal preparation with high activity (58 % recovered activity) and stability (around 86-fold more stable than the free enzyme) was obtained by DHTase immobilization on glyoxyl agarose for 24 h at 25 °C and pH 10.05, and a borohydride reduction step in the presence of 10 mM Zn2+ (DHTase-Glx). The enzyme was almost fully immobilized on glyoxyl agarose (19.8 mg/g of support) when offering 20 mg/g. This immobilized biocatalyst was used to catalyze the hydrolysis of d,l-phenylhydantoin under substrate racemization conditions, which produced 99 % of N-carbamoyl-d-phenylglycine after 9 h reaction.
Several enzymes of this superfamily are known to present broad substrate promiscuity, such as imidases [34–36], hydantoinases [33,37,38] or d-aminoacylases [39]. They are also known by a marked enantioselectivity toward their substrates [33,37–39], making them interesting candidates by their biotechnological applications [40–43]. Both the substrate promiscuity [18,19,44] and the enantioselectivity have been shown for allantoinases; although several allantoinases have been described as aspecific [12,18], the preference for the S-(+)-enantiomer has been demonstrated for several enzymes [9,12,13,18,32,45–48].
Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent Tm of 62 ± 1 °C. Optimal parameters for the enzyme activity were pH 7.5 and 50 °C, showing apparent Km and kcat values of 17.7 ± 2.7 mM and 24.4 ± 1.5 s−1, respectively. Co2+ proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3–4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H2O2 toward allantoinase.
Hydantoinase and carbamoylase are key biocatalysts for the production of optically pure amino acids from dl-5-substituted hydantoins (SSH). Out of 364 isolated strains with hydantoinase and carbamoylase at 45 °C, 24 strains showed efficient hydantoinase and carbamoylase activities. Among them, 17 produced l-amino acids, and 7 produced d-amino acids from both aromatic dl-5-benzylhydantoin and aliphatic dl-5-isopropylhydantoin. Most of the strains that were able to form l-amino acid belonged to genera Bacillus, Geobacillus, Brevibacillus, Aneurinibacillus, Microbacterium, and Kurthia. Phylogenetic relationships were investigated based on 16S rRNA from the hydantoinase-producing bacteria. Distinct tendencies toward certain genera were observed between most of the strains forming l-amino acids and d-amino acids from SSH. The results from this study can be utilized to develop new isolation technology of hydantoinase-producing microorganisms, and to understand metabolism and evolutionary origins of hydantoinase and carbamoylase among different bacteria.
Hydantoin cleaving bacterial isolates were recovered from terrestrial soil samples originating from different geographic sources (Antarctica, South Africa and China) using culture-based screening methods (selective agar plates and shake flask cultures supplemented with hydantoins). Thirty-two bacterial isolates possessing the capability to transform the model substrates benzylhydantoin and dihydrouracil to the corresponding N-carbamoyl-amino acids were successfully cultured. Amplification and sequencing of the 16S rDNA revealed that the isolates belonged to the genera Arthrobacter, Burkholderia, Bacillus, Delftia, Enterobacter, Flavobacterium, Ochrobactrum, Pseudomonas and Stenotrophomonas, with one isolate assigned to the family Microbacteriacae. We have shown that microorganisms with hydantoinase activity are: (i) distributed in various geographically distinct environmental habitats, (ii) distributed worldwide and (iii) found in certain bacterial genera. Furthermore, we have demonstrated the presence of hydantoinase activity in genera in which hydantoinase activity has not previously been reported.
