A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing l-amino acids

doi:10.1016/S1381-1177(00)00036-9

Journal of Molecular Catalysis B: Enzymatic

Volume 11, Issues 4–6, 22 January 2001, Pages 397-406

https://doi.org/10.1016/S1381-1177(00)00036-9 Get rights and content

Abstract

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding l-amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has l-selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of l-selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.

Introduction

Optically pure amino acids have numerous commercial applications such as the synthesis of pesticides, peptide hormones and semi-synthetic antibiotics. One method for the production of optically pure amino acids that has received considerable attention recently is the stereospecific conversion of 5-substituted hydantoins [1], [2]. The enzymatic hydrolysis of 5-substituted hydantoins to their corresponding amino acids is a two-step reaction catalysed by two sequential enzymes: first a hydantoinase (E.C. 3.5.2.2) converts hydantoins to N-carbamyl amino acids (NCAs), and then an N-carbamyl amino acid amidohydrolase (NCAAH), converts NCAs to amino acids [2]. One problem in the industrial application of such enzyme systems is that the NCAAHs frequently have relatively low activity. In addition, the use of the microorganisms producing these enzymes in an industrial process is complicated by the fact that hydantoinases and NCAAHs typically require different conditions for optimal activity and stability. Thus the search for novel strains with improved activity remains an active field.

A wide variety of microorganisms have hydantoinase activity which enables them to produce N-carbamyl-d-amino acids and/or d-amino acids [3]. These include Pseudomonas desmolyticum producing d-phenylglycine [4], a P. putida [5], [6], and an halophilic Pseudomonas strain producing N-carbamyl-d-phenylglycine [7]. La Pointe et al. [8] have reported several d-specific hydantoinase-producing P. putida strains that exhibit wide genetic diversity.

More unusual are the group of microorganisms that are able to convert hydantoins to l-amino acids. Those reported include a Flavobacterium sp. converting only aromatic substrates, with a non-selective hydantoinase and an l-selective NCAAH [9], and a Bacillus brevis with l-specific hydantoinase and NCAAH [10]. Pseudomonas strain NS671 is able to catalyse the conversion of (2-methylthioethyl)hydantoin to l-methionine by an ATP-dependent mechanism involving a non-selective hydantoinase, l-selective NCAAH and a racemase which converts d-hydantoins to the l-enantiomers. [11], [12], [13]. Several hydantoin-hydrolysing Arthrobacter isolates have been reported: Arthrobacter sp. strain DSM 7350 produces a non-selective hydantoinase and l-selective N-carbamyl amino acid amidohydrolase, but no racemase activity [14]. The hydantoin-hydrolysing enzyme system in A. auresscens DSM 3747 consists of an l-selective hydantoinase, an N-carbamyl-l-amino acid amidohydrolase and an hydantoin-racemase [15], while the enantioselectivity of an hydantoinase purified from A. aurescens DSM 3745, is substrate-dependent [16]. To date, the genes encoding a l-selective hydantoin-hydrolysing enzyme system have been cloned from only one bacterial strain, namely Pseudomonas sp. NS671 [11]. A thermophilic B. stearothermophilus strain, NS112A, which converted dl-5-(2-methylthioethyl)hydantoin to l-methionine by an ATP-independent mechanism, was also reported [17] and P. putida strain 77, was found to possess an N-methylhydantoin amidohydrolase with l-stereospecificity [18]. Thus there is considerable variation in the characteristics of the l-specific enzyme systems. In particular, the presence of racemases is variable, and the stereoselectivity of systems may arise from the activity of either the hydantoinase or NCAAH component or it may be substrate-dependent [19].

Of several bacterial strains exhibiting hydantoin-hydrolysing activity which we have isolated from local environmental samples [20], a strain designated RU-KM3_S was selected for further investigation, since it was found to produce significantly high amounts of NCA from a range of hydantoin substrates.

The most common method for catalysing the conversion of hydantoins into amino acids is the use of resting cell reactions, where bacterial cells producing hydantoinases are incubated together with substituted hydantoin and allowed to be converted to the desired amino acid derivatives. The factors that affect the efficiency of these reactions are variable and largely unexplained, and one aim of this study was to define the conditions for optimal enzyme activity. In addition, comparison of resting cell reactions with those utilising cell extracts was conducted with the aim of developing the most efficient biocatalytic system.

Section snippets

Chemicals

Hydantoin, N-carbamyglycine (NCG), methylhydantoin and N-carbamyalanine (NCA) were purchased from Sigma or Toronto Chemicals. Other 5-substituted hydantoins were synthesised by the Bucherer–Bergs method [21]. Spectrapor tubing and a molecular weight cut-off 6000–8000 was used for dialysis experiments.

Isolation and culture of bacterial cells

P. putida RU-KM3_S, which was among bacterial strains isolated from the environment previously, was identified by comparison of the DNA sequence of the 16S rRNA gene with 16S rRNA gene sequences in

Effects of reaction time, temperature and pH on biocatalytic activity of RU-KM3_S resting cells

Resting cells were incubated with 50 mM hydantoin as substrate, under biocatalytic assay conditions as described above, but for varying lengths of time up to 24 h. The highest levels of NCG and glycine were detected after 3 h of incubation, when total conversions under these conditions were typically found to be close to 100% (Fig. 1A). In subsequent experiments, resting cell biocatalytic assays were conducted over 3 h. These activity levels, giving yields of approximately 0.44 mg product per

Conclusions

This investigation demonstrates the presence, in P. putida RU-KM3_S, of a highly active hydantoin-hydrolysing enzyme system possessing hydantoinase and NCAAH activity. The presence of l-selective NCAAH activity in a pseudomonad is particulary useful with regard to the potential application of the strain for an industrial biotransformation process in view of the broad substrate selectivity we have observed. In our investigation, the production of l-leucine, l-nor-leucine, l-tert-leucine have been

Acknowledgements

This work was supported by research grants from, AECI, the National Research Foundation of South Africa and the THRIP program. The authors wish to acknowledge Murray Gardner who participated in the isolation of strain RU-KM3_S, and members of the Rhodes University Hydantoinase Group for their contributions in discussion of this work.

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A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing l-amino acids

Abstract

Introduction

Section snippets

Chemicals

Isolation and culture of bacterial cells

Effects of reaction time, temperature and pH on biocatalytic activity of RU-KM3S resting cells

Conclusions

Acknowledgements

TIBTECH

Enzyme Microb. Technol.

J. Biotechnol.

J. Biotechnol.

J. Biotechnol.

J. Mol. Catal. (Enzymic)

J. Biotechnol.

Enzyme Microbiol. Technol.

Adv. Biochem. Eng./Biotechnol.

Biochem. J.

Biotechnol. Tech.

Biochem. Biophys. Acta

Appl. Microbiol. Biotechnol.

Appl. Environ. Microbiol.

Effects of reaction time, temperature and pH on biocatalytic activity of RU-KM3_S resting cells