Review
Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids

https://doi.org/10.1016/S0958-1669(01)00263-4Get rights and content

Abstract

A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral d- and l-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts.

Introduction

Many hydantoin derivatives are substrates for enzymatic reactions. Since the 1940s it has been known that some microorganisms are able to grow on d,l-5-monosubstituted hydantoins when supplied as sole carbon and/or nitrogen source in a mineral salt medium, often hydrolyzing only one enantiomer of a racemic mixture. It is also well established that enzymes from plant and animal sources can be used to hydrolyze and close the hydantoin ring. Various enzymes, the so-called hydantoinases, enable the hydrolysis of the hydantoin ring system in a first reaction step. The biosynthesis of these enzymes in microorganisms often has to be induced by adding specific compounds during growth. These enzymes may have different substrate specificities and, in general, are selective in forming l- or d-N-carbamoyl amino acids (i.e. hydantoic acids). Hydantoin-cleaving enzymes can often be found in combination with highly stereoselective N-carbamoylamino acid amidohydrolases (N-carbamoylases), which catalyze the further hydrolysis of the hydantoic acids to the free d- or l-amino acids in an irreversible reaction. In some cases, hydantoin racemases are involved as a third enzyme. Together, these enzymes accomplish the total conversion of racemic d,l-5-monosubstituted hydantoin derivatives to the corresponding enantiomerically pure d- or l-amino acids. This cascade of reactions, whether located in whole cells or carried out using isolated enzymes, is called the ‘hydantoinase process’ (Fig. 1).

The great advantages of an industrial hydantoinase process are that, potentially, 100% conversion and 100% optically pure amino acid can be obtained at the same time if racemic substrates are used. Until the mid-90s mainly wild-type strains, resulting from traditional screening methods (for a recent review see [1]), were used as biocatalysts. Detailed reviews on the use of free or immobilized whole-cell systems for hydantoin-cleavage have also been published 2., 3., 4..

More recent activities are summarized in this review and concentrate on new enzymes and studies on structure elucidation of hydantoinases and N-carbamoylases. We also discuss the use of improved recombinant free or immobilized enzymes, fusion proteins and especially designed recombinant whole-cell biocatalysts, including studies on the optimization of enzyme properties by directed evolution.

Section snippets

New enzymes

For hydantoinase processes, improvements can be achieved by isolating new hydantoin-metabolizing enzymes with new substrate specificities, higher enantioselectivity, higher stability and so on. The conventional way of purifying enzymes from natural sources, like microorganisms, plants or organs of higher organisms, is still a promising way to obtain such new enzymes. For example, Su and Yang [5] purified an imidase (an enzyme related to hydantoinases) from pig liver, which is also able to

Structure elucidation of hydantoinases and N-carbamoylases

Progress has been made to elucidate the molecular structure of hydantoin-degrading enzymes in order to understand their catalytic mechanisms in detail. Crystal structures have been determined for a d-carbamoylase of Agrobacterium sp. [10••] and hydantoinases from A. aurescens [11] and Thermus sp. [12]. The complete protein structure of the d-carbamoylase at a high-resolution of 1.7Å was described by Nakai et al. [10••]. The enzyme forms a homotetramer and each monomer consists of a variant of

Improvement in enzyme properties or enzyme purification

Instead of isolating new enzymes, already known and well-characterized enzymes can be improved by new and powerful directed evolution methods. A hydantoinase process for the production of l-methionine was improved by employing random mutagenesis, saturated mutagenesis and high-throughput screening systems on a hydantoinase from A. aurescens. The preference of this hydantoinase for d-5(2-methylthioethyl)hydantoin was inverted to a higher selectivity for the l-enantiomer and the overall specific

Enzyme immobilization

It is common place that most purified enzymes are only stable for hours under reaction conditions and are difficult to recover from the reaction solution for further use. The method of choice is to immobilize enzymes on a solid support. Immobilization can increase enzyme half-life from hours to several months and allows a continuous production process. Immobilization conditions were optimized for the l-hydantoinase and l-N-carbamoylase from A. aurescens by Ragnitz et al. [23]. Several carrier

Tailor-made whole cell biocatalysts

The alternative to using immobilized enzymes for amino acid production from hydantoins is the use of resting cells that express hydantoinase and carbamoylase activity. Most of the bacterial isolates that are able to use hydantoins as carbon and nitrogen source seem to have in addition a hydantoin racemase, as racemic hydantoins are often enantiospecifically converted to d- or l-amino acids at an efficiency of nearly 100%. So far, hydantoin racemases have only been cloned and sequenced from l

Conclusions

There are many factors that influence the competitiveness of enzymatic and chemical processes, for example, the cost of substrates, costs for production, purification and immobilization of enzymes, possible space-time yields and the cost of product purification. These factors are strongly dependent on the desired products, which means there is no single ‘best process’. The increasing availability of hydantoin-degrading enzymes produced by recombinant DNA technology will also increase the

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • •of special interest

  • ••of outstanding interest

References (27)

  • C. Syldatk et al.
  • C. Syldatk et al.
  • C. Syldatk et al.
  • Cited by (151)

    View all citing articles on Scopus
    View full text