Dong-Cheol Lee

Production of d-p-hydroxyphenylglycine from d,l-5-(4-hydroxyphenyl)hydantoin using immobilized thermostable d-hydantoinase from Bacillus stearothermophilus SD-1

Abstract Thermostable d -hydantoinase from thermophilic Bacillus stearothermophilus SD-1 was used to produce N -carbamoyl- d - p -hydroxyphenylglycine (NC-HPG) from d,l -5-(4-hydroxyphenyl)hydantoin ( dl -5-HPH). Culture conditions for the production of the enzyme from B. stearothermophilus SD-1 were optimized. The d -hydantoinase was immobilized on various support matrices by adsorption, and DEAE-cellulose resin was found to be most effective in terms of the activity recovery and the amount of protein bound. The activity of enzyme immobilized on DEAE-cellulose was retained >90%, and the optimal reaction conditions for the immobilized enzyme were determined to be 55°C and pH 9.0, respectively. Immobilized enzyme was applied to the production of NC-HPG from dl -5-HPH in repeated batch, and the production rate was maintained constantly over nine successive operations.

Mass production of thermostable D‐hydantoinase by batch culture of recombinant Escherichia coli with a constitutive expression system

D-Hydantoinase is an industrial enzyme widely used for the synthesis of optically active D-amino acids. A gene encoding thermostable D-hydantoinase of Bacillus stearothermophilus SD-1 has previously been cloned and constitutively expressed by its native promoter in Escherichia coli XL1-Blue (Lee et al., 1996b). In this work, we attempted mass production of the D-hydantoinase by batch culture of the recombinant E. coli using glycerol as a carbon source. The plasmid content in cells increased in proportion to the culture temperature, which resulted in a two- or three-fold increase of the specific D-hydantoinase activity at 37 degrees C compared with that at 30 degrees C. The plasmid was stably maintained over 80 generations. When glycerol was initially added to a concentration of 100 g/L, the final biomass concentration reached about 50 g-dry cell weight/L in a 50 L-scale fermentation, resulting in the specific enzyme production of 3.8 x 10(4) unit/g-dry cell weight in a soluble form. Glycerol-using batch cultivation of recombinant E. coli was found to be a cost-effective process for the mass production of industrially useful D-hydantoinase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 449-455, 1997.

Isolation of thermostable D-hydantoinase-producing thermophilicBacillus sp SD-1

SummaryA thermophilic bacterium which showed highest thermostability and activity of the hydantoinase was isolated from 1m000 thermophiles and identified to beBacillus sp. SD-1 according to morphological and physiological characteristics. The optimal growth temperature of the bacterium was about 60°C. The hydantoinase ofBacillus sp. SD-1 was strictly D-specific, and optimal pH and temperature were determined to be about 8.0 and 70°C, respectively. The D-hydantoinase was stable upto 70°C, and half-life of the enzyme was about 20 min at 80°C.

Stabilization of papain and lysozyme for application to cosmetic products

For cosmetic applications, papain or lysozyme was conjugated to a soluble biopolymer produced by Schizophyllum commune. Stability of the conjugated enzymes were significantly enhanced, such that more than 90% of the initial activity remained after a month storage at 45 °C, even in a cosmetic formulation including various oils and surfactants. Cosmetic lotion containing 1% papain conjugate was more effective in exfoliating stratum corneum of skin than the lotion containing 5% lactic acid, one of the popular exfoliating agents.

Optimization of a heterogeneous reaction system for the production of optically active D-amino acids using thermostable D-hydantoinase.

A thermostable D-hydantoinase from Bacillus stearothermophilus SD-1 was previously mass-produced by batch cultivation of the recombinant E. coli harboring the gene encoding the enzyme (Lee et al., 1997). In this work, we attempted to optimize the process for the production of N-carbamoyl-D-p-hydroxyphenylglycine, which is readily hydrolyzed to D-p-hydroxyphenylglycine under acidic conditions, from 5-(4-hydroxyphenyl)hydantoin using the mass-produced D-hydantoinase. In an effort to overcome the low solubility of the substrate, enzyme reaction was carried out in a heterogeneous system consisting of a high substrate concentration up to 300 g/L. In this reaction system, most of substrate is present in suspended particles. Optimal temperature and pH were determined to be 45 degrees C and 8.5, respectively, by taking into account the reaction rate and conversion yield. When the free enzyme was employed as a biocatalyst, enzyme loading higher than 300 unit/g-substrate was required to achieve maximum conversion. Use of whole cell enzyme resulted in maximum conversion even at lower enzyme loadings than the free enzyme, showing 96% conversion yield at 300 g/L substrate. The heterogeneous reaction system used in this work might be applied to the enzymatic production of other valuable compounds from a rarely water-soluble substrate.

Purification and characterization of thermostableD-hydantoinase from thermophilicbacillus stearothermophilus SD-1

A thermostable D-hydantoinase of thermophilicBacillus stearothermophilus SD-1 was purified to homogeneity using an immuno-affinity chromatography. The affinity chromatography that employed polyclonal antibody immobilized on Sepharose 4B was simple to operate and gave a purification yield of 60% of enzyme activity. Molecular mass of the enzyme was determined to be about 133.9 kDa by gel filtration chromatography and the molecular mass of the subunit was 54 kDa on SDS-PAGE. Mass spectrometric analyses were also performed for the determination of the molecular mass of the native enzyme and its subunit. The apparent molecular masses were 51.1 and 102.1 kDa for the subunit and native enzyme, respectively. Based on the molecular masses determined by these two methods, it is suggested that the D-hydantoinase exists as a dimeric conformation in the cell. Isoelectric pH of the enzyme was observed to be 4.47. It was found that the enzyme requires one manganese ion per molecule of enzyme for the activity. The optimal pH and temperature for the catalytic activity were about 8.0 and 65‡C., respectively. The half-life of the enzyme was estimated to be 30 min at 80‡C., confirming that the enzyme purified is one of the most thermostable D-hydantoinase reported so far. Kinetic constants of the enzyme for different substrates were also determined.

Cloning and Overexpression of Thermostable D‐Hydantoinase from Thermophile in E. coli and Its Application to the Synthesis of Optically Active D‐Amino Acids

We cloned the thermostable D-hydantoinase gene from B. stearothermophilus SD-1 into E. coli. The cloned gene was constitutively expressed by its own promoter, and the enzyme was produced in its soluble form. The specific activity of the recombinant E. coli was 30 times higher than that of B. stearothermophilus SD-1. The cultivation conditions were investigated for the overproduction of the enzyme, and the temperature was found to affect the plasmid content and the expression level of the enzyme. Recombinant E. coli was cultivated in 30-L batch fermentation, the cell concentration reached 25 g-DCW/L, and the specific activity was about 20,000 units/g-DCW. D-Hydantoinase produced from the recombinant E. coli could be successfully applied to the synthesis of N-carbamoyl-D-amino acid from the 5-monosubstituted hydantoin derivative.

Purification and characterization of thermostable d-hydantoinase from Bacillus thermocatenulatus GH-2

A thermostable d-hydantoinase was isolated from thermophilic Bacillus thermocatenulatus GH-2 and purified to homogeneity by using immunoaffinity chromatography. The molecular mass of the enzyme was determined to be about 230 kDa, and a value of 56 kDa was obtained as a molecular mass of the subunit on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that oligomeric structure of the enzyme is tetrameric. Isoelectric pH of the enzyme was found to be approx 4.3. The enzyme required Mn2+ for the activity and exhibited its highest activity with phenylhydantoin as a substrate. The optimal pH and temperature for catalytic activity were about 7.5 and 65°C, respectively. The half-life of the enzyme was estimated to be about 45 min at 80°C.

Use of methicillin and ampicillin mixture as a selective pressure in the cultivation of recombinant E. coli

Ampicillin was rapidly degraded by an extracellular β-lactamase, and was therefore ineffective to isolate plasmid-harboring cells in the cultivation of recombinant E. coli for the production of thermostable d-hydantoinase. An effective way of preventing the degradation of ampicillin, methicillin was employed as a β-lactamase inhibitor. A mixture of methicillin and ampicillin was observed to effectively function as a selective pressure, and consequently plasmid stability and enzyme productivity of recombinant E. coli were highly maintained.

Engineering the Thermostable d‐Hydantoinases from Two Thermophilic Bacilli Based on Their Primary Structures

Thermostable enzymes find wide applications to basic studies concerning protein stability as well as to development of industrial and specialty bioprocesses. In the process for the production of optically pure D-amino acids, obtained from the corresponding hydantoin derivatives using a microbial D-hydantoinase,1 limited substrate solubility and enzyme stability were posed as problems. In this context, we have isolated and characterized a thermostable D-hydantoinase from Bacillus stearothermophilus SD1,2 and the gene encoding the enzyme was cloned and its nucleotide sequence was determined.3 However, the low specificity of the enzyme from B. stearothermophilus SD1 toward hydantoin derivatives with an aromatic group at the 5 -position prompted us to isolate an enzyme with improved substrate specificity from B. thermocatenulatus GH2.4 The enzyme of B. thermocatenulatus GH2 was a tetramer and showed high specific activity toward aromatic substrates. On the other hand, previously isolated enzyme was composed of two identical subunits and had a low specific activity for aromatic substrates. In order to get some insights into the difference in the biochemical characteristics of the two enzymes, the gene coding for the enzyme from B. thermocatenulatus GH2 was cloned and expressed in E. coli, and its nucleotide sequence was determined. Based on the primary structures of the two enzymes, hybrid and truncated mutant enzymes were constructed, and their catalytic properties were compared with wild-type enzymes. Details are reported here.