Moon-Hee Sung

Geobacillus toebii sp. nov., a novel thermophilic bacterium isolated from hay compost

A thermophilic, spore-forming rod isolated from hay compost in Korea was subjected to a taxonomic study. The micro-organism, designated strain SK-1(T), was identified as being aerobic, Gram-positive, motile and rod-shaped. Growth of the isolate was observed at 45-70 degrees C (optimum 60 degrees C) and pH 6.0-9.0 (optimum pH 7.5). The G+C content of the genomic DNA was 43.9 mol%. Chemotaxonomic characteristics of the isolate included the presence of mesodiaminopimelic acid in the cell wall and iso-C15:0 and iso-C17:0 as the major cellular fatty acids. The predominant isoprenoid quinone was MK-7. The chemotaxonomic characteristics of strain SK-1(T) were the same as those of the genus Geobacillus. Phylogenetic analysis based on 16S rDNA sequences showed that strain SK-1(T) is most closely related to Geobacillus thermoglucosidasius. However, the phenotypic properties of strain SK-1(T) were clearly different from those of G. thermoglucosidasius. The level of DNA-DNA relatedness between strain SK-1(T) and the type strain of G. thermoglucosidasius was 27%. On the basis of the phenotypic traits and molecular systematic data, strain SK-1(T) represents a novel species within the genus Geobacillus, for which the name Geobacillus toebii sp. nov. is proposed. The type strain is strain SK-1(T) (= KCTC 0306BP(T) - DSM 14590(T)).

Thermostable D-hydantoinase from thermophilic Bacillus stearothermophilus SD-1 : characteristics of purified enzyme

One thousand thermophiles isolated from soils were screened for hydantoinase and its thermostability. One thermophilic bacterium that showed the highest thermostability and activity of hydantoinase was identified to be Bacillus stearothermophilus SD-1 according to morphological and physiological characteristics. The hydantoinase of B. stearothermophilus SD-1 was purified to homogeneity via ammonium sulfate fractionation, anion-exchange chromatography, heat treatment, hydrophobic-interaction chromatography, and preparative gel electrophoresis. The relative molecular mass of the hydantoinase was determined to be 126 kDa by gel-filtration chromatography, and a value of 54 kDa was obtained as a molecular mass of the subunit on analytical sodiumdodecylsulfate/polyacrylamide gel electrophoresis. The hydantoinase was strictly d-specific and metal-dependent. The optimal pH and temperature were about 8.0 and 65°C respectively, and the half-life of the d-hydantoinase was estimated to be 30 min at 80°C, indicating the most thermostable enzyme so far.

Novel high-level constitutive expression system, pHCE vector, for a convenient and cost-effective soluble production of human tumor necrosis factor-α

A new E. coli expression system, the pHCE-IIB vector, using a constitutively expressing HCE promoter derived upstream from the d-amino acid aminotransferase (d-AAT) gene of Geobacillus toebii, was developed for the high-level expression of foreign proteins without induction. The expression efficiency of recombinant human tumor necrosis factor-α (rhTNFα) using pHCE-IIB was compared with that of the pET14B vector under the control of a T7 promoter induced by IPTG. The total amount of rhTNFα produced by the pHCE system was approximately twice that produced by the pET system under optimal fermentation conditions thereby demonstrating the convenience and economical advantage of the new constitutive pHCE-IIB expression vector over the conventional pET expression vector.

Development of an enzymatic system for the production of dopamine from catechol, pyruvate, and ammonia

To produce dopamine from catechol, pyruvate, and ammonia, an enzymatic process consisting of a two-step reaction, catechol → l-DOPA → dopamine, was developed. For the first reaction step to synthesize l-DOPA, tyrosine phenol-lyase of Symbiobacterium sp. SC-1 was used successfully as a biocatalyst, resulting in the high conversion yield of 92%. Two aromatic amino acid decarboxylases, rat liver l-DOPA decarboxylase and Streptoccus faecalis tyrosine decarboxylase (TDC), were tested for the subsequent step to produce dopamine. In investigating the effect of l-DOPA concentration, a serious substrate inhibition of l-DOPA decarboxylase activity was observed at concentrations over 1 mM, while no inhibition was detected for TDC up to 40 mM l-DOPA. Therefore, the TDC of S. faecalis was selected as the biocatalyst for the second reaction step. Enzymatic conversion of l-DOPA to dopamine was carried out in a reactor controlling the reaction pH with an HCl solution containing pyridoxal 5′-phosphate, to compensate for the loss of pyridoxal 5′-phosphate by an enzyme-catalyzed side reaction, i.e. decarboxylation-dependent transamination. When the enzyme reactor was operated at 37°C for 12 h, 100 mM of l-DOPA was converted to dopamine with the conversion yield of 100%. Simultaneous reactions of tyrosine phenol-lyase and TDC were tested for direct synthesis of dopamine, but the productivity was much lower than the separated two-step reactions.

A novel microbial interaction: obligate commensalism between a new gram- negative thermophile and a thermophilic Bacillus strain

Abstract Obligately commensal interaction between a new gram-negative thermophile and a thermophilic Bacillus strain was investigated. From compost samples, a mixed culture showing tyrosine phenol-lyase activity was enriched at 60°C. The mixed culture consisted of a thermophilic gram-negative strain, SC-1, and a gram-positive spore-forming strain, SK-1. In mixed cultures, strain SC-1 started to grow only when strain SK-1 entered the stationary phase. Although strain SC-1 showed tyrosine phenol lyase activity, we could not isolate a colony with any nutrient medium. For the isolation and cultivation of strain SC-1, we added culture supernatant and cell extract of the mixed culture to the basal medium. The supernatant and cell extract of the mixed culture contained heat-stable and heat-labile factors, respectively, that are essential to the growth of strain SC-1. During pure cultures of strain SK-1, the heat-stable growth factors were released during the growth phase and the heat-labile growth factors were produced intracellularly at the early stationary phase. Strain SC-1 was gram-negative and microaerophilic, and grows optimally at 60°C. Based on these results, we propose a novel commensal interaction between a new gram-negative thermophile, strain SC-1, and Bacillus sp. strain SK-1.

Removal and bioconversion of phenol in wastewater by a thermostable β-tyrosinase

Abstract This study explores an enzymatic method for removing phenol from the wastewater created during the manufacture of phenolic resin. The enzyme used was a thermostable β-tyrosinase catalyzing the synthesis of l -tyrosine from phenol, pyruvate, and ammonia. As the reaction proceeds, l -tyrosine precipitates as insoluble aggregates because l -tyrosine is barely soluble in water. The enzymatic removal of phenol was effective at pH values ranging from 6.5–9.0 and temperatures below 70°C. The optimal concentration of each substrate was determined as 60 m m phenol, 0.1 m pyruvate, and 0.4 m ammonia. When the enzyme was used in an intact cell or acetone-dried cell state instead of the cell-free extract, the optimal concentration of phenol was increased up to 120 m m . By treating wastewater containing 100 m m phenol with acetone-dried cells at 37°C, we could reduce the concentration of phenol to 8 m m within 24 h.

Site-directed mutagenesis of the amino acid residues in β-strand III [Val30-Val36] of d-amino acid aminotransferase of Bacillus sp. YM-1

The β‐strand III formed by amino acid residues Val30‐Val36 is located across the active site of the thermostable d‐amino acid aminotransferase (d‐AAT) from thermophilic Bacillus sp. YM‐1, and the odd‐numbered amino acids (Tyr31, Val33, Lys35) in the strand are revealed to be directed toward the active site. Interestingly, Glu32 is also directed toward the active site. We first investigated the involvement of these amino acid residues in catalysis by alanine scanning mutagenesis. The Y31A and E32A mutant enzymes showed a marked decrease in k cat value, retaining less than 1% of the wild‐type enzyme activity. The k cat values of V33A and K35A were changed slightly, but the K m of K35A for α‐ketoglutarate was increased to 35.6 mM, compared to the K m value of 2.5 mM for the wild‐type enzyme. These results suggested that the positive charge at Lys35 interacted electrostatically with the negative charge at the side chain of α‐ketoglutarate. Site‐directed mutagenesis of the Glu32 residue was conducted to demonstrate the role of this residue in detail. From the kinetic and spectral characteristics of the Glu32‐substituted enzymes, the Glu32 residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5′‐phosphate (PLP) and the ε‐amino group of the Lys145 residue.

Simple and rapid screening method for microbial D-stereospecific peptidase and esterase

The use of a turbid plate containing Z-D-Ala-D-AlaOBzl was a convenient method for the screening of microorganisms with D-stereospecific peptidase activity as well as D-stereospecific esterase activity. This paper discusses the suitability of the developed method and its application to molecular cloning of D-stereospecifc peptidase and esterase genes.