scholarly journals Molecular Cloning and Characterization of Fengycin Synthetase Gene fenB from Bacillus subtilis

1998 ◽  
Vol 180 (5) ◽  
pp. 1338-1341 ◽  
Author(s):  
Guang-Huey Lin ◽  
Chyi-Liang Chen ◽  
Johannes Scheng-Ming Tschen ◽  
San-San Tsay ◽  
Yu-Sun Chang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Optimum Temperature ◽  
Turnover Number ◽  
Optimum Ph

ABSTRACT A fengycin synthetase gene, fenB, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed inEscherichia coli and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25°C, an optimum pH of 4.5, aKm value of 922 μM, and a turnover number of 236 s−1. FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.

Properties of a non collagen-degradingBacillus subtiliskeratinase

2008 ◽  
Vol 54 (3) ◽  
pp. 180-188 ◽  
Author(s):  
Alexandre José Macedo ◽  
Walter Orlando Beys da Silva ◽  
Carlos Termignoni
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Detectable Effect ◽  
Amino Acid Residues ◽  
Keratinolytic Activity ◽  
Biotechnological Potential ◽  
Optimum Ph

Bacillus subtilis S14 produces a keratinase (KerS14) with non collagen-degrading activity. Indeed, this is the first keratinase described so far that does not have any detectable effect on collagen, which is a crucial property for an enzyme intended to be used in skin dehairing. Because of its importance as an industrial tanning enzyme, we report the biochemical characterization of KerS14. This protein exhibited an apparent molecular mass of 27 kDa, a pI of 6.5, and an optimum pH in the range of 8.0–9.0. The enzyme’s activity was stimulated by Mn2+(7.7-fold), Ca2+(6.1-fold), Mg2+(4.9-fold), and Co2+(4.0-fold) but was inhibited by Cu2+and Zn2+. Using p-nitroanilide and methylcoumarine derivatized peptides, we observed that KerS14 prefered Arg at subsite P1, small amino acid residues at subsite P2, and Gln or Glu at subsite P3. KerS14 presented higher keratin degradation specificity than other commercial proteases. Its high keratinolytic activity and the absence of virtually any activity against collagen remark the biotechnological potential of this enzyme to be used at larger scales in tannery dehairing processes.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals Characterization of an Agrobacterium tumefaciensd-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

2006 ◽  
Vol 72 (2) ◽  
pp. 981-985 ◽  
Author(s):  
Hye-Jung Kim ◽  
Eun-Kyung Hyun ◽  
Yeong-Su Kim ◽  
Yong-Joo Lee ◽  
Deok-Kun Oh
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Molecular Mass ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Turnover Number ◽  
Conversion Yield ◽  
Equilibrium Ratio

ABSTRACT The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (k cat) and catalytic efficiency (k cat/Km ) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%.

scholarly journals Isolation, Purification, and Characterization of the PR Oxidase from Penicillium roqueforti

1998 ◽  
Vol 64 (12) ◽  
pp. 5012-5015 ◽  
Author(s):  
Shenq-Chyi Chang ◽  
Wen-Yee Lei ◽  
Ying-Chieh Tsai ◽  
Yau-Huei Wei
Keyword(s):  
Molecular Mass ◽  
Extracellular Enzyme ◽  
Culture Broth ◽  
Penicillium Roqueforti ◽  
Optimum Temperature ◽  
Purification And Characterization ◽  
Optimum Ph

ABSTRACT The PR oxidase, an extracellular enzyme, involved in the conversion of PR toxin into PR acid, was purified from the culture broth ofPenicillium roqueforti ATCC 48936. The enzyme has a pI of 4.5 and a molecular mass of approximately 88 kDa, and it is a monomer. The optimum pH for this enzyme is ca. 4.0, and the optimum temperature is 50°C.

Identification and characterization of novel broad-spectrum amino acid racemases from Escherichia coli and Bacillus subtilis

Amino Acids ◽  
2017 ◽  
Vol 49 (11) ◽  
pp. 1885-1894 ◽  
Author(s):  
Tetsuya Miyamoto ◽  
Masumi Katane ◽  
Yasuaki Saitoh ◽  
Masae Sekine ◽  
Hiroshi Homma
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Broad Spectrum ◽  
Identification And Characterization

scholarly journals Molecular Cloning, Purification, and Biochemical Characterization of Hydantoin Racemase from the Legume Symbiont Sinorhizobium meliloti CECT 4114

2004 ◽  
Vol 70 (1) ◽  
pp. 625-630 ◽  
Author(s):  
Sergio Martínez-Rodríguez ◽  
Francisco Javier Las Heras-Vázquez ◽  
Lydia Mingorance-Cazorla ◽  
Josefa María Clemente-Jiménez ◽  
Felipe Rodríguez-Vico
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Molecular Cloning ◽  
Sinorhizobium Meliloti ◽  
Biochemical Characterization ◽  
Competitive Inhibition ◽  
Optimum Temperature ◽  
Aromatic Rings ◽  
Native Form

ABSTRACT Hydantoin racemase from Sinorhizobium meliloti was functionally expressed in Escherichia coli. The native form of the enzyme was a homotetramer with a molecular mass of 100 kDa. The optimum temperature and pH for the enzyme were 40°C and 8.5, respectively. The enzyme showed a slight preference for hydantoins with short rather than long aliphatic side chains or those with aromatic rings. Substrates, which showed no detectable activity toward the enzyme, were found to exhibit competitive inhibition.

scholarly journals Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity.

1987 ◽  
Vol 262 (8) ◽  
pp. 3754-3761
Author(s):  
A.J. Ganzhorn ◽  
D.W. Green ◽  
A.D. Hershey ◽  
R.M. Gould ◽  
B.V. Plapp
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residue ◽  
Kinetic Characterization ◽  
Alcohol Dehydrogenases

Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis

2021 ◽  
Author(s):  
Ryushi Kawakami ◽  
Chinatsu Kinoshita ◽  
Tomoki Kawase ◽  
Mikio Sato ◽  
Junji Hayashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Enzyme Stability ◽  
Hyperthermophilic Archaea ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
Aminobutyrate Aminotransferase ◽  
Chaperone Protein ◽  
Amino Acid Racemase

Abstract The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by co-expression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and non-substrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

Immobilization and Characterization of Tannase and its Haze-removing

2009 ◽  
Vol 15 (6) ◽  
pp. 545-552 ◽  
Author(s):  
Erzheng Su ◽  
Tao Xia ◽  
Liping Gao ◽  
Qianying Dai ◽  
Zhengzhu Zhang
Keyword(s):  
Kinetic Parameter ◽  
High Activity ◽  
Green Tea ◽  
Storage Stability ◽  
Residual Activity ◽  
Black Tea ◽  
Optimum Temperature ◽  
Oolong Tea ◽  
Optimum Ph

Tannase was effectively immobilized on alginate by the method of crosslinking-entrapment-crosslinking with a high activity recovery of 76.6%. The properties of immobilized tannase were investigated. Its optimum temperature was determined to be 35 ° C, decreasing 10 °C compared with that of free enzyme, whereas the optimum pH of 5.0 did not change. The thermal and pH stabilities of immobilized tannase increased to some degree. The kinetic parameter, Km, for immobilized tannase was estimated to be 11.6 × 10-4 mol/L. Fe2+ and Mn2+ could activate the activity of immobilized tannase. The immobilized tannase was also applied to treat the tea beverage to investigate its haze-removing effect. The content of non-estern catechins in green tea, black tea and oolong tea increased by 52.17%, 12.94% and 8.83%, respectively. The content of estern catechins in green tea, oolong tea and black tea decreased by 20.0%, 16.68% and 5.04%, respectively. The anti-sediment effect of green tea infusion treated with immobilized tannase was significantly increased. The storage stability and reusability of the immobilized tannase were improved greatly, with 72.5% activity retention after stored for 42 days and 86.9% residual activity after repeatedly used for 30 times.

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