scholarly journals Highly Active and Stable Large Catalase Isolated from a Hydrocarbon Degrading Aspergillus terreus MTCC 6324

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Preety Vatsyayan ◽  
Pranab Goswami
Keyword(s):  
Molecular Mass ◽  
Aspergillus Terreus ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Highly Active ◽  
Peptide Mass ◽  
Tandem Mass Spectroscopy ◽  
Isoelectric Ph ◽  
Alkaline Ph Stability ◽  
High Level

A hydrocarbon degrading Aspergillus terreus MTCC 6324 produces a high level of extremely active and stable cellular large catalase (CAT) during growth on n-hexadecane to combat the oxidative stress caused by the hydrocarbon degrading metabolic machinery inside the cell. A 160-fold purification with specific activity of around 66 × 105 U mg−1 protein was achieved. The native protein molecular mass was 368 ± 5 kDa with subunit molecular mass of nearly 90 kDa, which indicates that the native CAT protein is a homotetramer. The isoelectric pH (pI) of the purified CAT was 4.2. BLAST aligned peptide mass fragments of CAT protein showed its highest similarity with the catalase B protein from other fungal sources. CAT was active in a broad range of pH 4 to 12 and temperature 25°C to 90°C. The catalytic efficiency (Kcat/Km) of 4.7 × 108 M−1 s−1 within the studied substrate range and alkaline pH stability (half-life, t1/2 at pH 12~15 months) of CAT are considerably higher than most of the extensively studied catalases from different sources. The storage stability (t1/2) of CAT at physiological pH 7.5 and 4°C was nearly 30 months. The haem was identified as haem b by electrospray ionization tandem mass spectroscopy (ESI-MS/MS).

scholarly journals Genetic and Functional Analysis of the Soluble Oxaloacetate Decarboxylase from Corynebacterium glutamicum

2010 ◽  
Vol 192 (10) ◽  
pp. 2604-2612 ◽  
Author(s):  
Simon Klaffl ◽  
Bernhard J. Eikmanns
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Peptide Mass Fingerprinting ◽  
Transcriptional Analysis ◽  
Lysine Production ◽  
Ionization Time ◽  
Peptide Mass ◽  
Methyltransferase Gene ◽  
High Level

ABSTRACT Soluble, divalent cation-dependent oxaloacetate decarboxylases (ODx) catalyze the irreversible decarboxylation of oxaloacetate to pyruvate and CO2. Although these enzymes have been characterized in different microorganisms, the genes that encode them have not been identified, and their functions have been only poorly analyzed so far. In this study, we purified a soluble ODx from wild-type C. glutamicum about 65-fold and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and peptide mass fingerprinting for identification of the corresponding odx gene. Inactivation and overexpression of odx led to an absence of ODx activity and to a 30-fold increase in ODx specific activity, respectively; these findings unequivocally confirmed that this gene encodes a soluble ODx. Transcriptional analysis of odx indicated that there is a leaderless transcript that is organized in an operon together with a putative S-adenosylmethionine-dependent methyltransferase gene. Biochemical analysis of ODx revealed that the molecular mass of the native enzyme is about 62 ± 1 kDa and that the enzyme is composed of two ∼29-kDa homodimeric subunits and has a Km for oxaloacetate of 1.4 mM and a V max of 201 μmol of oxaloacetate converted per min per mg of protein, resulting in a k cat of 104 s−1. Introduction of plasmid-borne odx into a pyruvate kinase-deficient C. glutamicum strain restored growth of this mutant on acetate, indicating that a high level of ODx activity redirects the carbon flux from oxaloacetate to pyruvate in vivo. Consistently, overexpression of the odx gene in an l-lysine-producing strain of C. glutamicum led to accumulation of less l-lysine. However, inactivation of the odx gene did not improve l-lysine production under the conditions tested.

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 High-Level Formation of Active Pseudomonas cepaciaLipase after Heterologous Expression of the Encoding Gene and Its Modified Chaperone in Escherichia coli and Rapid In Vitro Refolding

1999 ◽  
Vol 65 (2) ◽  
pp. 787-794 ◽  
Author(s):  
Dinh Thi Quyen ◽  
Claudia Schmidt-Dannert ◽  
Rolf D. Schmid
Keyword(s):  
Escherichia Coli ◽  
Burkholderia Cepacia ◽  
Specific Activity ◽  
Gc Content ◽  
Pseudomonas Cepacia ◽  
Membrane Anchor ◽  
E Coli ◽  
Highly Active ◽  
High Level

ABSTRACT The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible λPRL promoter. However, no overexpression of the lipase chaperone was achieved in E. coli. Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P. cepacia per mg. Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5′ region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5′ region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids. Only truncation of the gene led to overexpression of the chaperone (up to 60%) in E. coli. With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active Pseudomonas lipase (classes I and II) expressed inE. coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E. coli wet cells.

Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Biosynthesis in Yeasts

2019 ◽  
pp. 30-41 ◽  
Author(s):  
E.P. Sannikova ◽  
A.V. Malysheva ◽  
F.A. Klebanov ◽  
D.G. Kozlov
Keyword(s):  
New Technologies ◽  
Specific Activity ◽  
Point Mutations ◽  
Egg Yolk ◽  
Enzyme Preparations ◽  
Highly Active ◽  
High Calcium ◽  
Pla2 Enzyme ◽  
Streptomyces Violaceoruber ◽  
The Cost

The capacity of yeast to produce the highly active variants of PLA2 has been confirmed. The high-active variants were based on the original enzyme from the strain А-2688 of Streptomyces violaceoruber. To reduce the enzyme toxicity and to increase its expression, various approaches were tested including point mutations, construction of artificial N- and/or C-end pro-regions, hybridization with other proteins and engineering or inactivation of glycosylation sites. As a main result, the modified PLA2 enzymes were obtained which have the same secretion level as their low-active predecessors, but specific activity of which was at least tenfold higher. As the main feature, the selected mutants were characterized by a lower affinity for Ca2+ that probably accounts for their low toxicity (and high expression capacity) at the stage of biosynthesis and their ability to activate under special conditions, e.g. during the egg yolk fermentation. The data obtained can provide a basis for the cost reduction of highly active PLA2 enzyme preparations in industries where the application of high calcium concentrations is allowed. recombinant phospholipase А2, Streptomyces violaceoruber, yeasts, secretion, producer strain The work was initiated by the Innovation Center Biriuch - New Technologies, Ltd., and was supported within the framework of the State Assignment no. 595-00004-18 PR.

scholarly journals Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Hui-Hui Su ◽  
Fei Peng ◽  
Pei Xu ◽  
Xiao-Ling Wu ◽  
Min-Hua Zong ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Rational Design ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Value Added ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Glucaric Acid ◽  
Triple Mutant ◽  
Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

scholarly journals Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears

2002 ◽  
Vol 366 (2) ◽  
pp. 511-520 ◽  
Author(s):  
Benjamin L. SCHULZ ◽  
David OXLEY ◽  
Nicolle H. PACKER ◽  
Niclas G. KARLSSON
Keyword(s):  
Molecular Mass ◽  
Peptide Mass Fingerprinting ◽  
High Molecular Mass ◽  
Tear Fluid ◽  
Protein Variant ◽  
Peptide Mass ◽  
Composite Gel ◽  
Molecular Masses ◽  
Protein Components ◽  
Sialyl Lea

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex0→2NeuAc1→xHexxHexNAcx(-ol), x = 1–6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Lea epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

scholarly journals RamB, a Novel Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

2004 ◽  
Vol 186 (9) ◽  
pp. 2798-2809 ◽  
Author(s):  
Robert Gerstmeir ◽  
Annette Cramer ◽  
Petra Dangel ◽  
Steffen Schaffer ◽  
Bernhard J. Eikmanns
Keyword(s):  
Corynebacterium Glutamicum ◽  
Isocitrate Lyase ◽  
Transcriptional Regulator ◽  
Regulatory Elements ◽  
Malate Synthase ◽  
Acetate Kinase ◽  
Acetate Metabolism ◽  
Peptide Mass ◽  
Specific Activities ◽  
High Level

ABSTRACT The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions. Directed deletion of the ramB gene in the genome of C. glutamicum resulted in mutant strain RG1. Whereas the wild type of C. glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum.

Protein hydrolysates from silkworm (Bombyx mori) pupae protein treated with a novel neutral protease

2021 ◽  
pp. 1-18
Author(s):  
R.A. Herman ◽  
Z.-N. Li ◽  
C. Xie ◽  
J.-Z. Wang ◽  
Y. Hu ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Water Solubility ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Apparent Molecular Weight ◽  
Nutritional Composition ◽  
Neutral Protease ◽  
Degree Of Hydrolysis ◽  
Silkworm Pupae ◽  
Silkworm Bombyx Mori

Edible insects, regarded as a potential contributor to food security are currently given wide consideration due to their rich protein and other micronutrients contents. In this study, protease-assisted hydrolysis proposes an economically effective approach to hydrolyse proteins from silkworm (Bombyx mori) pupae to improve its functional properties. The proteolytic activity of a novel neutral protease (265.14 U/ml) with appreciable thermal activities, was identified using 16S rDNA as Stenotrophomonas maltophilia JW20 (SmNP20). The neutral protease with an apparent molecular weight of 28 kDa emerged active at pH 7 and maintained stability in pH range 6.0-8.0. The optimum temperature was 60 °C and stable at 55-60 °C, maintaining over 80% of its initial activity, with a half-life of 78.75, 89, 66.8 and 44 min at 50, 60, 70 and 80 °C. It was purified to 9.98-fold with a specific activity of 455.06 U/mg and 63.73% yield. The Km and Vmax values were 0.70 mg/ml and 9.48 μmol/min/mg, respectively. Enzymolysis with neutral protease enhanced the degree of hydrolysis (97.46±4.87%), increased water solubility over 50%, and a significant protein solubility of 63.44±0.65%. The Km and Vmax of the protein yield were 0.24 mg/ml and 165.63 μmol/min/mg respectively. A total of 17 amino acids have been detected in the hydrolysates obtained from the silkworm pupae protein. In comparison with neutrase and flavorzyme®, the enzyme possesses an elevated hydrolytic and catalytic efficiency. Emulsion activity and foam capacity ranged from 8-48 m2/g and 6-25% respectively. Hence, this study confirms the unique and efficient characteristics of an insect-enzyme correlation that is practically significant with potential improvement in nutritional composition and functional quality of insect proteins.

scholarly journals [Phosphotyrosine]protein phosphatase in rat brain. A major [phosphotyrosine]protein phosphatase is a 23 kDa protein distinct from acid phosphatase

1986 ◽  
Vol 239 (1) ◽  
pp. 155-162 ◽  
Author(s):  
M Okada ◽  
K Owada ◽  
H Nakagawa
Keyword(s):  
Acid Phosphatase ◽  
Molecular Mass ◽  
Rat Brain ◽  
Column Chromatography ◽  
Protein Phosphatase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Sodium Vanadate ◽  
Nitrophenyl Phosphate ◽  
Phosphotyrosine Protein Phosphatase

A [phosphotyrosine]protein phosphatase (PTPPase) was purified almost to homogeneity from rat brain, with [32P]p130gag-fps, an oncogene product of Fujinami sarcoma virus, as substrate. The characteristics of the purified preparation of PTPPase were as follows: the enzyme was a monomer with a molecular mass of 23 kDa; its optimum pH was 5.0-5.5; its activity was not dependent on bivalent cations; its activity was strongly inhibited by sodium vanadate, but was not inhibited by ZnCl2, L(+)-tartrate or NaF; it catalysed the dephosphorylation of [32P]p130gag-fps, [[32P]Tyr]casein, p-nitrophenyl phosphate and L-phosphotyrosine, but did not hydrolyse [[32P]Ser]tubulin, L-phosphoserine, DL-phosphothreonine, 5′-AMP, 2′-AMP or beta-glycerophosphate significantly. During the purification, most of the PTPPase activity was recovered in distinct fractions from those of conventional low-molecular-mass acid phosphatase (APase), which was reported to be a major PTPPase [Chernoff & Li (1985) Arch. Biochem. Biophys. 240, 135-145], from DE-52 DEAE-cellulose column chromatography, and those two enzymes could be completely separated by Sephadex G-75 column chromatography. APase also showed PTPPase activity with [32P]p130gag-fps, but the specific activity was lower than that of PTPPase with molecular mass of 23 kDa, and it was not sensitive to sodium vanadate. These findings suggested that PTPPase (23 kDa) was the major and specific PTPPase in the cell.

Purification and Partial Amino Acid Sequence of Pentocin 31-1, an Anti-Listeria Bacteriocin Produced by Lactobacillus pentosus 31-1

2009 ◽  
Vol 72 (12) ◽  
pp. 2524-2529 ◽  
Author(s):  
JINLAN ZHANG ◽  
GUORONG LIU ◽  
NAN SHANG ◽  
WANPENG CHENG ◽  
SHANGWU CHEN ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mass Spectrometry Analysis ◽  
Gel Chromatography ◽  
Original Activity ◽  
Lactobacillus Pentosus ◽  
Spectrometry Analysis

Pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1 from the traditional Chinese fermented Xuan-Wei ham, was successfully purified by the pH-mediated cell adsorption-desorption method and then purified by gel chromatography with Sephadex G-10. The purification resulted in a 1,381.9-fold increase in specific activity with a yield of 76.8% of the original activity. Using Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of the purified peptide was found to be between 3,500 and 6,400 Da, and bacteriocin activity was confirmed by overlayer techniques. When subjected to mass spectrometry analysis, the protein was highly pure and its molecular mass was 5,592.225 Da. The partial N-terminal sequence of pentocin 31-1 was the following: NH2-VIADYGNGVRXATLL. Compared with the sequence of other bacteriocins, pentocin 31-1 has the consensus sequence YGNGV in its N-terminal region, and therefore it belongs to the class IIa of bacteriocins.

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