scholarly journals Biochemical and Structural Characterization of a Novel Bacterial Tannase From Lachnospiraceae bacterium in Ruminant Gastrointestinal Tract

2021 ◽  
Vol 9 ◽  
Author(s):  
Lijun Guan ◽  
Kunlun Wang ◽  
Yang Gao ◽  
Jialei Li ◽  
Song Yan ◽  
...  
Keyword(s):  
Tannic Acid ◽  
Md Simulation ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Relative Activity ◽  
Hydrolyzable Tannins ◽  
Industrial Use ◽  
Hydrolysis Of ◽  
Lauryl Gallate

Tannases are a family of esterases that catalyze the hydrolysis of ester and depside bonds present in hydrolyzable tannins to release gallic acid. Here, a novel tannase from Lachnospiraceae bacterium (TanALb) was characterized. The recombinant TanALb exhibited maximal activity at pH 7.0 and 50°C, and it maintained more than 70% relative activity from 30°C to 55°C. The activity of TanALb was enhanced by Mg2+ and Ca2+, and was dramatically reduced by Cu2+ and Mn2+. TanALb is capable of degrading esters of phenolic acids with long-chain alcohols, such as lauryl gallate as well as tannic acid. The Km value and catalytic efficiency (kcat /Km) of TanALb toward five substrates showed that tannic acid (TA) was the favorite substrate. Homology modeling and structural analysis indicated that TanALb contains an insertion loop (residues 341–450). Based on the moleculer docking and molecular dynamics (MD) simulation, this loop was observed as a flap-like lid to interact with bulk substrates such as tannic acid. TanALb is a novel bacterial tannase, and the characteristics of this enzyme make it potentially interesting for industrial use.

Partial characterization of proteinase activity in the larval midgut of the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae)

1989 ◽  
Vol 67 (4) ◽  
pp. 864-868 ◽  
Author(s):  
Jon G. Houseman ◽  
B. J. R. Philogène ◽  
A. E. R. Downe
Keyword(s):  
Ethyl Ester ◽  
Ostrinia Nubilalis ◽  
European Corn Borer ◽  
Alimentary Tract ◽  
Maximal Activity ◽  
Corn Borer ◽  
Lepidoptera Pyralidae ◽  
Chymotryptic Activity ◽  
Hydrolysis Of

Protease activity in the alimentary tract of the European corn borer, Ostrinia nubilalis, can be attributed to at least three endoproteinases. A high alkaline trypsin with maximal hydrolysis of benzoyl arginine p-nitroanilide at pH values higher than 10.0, a low alkaline trypsin with maximal activity against benzoyl arginine ethyl ester at pH 9.0, and chymotrypsin, which hydrolyzed benzoyl tryosine ethyl ester at pH 7.5–8.0, were detected in gut homogenates. Total proteolysis, measured using azocasein, had maximal activity at pH 10.0 or higher. Corn borer chymotryptic activity had characteristics similar to the vertebrate enzyme. Both tryptic activities differed from vertebrate trypsin by being insensitive to ovomucoid trypsin inhibitor. High and low alkaline trypsin differed from each other by their pH optima. High alkaline trypsin was activated by magnesium and calcium, and low alkaline trypsin was not affected by inclusion of either chemical in the assay mixture.

scholarly journals Characterization of a Mannose-6-Phosphate Isomerase fromThermus thermophilusand Increased l-Ribose Production by Its R142N Mutant

2010 ◽  
Vol 77 (3) ◽  
pp. 762-767 ◽  
Author(s):  
Soo-Jin Yeom ◽  
Eun-Sun Seo ◽  
Bi-Na Kim ◽  
Yeong-Su Kim ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Wild Type ◽  
Active Site Residues ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Mannose 6 Phosphate

ABSTRACTAn uncharacterized gene fromThermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed inEscherichia coli. The maximal activity of the recombinant enzyme forl-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion ofl-ribulose tol-ribose, a potential starting material for manyl-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase inl-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. Thekcat/Kmof the R142N mutant was 3.8-fold higher than that ofGeobacillus thermodenitrificansmannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reportedkcat/Km. The R142N mutant enzyme produced 213 g/literl-ribose from 300 g/literl-ribulose for 2 h, with a volumetric productivity of 107 g liter−1h−1, which was 1.5-fold higher than that of the wild-type enzyme.

scholarly journals Glucosylglycerate Biosynthesis in the Deepest Lineage of the Bacteria: Characterization of the Thermophilic Proteins GpgS and GpgP from Persephonella marina

2006 ◽  
Vol 189 (5) ◽  
pp. 1648-1654 ◽  
Author(s):  
Joana Costa ◽  
Nuno Empadinhas ◽  
Milton S. da Costa
Keyword(s):  
Escherichia Coli ◽  
High Temperatures ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Thermophilic Bacterium ◽  
Cold Adapted ◽  
Dimeric Protein ◽  
Hydrolysis Of

ABSTRACT The pathway for the synthesis of glucosylglycerate (GG) in the thermophilic bacterium Persephonella marina is proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (GPG) synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). The sequences of gpgS and gpgP from the cold-adapted bacterium Methanococcoides burtonii were used to identify the homologues in the genome of P. marina, which were separately cloned and overexpressed as His-tagged proteins in Escherichia coli. The recombinant GpgS protein of P. marina, unlike the homologue from M. burtonii, which was specific for GDP-glucose, catalyzed the synthesis of GPG from UDP-glucose, GDP-glucose, ADP-glucose, and TDP-glucose (in order of decreasing efficiency) and from d-3-phosphoglycerate, with maximal activity at 90°C. The recombinant GpgP protein, like the M. burtonii homologue, dephosphorylated GPG and mannosyl-3-phosphoglycerate (MPG) to GG and mannosylglycerate, respectively, yet at high temperatures the hydrolysis of GPG was more efficient than that of MPG. Gel filtration indicates that GpgS is a dimeric protein, while GpgP is monomeric. This is the first characterization of genes and enzymes for the synthesis of GG in a thermophile.

scholarly journals Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity

2020 ◽  
Vol 21 (5) ◽  
pp. 1683 ◽  
Author(s):  
Yoko Suzumoto ◽  
Orly Dym ◽  
Giovanni N. Roviello ◽  
Franz Worek ◽  
Joel L. Sussman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Nerve Agents ◽  
Process Step ◽  
Homologous Genes ◽  
New Variant ◽  
Genes Encoding ◽  
Hydrolysis Of

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M−1 s−1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

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 Identification and partial characterization of an adenosine(5′)tetraphospho(5′)adenosine hydrolase on intact bovine aortic endothelial cells

1989 ◽  
Vol 259 (1) ◽  
pp. 97-103 ◽  
Author(s):  
A Ogilvie ◽  
J Lüthje ◽  
U Pohl ◽  
R Busse
Keyword(s):  
Endothelial Cells ◽  
Extracellular Fluid ◽  
Maximal Activity ◽  
Biologically Active ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Endothelial Surface ◽  
Hydrolysis Of ◽  
Aortic Endothelial

The biologically active dinucleotides adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and adenosine(5′)-triphospho(5′)adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.

scholarly journals CHARACTERIZATION OF THERMOSTABLE BETA-1,4-GALACTANASE AND ITS APPLICATION IN HYDROLYSIS OF PECTIN FROM SWEET POTATO (Ipomoea batatas (L.) Lam) PEELS

2021 ◽  
Vol 83 (5) ◽  
pp. 57-65
Author(s):  
Noor Faizah Ismail ◽  
Dayang Norulfairuz Abang Zaidel ◽  
Mohd Noor Mat Isa
Keyword(s):  
Sweet Potato ◽  
High Performance ◽  
Ipomoea Batatas ◽  
Catalytic Efficiency ◽  
Tween 20 ◽  
E Coli ◽  
Chromatography Analysis ◽  
Potato Peels ◽  
Hydrolysis Of

Galactooligosaccharides (GOS) synthesis has received much attention due to its prebiotic function. Beta-1,4-galactanase responsible for the hydrolysis of galactan plays an important role in producing GOS from biodegradation of this pectin component. In this study, beta-1,4-galactanase (BgcGC) from a thermophilic Geobacillus mahadii Geo-05 was heterologously expressed in Escherichia coli (E. coli) and characterized. The optimum temperature of BgcGC was at 60°C and stable from 20-60°C while optimum pH was at 6 and stable from pH 4-10. BgcGC showed high catalytic efficiency towards potato galactan (873.8 ml mg-1 s-1) and lupin galactan (1694.4 ml mg-1 s-1). The activity of BgcGC was not significantly affected with the presence of 100 mM K+, Tween-20 and 2-mercaptoethanol. Application of BgcGC towards pectin-containing galactan oligomer extracted from sweet potato peels resulted in galactose and GOS synthesis as revealed by high performance liquid chromatography analysis. Thus, this enzyme has a potential to be one of the enzyme candidates involves in pectin complex degradation to produce GOS.

scholarly journals Mutational effects on carbapenem hydrolysis of YEM-1, a new sub-class B2 metallo-β-lactamase from Yersinia mollaretii

2020 ◽  
Author(s):  
P. S. Mercuri ◽  
R. Esposito ◽  
S. Blétard ◽  
S. Di Costanzo ◽  
M. Perilli ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Catalytic Efficiency ◽  
The Other ◽  
Kinetic Characterization ◽  
Activity Profile ◽  
Gene Coding ◽  
Hydrolysis Of

ABSTRACTThe analysis of the genome sequence of Yersinia mollaretii (Y. mollaretii) ATCC 43969 indicates the presence of the blaYEM gene coding for YEM-1, a putative subclass B2 metallo-β-lactamase. The objectives of our work were to produce, purify and complete the kinetic characterization of YEM-1. Compared to the known subclass B2 metallo–β-lactamases, YEM-1 displayed a narrowest substrate profile since it is only able to hydrolyse imipenem with a high catalytic efficiency but not all the other carbapenems tested such as biapenem, meropenem, doripenem and ertapenem. A possible explanation of this peculiar activity profile is the presence of tyrosine 67 (loop L1), threonine 156 (loop L2) and serine 236 (loop L3) respectively. We showed that the substitution of Y67 broadened the activity profile of the enzyme for all carbapenems but still displayed a poor activity toward the other β-lactam classes.

scholarly journals Mineralization of the herbicide swep by a two-strain consortium and characterization of a new amidase for hydrolyzing swep

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Long Zhang ◽  
Ping Hang ◽  
Xiyi Zhou ◽  
Chen Dai ◽  
Ziyi He ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Maximal Activity ◽  
Contaminated Sites ◽  
Bacterial Strains ◽  
Catalytic Sites ◽  
Structural Analogues ◽  
Microbial Resources ◽  
Hydrolysis Of ◽  
Amidase Gene

Abstract Background Swep is an excellent carbamate herbicide that kills weeds by interfering with metabolic processes and inhibiting cell division at the growth point. Due to the large amount of use, swep residues in soil and water not only cause environmental pollution but also accumulate through the food chain, ultimately pose a threat to human health. This herbicide is degraded in soil mainly by microbial activity, but no studies on the biotransformation of swep have been reported. Results In this study, a consortium consisting of two bacterial strains, Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34, was enriched from a contaminated soil sample and shown to be capable of mineralizing swep. Swep was first transformed by Comamonas sp. SWP-3 to the intermediate 3,4-dichloroaniline (3,4-DCA), after which 3,4-DCA was mineralized by Alicycliphilus sp. PH-34. An amidase gene, designated as ppa, responsible for the transformation of swep into 3,4-DCA was cloned from strain SWP-3. The expressed Ppa protein efficiently hydrolyzed swep and a number of other structural analogues, such as propanil, chlorpropham and propham. Ppa shared less than 50% identity with previously reported arylamidases and displayed maximal activity at 30 °C and pH 8.6. Gly449 and Val266 were confirmed by sequential error prone PCR to be the key catalytic sites for Ppa in the conversion of swep. Conclusions These results provide additional microbial resources for the potential remediation of swep-contaminated sites and add new insights into the catalytic mechanism of amidase in the hydrolysis of swep.

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