Gene cloning, functional expression and characterization of a novel GH46 chitosanase from Streptomyces avermitilis (SaCsn46A)

2021 ◽  
Author(s):  
Jing Guo ◽  
Yi Wang ◽  
Wenjun Gao ◽  
Xinrou Wang ◽  
Xin Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Functional Expression ◽  
Streptomyces Avermitilis ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Glycosidic Bonds ◽  
Sds Page ◽  
Mature Enzyme ◽  
Layer Chromatography

Abstract A novel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acids signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 oC and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 oC. The Km and Vmax values of this enzyme were 1.32 mg∙mL− 1, 526.32 µM∙mg− 1∙min− 1, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3 mM concentration. The enzyme can hydrolyze a variety of polysaccharides which linked by β-1,4-glycosidic bonds such as chitin, xylan and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)2. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.

scholarly journals Functional Expression and Characterization of Trichome-Specific (-)-Limonene Synthase and (+)-α-Pinene Synthase from Cannabis sativa

2007 ◽  
Vol 2 (3) ◽  
pp. 1934578X0700200 ◽  
Author(s):  
Nils Günnewich ◽  
Jonathan E. Page ◽  
Tobias G. Köllner ◽  
Jörg Degenhardt ◽  
Toni M. Kutchan
Keyword(s):  
Phylogenetic Analysis ◽  
Natural Products ◽  
Cannabis Sativa ◽  
Functional Expression ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Limonene Synthase ◽  
Monoterpene Synthases

Two recombinant, stereospecific monoterpene synthases, a (-)-limonene synthase (CsTPS1) and a (+)-α-pinene synthase (CsTPS2), encoded by Cannabis sativa L. cv. ‘Skunk’ trichome mRNA, have been isolated and characterized. Recombinant CsTPS1 showed a Km value of 6.8 μM, a Vmax of 1.1 × 10−4 μmol/min and Vmax/Km of 0.016; the pH optimum was determined at pH 6.5, and a temperature optimum at 40°C. Recombinant CsTPS2 showed a Km value of 10.5 μM, a Vmax of 2.2 × 10−4 μmol/min and Vmax/Km of 0.021; the pH optimum was determined at pH 7.0, and a temperature optimum at 30°C. Phylogenetic analysis showed that both CsTPSs group within the angiosperms and belong to the Tpsb subgroup of monoterpene synthases. The enzymatic products (-)-limonene and (+)-α-pinene were detected as natural products in C. sativa trichomes.

scholarly journals Isolation and partial characterization of protease from Pseudomonas aeruginosa ATCC 27853

2010 ◽  
Vol 75 (8) ◽  
pp. 1041-1052 ◽  
Author(s):  
Lidija Izrael-Zivkovic ◽  
Gordana Gojgic-Cvijovic ◽  
Ivanka Karadzic
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Zinc Ion ◽  
Metal Chelators ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Sds Page ◽  
Potential Use

Enzymatic characteristics of a protease from medically important, referent strain of Pseudomonas aeruginosa ATCC 27853 were determined. According to SDS PAGE and gel filtration it was estimated that molecular mass of the purified enzyme was about 15 kDa. Other enzymatic properties were found to be: pH optimum 7.1, pH stability between pH 6.5 and pH 10; temperature optimum around 60?C while the enzyme was stable at 60?C for 30 min. The inhibition of the enzyme was observed with the metal chelators such as EDTA and 1,10- phenanthroline, suggesting that the protease is a metalloenzyme. Further more it was determined that enzyme contains one mole of zinc ion per mole of enzyme. The protease is stable in the presence of different organic solvents, which enable potential use for synthesis of peptides.

Production of Inulinase by Fusarium sp. and its Application for Fructo-oligosaccharide Production for use as Prebiotics

2017 ◽  
Vol 3 (1) ◽  
pp. 45
Author(s):  
Neera . ◽  
K. V. Ramana ◽  
N. Gopalan ◽  
R. K. Sharma
Keyword(s):  
Incubation Temperature ◽  
Carbon Substrate ◽  
Fusarium Spp ◽  
Ph Optimum ◽  
Sds Page ◽  
Initial Ph ◽  
Fusarium Sp ◽  
Optimization Of Fermentation ◽  
Layer Chromatography

Fructooligosaccharides (FOS) are useful due to their applications in food and pharmaceutical industry. Fusarium spp., isolated from dahlia rhizosphere, produced endoinulinases in a medium containing inulin or sucrose as carbon substrate. In this study, characterization of exo-inulinase and production of FOS were investigated. Temperature and pH optimum of the enzyme was found to be 60°C and pH 6.0, respectively. The optimization of fermentation conditions for inulinase production was carried out using one at a time approach. The optimized medium with sucrose as a carbon source and yeast extract as a nitrogen source were found to be optimal for inulinase production at an initial pH 5.0 and incubation temperature 30 °C for 120 h. Under these conditions, the maximum inulinase concentration of 131.6 U/ml was achieved. SDS PAGE revealed that the molecular weight of the enzyme was around 90 kDa. Further study revealed that Fusarium sp. could produce inulinase as well as invertase. Thin layer chromatography was carried out to analyze the FOS production and their oligomeric properties. Inulin as well FOS can be used as prebiotics as they are selectively fermented by Bifidobacteria and Lactic acid bacteria and thus imparting health benefits.

scholarly journals Purification and characterization of cytosolic pyruvate kinase from banana fruit

2000 ◽  
Vol 352 (3) ◽  
pp. 875-882 ◽  
Author(s):  
William L. TURNER ◽  
William C. PLAXTON
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Banana Fruit ◽  
Ph Optimum ◽  
Cytosolic Ph ◽  
Pep Carboxylase ◽  
Sds Page ◽  
Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

scholarly journals Characterization of an Acid-Dependent Arginine Decarboxylase Enzyme from Chlamydophila pneumoniae

2007 ◽  
Vol 189 (20) ◽  
pp. 7376-7383 ◽  
Author(s):  
Teresa N. Giles ◽  
David E. Graham
Keyword(s):  
Acid Resistance ◽  
Arginine Decarboxylase ◽  
Chlamydophila Pneumoniae ◽  
Host Cells ◽  
Respiratory Pathogen ◽  
Ph Optimum ◽  
Polyamine Biosynthesis ◽  
Mature Enzyme ◽  
Oxide Synthase

ABSTRACT Genome sequences from members of the Chlamydiales encode diverged homologs of a pyruvoyl-dependent arginine decarboxylase enzyme that nonpathogenic euryarchaea use in polyamine biosynthesis. The Chlamydiales lack subsequent genes required for polyamine biosynthesis and probably obtain polyamines from their host cells. To identify the function of this protein, the CPn1032 homolog from the respiratory pathogen Chlamydophila pneumoniae was heterologously expressed and purified. This protein self-cleaved to form a reactive pyruvoyl group, and the subunits assembled into a thermostable (αβ)3 complex. The mature enzyme specifically catalyzed the decarboxylation of l-arginine, with an unusually low pH optimum of 3.4. The CPn1032 gene complemented a mutation in the Escherichia coli adiA gene, which encodes a pyridoxal 5′-phosphate-dependent arginine decarboxylase, restoring arginine-dependent acid resistance. Acting together with a putative arginine-agmatine antiporter, the CPn1032 homologs may have evolved convergently to form an arginine-dependent acid resistance system. These genes are the first evidence that obligately intracellular chlamydiae may encounter acidic conditions. Alternatively, this system could reduce the host cell arginine concentration and produce inhibitors of nitric oxide synthase.

scholarly journals Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease

1996 ◽  
Vol 313 (2) ◽  
pp. 423-429 ◽  
Author(s):  
Rajamma USHA ◽  
Manoranjan SINGH
Keyword(s):  
Physiological Role ◽  
Immunoblot Analysis ◽  
Protein Band ◽  
Winged Bean ◽  
Psophocarpus Tetragonolobus ◽  
Ph Optimum ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Storage Protein Mobilization

Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca2+-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

Partial Purification and Characterization of S-Adenosyl-ʟ-Methionine: Norreticuline N-Methyltransferases from Berberis Cell Suspension Cultures

1986 ◽  
Vol 41 (1-2) ◽  
pp. 126-134 ◽  
Author(s):  
Chi-Kit Wat ◽  
Paul Steffens ◽  
Meinhart H. Zenk
Keyword(s):  
Cell Suspension ◽  
Cell Suspension Cultures ◽  
Apparent Molecular Weight ◽  
Suspension Cultures ◽  
Partial Purification ◽  
Enzymatic System ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Ph Optimum

Abstract Two new N-methyltransferases (NMT-I and NMT-II) were found to occur in Berberis vulgaris cell suspension cultures. One of these enzymes (NMT-I) was partially purified (100-fold) and characterized. This enzyme is specific for tetrahydrobenzylisoquinoline alkaloids and S-adenosyl-ʟ-methionine serves as the methyl donor. The apparent molecular weight of the enzyme is 68,000. The pH optimum of the enzyme is 7.6, the temperature optimum 35 °C. Apparent KM values for (R)-tetrahydropapaverin as substrate were 0.2 mᴍ and for SAM 0.04 mᴍ. The preparation of the same type of enzyme from B. wilsoniae var. subcaulialata was utilized as an efficient enzymatic system for the synthesis of stereochemically pure (R)-as well as (S)-reticuline labelled with tritium or 14C at the N-CH3 group. Enzymes catalyzing this type of reactions are named S-adenosyl-ʟ-methionine: norreticuline N-methyltransferases.

Characterization of glycineN-methyltransferase from rabbit liver

2004 ◽  
Vol 82 (3) ◽  
pp. 369-374 ◽  
Author(s):  
Doris Kloor ◽  
Katrin Karnahl ◽  
Jost Kömpf
Keyword(s):  
Enzymatic Activity ◽  
Kinetic Data ◽  
Adenine Nucleotides ◽  
The Other ◽  
Rabbit Liver ◽  
Ph Optimum ◽  
Isolation And Purification ◽  
Endogenous Adenosine ◽  
Sds Page

The enzymatic properties of glycine N-methyltransferase from rabbit liver and the effects of endogenous adenosine nucleosides, nucleotides and methyltransferase inhibitors were investigated using a photometrical assay to detect sarcosine with o-dianisidine as a dye. After isolation and purification the denatured enzyme showed a two-banded pattern by SDS–PAGE. The enzyme was highly specific for its substrates with a pH-optimum at pH 8.6. Glycine N-methyltransferase exhibits Michaelis-Menten kinetics for its substrates, S-adenosylmethionine and glycine, respectively. The apparent Kmand Vmaxvalues were determined for both the substrates, the other substrate being present at saturating concentrations. The enzyme was strongly inhibited in the presence of S-adenosylhomocysteine, 3-deazaadenosine, and 5′-S-isobutylthio-5′-deoxyadenosine. All other inhibitors investigated, adenosine, 2′-deoxyadenosine, aciclovir, and 5′-N-ethylcarboxamidoadenosine were poor inhibitors of the methylation rection. Adenine nucleotides and vidarabin were without effect on the enzymatic activity. Based on the kinetic data glycine N-methyltransferase from rabbit liver exhibits appreciable activity at physiological S-adenosylmethionine and S-adenosylhomocysteine levels.Key words: glycine N-methyltransferase, S-adenosylhomocysteine, S-adenosylmethionine, sarcosine oxidase, peroxidase.

scholarly journals Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

1995 ◽  
Vol 308 (3) ◽  
pp. 733-741 ◽  
Author(s):  
S M Pitson ◽  
R J Seviour ◽  
B M McDougall ◽  
J R Woodward ◽  
B A Stone
Keyword(s):  
Filamentous Fungus ◽  
Gel Filtration ◽  
High Specificity ◽  
Major Product ◽  
Ph Optimum ◽  
Sds Page ◽  
Molecular Masses ◽  
Monomeric Proteins ◽  
Ph Range

Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

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