scholarly journals Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0256562
Author(s):  
Nagina Rafique ◽  
Saiqa Bashir ◽  
Muhammad Zubair Khan ◽  
Imran Hayat ◽  
Willium Orts ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Metal Ions ◽  
Enzyme Production ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pathogenic Strain ◽  
Pectobacterium Carotovorum ◽  
Iptg Induction

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

scholarly journals Metabolic engineering of Bacillus subtilis with an Endopolygalacturonase gene Isolated from Pectobacterium carotovorum; a Plant Pathogenic bacterial strain.

2021 ◽  
Author(s):  
Nagina Rafique ◽  
Saiqa Bashir ◽  
Muhammad Zubair Khan ◽  
Imran Hayat ◽  
Willium Orts ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Enzyme Production ◽  
Metal Ion ◽  
Paper Industry ◽  
Expression System ◽  
Pathogenic Strain ◽  
Pectobacterium Carotovorum ◽  
Iptg Induction ◽  
Competent Cells ◽  
Bacillus Subtilis Wb800n

Pectinolytic enzymes [pectinases] produced by microbes are highly important for their biotechnological use in processing of vegetables and fruits beverages and use in pulp and paper industry. A pectinases, namely endo-polygalacturonase [endo-PGase], encoding gene isolated from Pectobacterium carotovorum, a plant pathogenic strain of bacteria was successfully cloned into a secretion vector pHT43 having σ?-dependent promoter P grac . For enhanced expression analysis, competent cells of Bacillus subtilis (WB800N) were prepared at stationary phase using high salt medium. The recombinant B. subtilis competent cells, harboring the engineered pHT43 with the endo-PGase gene were cultured in 2X-yeast extract tryptone medium. The recombinant endo-PGase enzyme was secreted directly into the medium after 72 hours of the first IPTG induction. The recombinant endo-PGase was screened for its activity at various temperatures and pH ranges. Optimal activity was found at pH 5.0 and a temperature of 40°C with a stability ranging from pH 5.0-9.0. For detection of metal ion effect, recombinant enzyme was incubated with 1mM concentration of; Ca ++ , Mg ++ , Zn ++ , EDTA, K ++ for 45 minutes. Resultantly, Ca ++ , EDTA and Zn ++ strongly inhibited the enzyme activity. The chromatographic analysis of enzymatic hydrolysate of polygalacturonic acid [PGA] and pectin substrates using HPLC and TLC revealed that tri and tetra-galacturonates were the end products of hydrolysis. The study led to the conclusion that endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis and assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safe for commercial enzyme production as compared to yeast and fungi to escape endotoxins.

scholarly journals Expression of Aleutian Mink Disease Parvovirus Capsid Proteins in a Baculovirus Expression System for Potential Diagnostic Use

1994 ◽  
Vol 6 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Wai-Hong Wu ◽  
Marshall E. Bloom ◽  
Bradley D. Berry ◽  
Michael J. McGinley ◽  
Kenneth B. Platt
Keyword(s):  
Microscopic Examination ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Baculovirus Expression System ◽  
Immunoblot Analysis ◽  
Capsid Proteins ◽  
Electron Microscopic ◽  
Nuclear Polyhedrosis

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1 -infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23–25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.

scholarly journals The purification of a novel amylase from Bacillus subtilis and its inhibition by wheat proteins

1983 ◽  
Vol 209 (2) ◽  
pp. 561-564 ◽  
Author(s):  
A R Orlando ◽  
P Ade ◽  
D Di Maggio ◽  
C Fanelli ◽  
L Vittozzi
Keyword(s):  
Bacillus Subtilis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alpha Amylase ◽  
Wheat Protein ◽  
Wheat Proteins ◽  
Maximal Inhibition ◽  
Molecular Weights ◽  
Amylase Inhibitors

A new alpha-amylase (EC 3.2.1.1) from Bacillus subtilis was purified by affinity chromatography. The molecular weight of the purified enzyme, estimated from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 93000, which is very different from the molecular weights of two well-characterized amylases from B. subtilis. Electrofocusing showed an isoelectric point of 5. Amylase shows a broad maximum of activity between pH 6 and 7; maximal inhibition of enzyme by wheat-protein alpha-amylase inhibitors is displayed at pH 7.

scholarly journals Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis

2003 ◽  
Vol 185 (4) ◽  
pp. 1443-1454 ◽  
Author(s):  
Erh-Min Lai ◽  
Nikhil D. Phadke ◽  
Maureen T. Kachman ◽  
Rebecca Giorno ◽  
Santiago Vazquez ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Bacillus Anthracis ◽  
Proteomic Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Detection Methods ◽  
Coat Proteins ◽  
Bona Fide ◽  
Spore Proteins ◽  
Spore Coat Proteins

ABSTRACT The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.

scholarly journals Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus

1979 ◽  
Vol 177 (1) ◽  
pp. 49-62 ◽  
Author(s):  
C M Clarke ◽  
B S Hartley
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Dna Cleavage ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Activity Restriction

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).

Purification and some properties of the extracellular α-amylase from Aspergillus awamori

1985 ◽  
Vol 31 (2) ◽  
pp. 149-153 ◽  
Author(s):  
Resham S. Bhella ◽  
Illimar Altosaar
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Aspergillus Awamori ◽  
Original Activity

Alpha-amylase was purified from the extracellular culture medium of Aspergillus awamori by means of ethanol precipitation. Sephacryl-200 gel filtration and anion-exchange chromatography on Dowex (AG1-X4) resin. The enzyme preparation was found to be homogeneous by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 54 000 ± 2 500 and its isoelectric point was pH 4.2. The enzyme was found to be most active between pH 4.8 and 5.0 and was stable between pH 3.5 and 6.5. The optimal temperature for the enzyme activity was around 50 °C and the enzyme was stable for at least 1 h up to 45 °C retaining more than 80% of its original activity. The Km (37 °C, pH 5.3) for starch hydrolysis was 1.0 g∙L−1 and maltose inhibited the enzyme activity uncompetitively with a K1 value of 20.05 g∙L−1

scholarly journals Protease-producing microorganisms inhabiting salted fish (Moloha) with special reference to protease activity of Bacillus subtilis

2014 ◽  
Vol 63 (3-4) ◽  
pp. 303-307 ◽  
Author(s):  
M. H. Abd-Alla ◽  
S. A. Omar ◽  
M. A. El-Nagdy
Keyword(s):  
Bacillus Subtilis ◽  
Salt Tolerance ◽  
Metal Ions ◽  
Special Reference ◽  
Protease Activity ◽  
Enzyme Production ◽  
Effect Of Temperature ◽  
Protease Production ◽  
Salted Fish ◽  
Tanning Industry

The investigation was designed to isolate and identify the proteolytic microorganisms inhabiting salted fish. <i>Bacillus subtilis</i> was chosen as the most promising protease producer. Some properties of the crude protease are presented, the effect of metal ions on protease production has been studied. It was shown that Ca<sup>2+</sup> and Mg<sup>2+</sup> stimulated, while Co<sup>2+</sup> , Zn<sup>2+</sup> and Cu<sup>2+</sup> inhibited the enzyme production. The effect of temperature and pH and salt tolerance have also been studied. Protease activity was stable in 25% NaCl. The favourable characteristics of the enzyme might have extensive application in laundry detergents and in tanning industry.

scholarly journals Cymbopogon nardus Essential Oil as Protein Inducer in Bacillus subtilis ATCC21332

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Hairul Shahril Muhamad ◽  
Ismatul Nurul Asyikin Ismail ◽  
Nabilah Ahmad Alhadi ◽  
Salina Mat Radzi ◽  
Maryam Mohamed Rehan ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Essential Oil ◽  
Protein Production ◽  
Antimicrobial Agents ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extracellular Proteins ◽  
Specific Chemical ◽  
Cymbopogon Nardus

Protein production by bacteria might be increased in stressful conditions such as in the presence of antimicrobial agents. Many studies have proven that antibiotics or antimicrobial agents at low concentration are able to activate or repress gene transcription process in bacteria. However, there have been comparatively few studies on the potential of natural compounds in nature as a specific chemical signal that can trigger a variety of biological functions. An attempt was made to study the effect of essential oil from Cymbopogon nardus in regulating protein production by Bacillus subtilis ATCC21332. The bacterial cells were further exposed to the C. nardus essential oil at concentration of 0.02 % for 48 h at 37°C. The intracellular proteins were then isolated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile showed that a band with approximate size of 180 kDa appeared for the treated bacteria with C. nardus essential oil. An alignment of peptide sequences to the NCBI BLAST database revealed that B. subtilis ATCC21332 in stressful condition tend to produce intracellular protein recognized as respiratory nitrate reductase ? subunit enzyme. Besides, the extracellular proteins secreted by B. subtilis ATCC21332 after being subjected to 0.02% of C. nardus essential oil for 48 and 72 h at 30°C, were further analyzed on antimicrobial activity. The extracellular proteins secreted by B. subtilis ATCC21332 prior to enhancing with 0.02 % C. nardus essential oil at 30°C for 72 h exhibited antimicrobial activity towards two strains of bacteria, which are Bacillus cereus and Escherichia coli.

Effects of Some Vitamins on Lactoperoxidase Enzyme Activity

2009 ◽  
Vol 79 (3) ◽  
pp. 188-194 ◽  
Author(s):  
Melda Sisecioglu ◽  
Murat Cankaya ◽  
Hasan Ozdemir
Keyword(s):  
Ascorbic Acid ◽  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Exchange Chromatography ◽  
Inhibition Mechanism ◽  
Vitamin K3 ◽  
Vitro Effect

Objective: The present paper investigates the in vitro effect of L-ascorbic acid (vitamin C), menadione sodium bisulfate (vitamin K3), and folic acid on purified lactoperoxidase (LPO). Methods: This enzyme was purified from bovine milk by Amberlite CG 50 resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. Results: Rz (A412/A280) value for the purified LPO was found to be 0.8. Lactoperoxidase was purified 20.45-fold with a yield of 28.8 %. Purity of enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method and a single band was observed. All tested vitamins caused inhibition of the enzyme activity and displayed a competitive type of inhibition mechanism. IC50 values of these three vitamins were 2.03 µM, 0.025 mM, and 0.0925 mM, and the Ki constants were 0.508±0.257 µM, 0.0107±0.0044 mM, and 0.0218±0.0019 mM respectively. Conclusion: The vitamins discussed here displayed inhibition-type competition with LPO enzyme at varying concentrations. Our study showed that L-ascorbic acid exhibited a much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than other vitamins tested.

scholarly journals Characterization of a Thermostable d-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

2003 ◽  
Vol 69 (2) ◽  
pp. 980-986 ◽  
Author(s):  
Dae Heoun Baek ◽  
Seok-Joon Kwon ◽  
Seung-Pyo Hong ◽  
Mi-Sun Kwak ◽  
Mi-Hwa Lee ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Optimum Temperature ◽  
Gene Encoding ◽  
Ph Range

ABSTRACT A gene encoding a new thermostable d-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l-amino acid amides, d-amino acid-containing peptides, and NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+. The k cat/Km for d-alaninamide was measured as 544.4 ± 5.5 mM−1 min−1 at 50°C with 1 mM Co2+.

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