scholarly journals Isolation, heterologous expression, and purification of a novel antifungal protein from Bacillus subtilis strain Z-14

2020 ◽  
Author(s):  
Xuechao Zhang ◽  
Xiaojun Guo ◽  
Cuihong Wu ◽  
Chengcui Li ◽  
Dongdong Zhang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Pichia Pastoris ◽  
Fungal Infections ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteinase K ◽  
Antifungal Protein ◽  
Subtilis Strain ◽  
Affinity Column

Abstract Background: Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and was confirmed to have strong antifungal activity against R. cerealis. Results: An antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography, and reverse phase chromatography. Purified F2 had a molecular mass of approximately 8 kDa, as assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Edman degradation was used to determine the amino acid sequence of the N-terminus, which was NH2‑ASGGTVGIYGANMRS. This sequence is identical to a hypothetical protein RBAM_004680 (YP_001420098.1) synthesized by B. amyloliquefaciens FZB42. The recombinant F2 protein (rF2) was heterologously expressed in the yeast host Pichia pastoris, purified using a Ni‑affinity column, and demonstrated significant antifungal activity against R. cerealis. The purified rF2 demonstrated broad spectrum antifungal activity against different varieties of fungi such as Fusarium oxysporum, Verticillium dahliae, Bipolaris papendorfii, and Fusarium proliferatum. rF2 was thermostable, retaining 91.5% of its activity when incubated for 30 min at 100 °C. Meanwhile, rF2 maintained its activity under treatment by proteinase K and trypsin and over a wide pH range from 5 to 10.Conclusions: A novel antifungal protein, F2, was purified from biocontrol Bacillus subtilis Z-14 strain fermentation supernatant and heterologously expressed in Pichia pastoris to verify its antifungal activity against R. cerealis and the validity of the gene encoding F2. Considering its significant antifungal activity and stable characteristics, protein F2 presents an alternative compound to resist fungal infections caused by R. cerealis.

scholarly journals Purification and heterologous expression of a novel antifungal protein from Bacillus subtilis strain Z-14

2020 ◽  
Author(s):  
Xuechao Zhang ◽  
Xiaojun Guo ◽  
Cuihong Wu ◽  
Chengcui Li ◽  
Dongdong Zhang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Pichia Pastoris ◽  
Fungal Infections ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteinase K ◽  
Antifungal Protein ◽  
Subtilis Strain ◽  
Affinity Column

Abstract Background: Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and has been confirmed to have strong antifungal activity against R. cerealis. Results: An antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography, and reverse phase chromatography. Purified F2 had a molecular mass of approximately 9 kDa, as assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Edman degradation was used to determine the amino acid sequence of the N-terminus, which was NH2-ASGGTVGIYGANMRS. This sequence is identical to a hypothetical protein RBAM_004680 (YP_001420098.1) synthesized by B. amyloliquefaciens FZB42. The recombinant F2 protein (rF2) was heterologously expressed in the yeast host Pichia pastoris, purified using Ni‑affinity column, and demonstrated significant antifungal activity against R. cerealis. The purified rF2 was thermostable, retaining 91.5% of its activity when incubated for 30 min at 100 °C. rF2 maintained its activity under treatment by proteinase K and trypsin.Conclusions: A novel antifungal protein F2 was purified from biocontrol Bacillus subtilis Z-14 strain fermentation supernatant and heterologously expressed in Pichia pastoris to certificate the antifungal activity against R. cerealis and the validity of gene sequence of protein F2. Considering its significant antifungal activity and stable characteristics, protein F2 presents an alternative compound to deal with fungal infections caused by R. cerealis.

scholarly journals Isolation, heterologous expression, and purification of a novel antifungal protein from Bacillus subtilis strain Z-14

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Xuechao Zhang ◽  
Xiaojun Guo ◽  
Cuihong Wu ◽  
Chengcui Li ◽  
Dongdong Zhang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Pichia Pastoris ◽  
Fungal Infections ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Antifungal Protein ◽  
Subtilis Strain

Abstract Background Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and was confirmed to have strong antifungal activity against R. cerealis. Results An antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography, and reverse phase chromatography. Purified F2 had a molecular mass of approximately 8 kDa, as assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Edman degradation was used to determine the amino acid sequence of the N-terminus, which was NH2ASGGTVGIYGANMRS. This sequence is identical to a hypothetical protein RBAM_004680 (YP_001420098.1) synthesized by B. amyloliquefaciens FZB42. The recombinant F2 protein (rF2) was heterologously expressed in the yeast host Pichia pastoris, purified using a Niaffinity column, and demonstrated significant antifungal activity against R. cerealis. The purified rF2 demonstrated broad spectrum antifungal activity against different varieties of fungi such as Fusarium oxysporum, Verticillium dahliae, Bipolaris papendorfii, and Fusarium proliferatum. rF2 was thermostable, retaining 91.5% of its activity when incubated for 30 min at 100 °C. Meanwhile, rF2 maintained its activity under treatment by proteinase K and trypsin and over a wide pH range from 5 to 10. Conclusions A novel antifungal protein, F2, was purified from biocontrol Bacillus subtilis Z-14 strain fermentation supernatant and heterologously expressed in Pichia pastoris to verify its antifungal activity against R. cerealis and the validity of the gene encoding F2. Considering its significant antifungal activity and stable characteristics, protein F2 presents an alternative compound to resist fungal infections caused by R. cerealis.

scholarly journals An Antifungal Chitosanase from Bacillus subtilis SH21

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1863
Author(s):  
Yuanxiang Pang ◽  
Jianjun Yang ◽  
Xinyue Chen ◽  
Yu Jia ◽  
Tong Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Liquid Chromatography ◽  
Tertiary Structure ◽  
Fungal Infections ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Antifungal Protein ◽  
Selection Marker ◽  
Ms Analysis

Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC–MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.

scholarly journals Biosynthesis of Silver Chloride Nanoparticles Using Bacillus subtilis MTCC 3053 and Assessment of Its Antifungal Activity

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Kanniah Paulkumar ◽  
Shanmugam Rajeshkumar ◽  
Gnanadhas Gnanajobitha ◽  
Mahendran Vanaja ◽  
Chelladurai Malarkodi ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Silver Chloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cellular Membrane ◽  
X Ray Diffraction ◽  
Xrd Pattern ◽  
Transmission Electron ◽  
Crystalline Nature

The present investigation reported the synthesis of silver chloride nanoparticles using Bacillus subtilis. The adsorption of colloidal silver chloride nanoparticles showed an intense peak at the wavelength of 400 nm after 20 hrs of biomass incubation. The size of the silver nanoparticles ranges from 20 to 60 nm which was obtained from transmission electron microscope (TEM). The X-ray diffraction (XRD) pattern confirmed the crystalline nature of the nanoparticles. The bright circular spots of selected diffraction area (SAED) pattern also confirmed the good crystalline nature of the silver chloride nanoparticles with high magnification of TEM images. The presence of nitrate reductase enzyme in the cellular membrane of B. subtilis was confirmed by sodium dodecyl (SDS) polyacrylamide gel electrophoresis and it was found that the molecular weight is 37 kDa. The possible functional groups of the reductase enzyme in B. subtilis were identified by Fourier transform infrared spectroscopy (FTIR). Finally, antifungal activity of silver chloride nanoparticle was examined against Candida albicans, Aspergillus niger, and Aspergillus flavus. We conclude that the synthesis of silver chloride nanoparticles using microorganisms is more economical and simple. The antifungal property of silver chloride nanoparticles will play a beneficial role in biomedical nanotechnology.

scholarly journals Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

2007 ◽  
Vol 73 (7) ◽  
pp. 2247-2250 ◽  
Author(s):  
Sirinat Srionnual ◽  
Fujitoshi Yanagida ◽  
Li-Hsiu Lin ◽  
Kuang-Nan Hsiao ◽  
Yi-sheng Chen
Keyword(s):  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Proteinase K ◽  
Mass Spectrometry Analysis ◽  
Fermented Fish ◽  
Spectrometry Analysis ◽  
Weissella Cibaria ◽  
Fish Product

ABSTRACT Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.

Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements

1990 ◽  
Vol 10 (10) ◽  
pp. 5177-5186
Author(s):  
J Zhang ◽  
S T Jacob
Keyword(s):  
Ribosomal Dna ◽  
Core Promoter ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Column ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Molecular Weights ◽  
The Core ◽  
Pol I

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

Isolation and Preliminary Characterization of a Bacteriocin Produced by Lactobacillus plantarum N014 Isolated from Nham, a Traditional Thai Fermented Pork

2006 ◽  
Vol 69 (8) ◽  
pp. 1937-1943 ◽  
Author(s):  
PONGSAK RATTANACHAIKUNSOPON ◽  
PARICHAT PHUMKHACHORN
Keyword(s):  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteinase K ◽  
Exponential Growth Phase ◽  
Plasmid Isolation ◽  
Inhibitory Spectrum ◽  
Lipase A

Lactobacillus plantarum N014 was isolated from nham, a traditional Thai fermented pork, and exhibited antimicrobial activity against Listeria monocytogenes. Its bacteriocin had a broad inhibitory spectrum toward both gram-positive and gram-negative bacteria. The bacteriocin activity was sensitive to all proteolytic enzymes used in this study, including papain, pepsin, pronase E, proteinase K, and trypsin, but was resistant to the other enzymes, such as α-amylase, lipase A, and lysozyme. Furthermore, activity was stable over various heat treatments and pH values. The bacteriocin exerted a bacteriolytic mode of action. It was produced during the exponential growth phase and reached its highest level as producer cells entered the stationary phase. Adsorption of the bacteriocin onto producer cells was pH-dependent. No bacteriocin adsorption was detected at pH 1 to 3, whereas 100% bacteriocin adsorption was found at pH 7. Plasmid isolation revealed that L. plantarum N014 contained no plasmids. From Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and growth inhibition testing against L. monocytogenes, the estimated molecular mass of L. plantarum N014 bacteriocin was 8 kDa.

scholarly journals Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements.

1990 ◽  
Vol 10 (10) ◽  
pp. 5177-5186 ◽  
Author(s):  
J Zhang ◽  
S T Jacob
Keyword(s):  
Ribosomal Dna ◽  
Core Promoter ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Column ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Molecular Weights ◽  
The Core ◽  
Pol I

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

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.

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