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 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 Caseinase Production and Media Optimization from Bacillus subtilis

2020 ◽  
Vol 71 (11) ◽  
pp. 1-9
Author(s):  
Noor Nihad Abdul Hussein ◽  
Aseel I. Ibrahim ◽  
Firas Hashim Kamar ◽  
Arelia Cristina Nechifor
Keyword(s):  
Bacillus Subtilis ◽  
Enzyme Production ◽  
Paper Industry ◽  
Nitrogen Sources ◽  
The Body ◽  
Biochemical Tests ◽  
Isolation And Identification ◽  
Carbon And Nitrogen Sources ◽  
Carbon And Nitrogen ◽  
Sequencing Result

Caseinase is involved in the breakdown of milk protein casein and converts casein into smaller simple sugars which can be easily utilized by the body for the production of ATP and Fat. Casein can be an instant energy source to the body and involves in muscle building. Caseinase enzyme can be extensively used at the industrial scale for Milk, Textile, Dairy, Paper industry and several other medical purposes. In view of the importance of caseinase, the current research deals with the isolation and identification of caseinase producing bacteria from soil. This is followed by the production of enzyme and its purification. The study also includes its kinetic characterization using the parameters Temperature, pH as well as Carbon and Nitrogen Sources. The organism which was isolated from soil and capable of producing the caseinase enzyme was identified to be Bacillus subtilis based on the Biochemical tests and 16S rRNA sequencing result. The optimal carbon and nitrogen sources were identified to be Glucose and casein respectively. Regarding the optimal conditions, the suitable temperature for maximum enzyme production was found to be 40 0C and pH was 9. When the organism was cultured under the optimal condition using casein as a nitrogen source and glucose as the carbon source, at 40 0C and pH 9, 1590 ng/mL of enzyme production was estimated.

scholarly journals Using cellulose binding module of CBM3A as the purification tag for secreted Endoglucanase A (CelA) in Bacillus subtilis

2016 ◽  
Vol 19 (2) ◽  
pp. 5-14
Author(s):  
Phuong Hoang Ngoc Nguyen ◽  
Phuoc Thanh Nguyen ◽  
Thang Luong Pham ◽  
Hoang Duc Nguyen ◽  
Thuoc Linh Tran ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Protein Purification ◽  
Clostridium Thermocellum ◽  
Expression System ◽  
Secreted Proteins ◽  
Amorphous Cellulose ◽  
Recombinant Protein Purification ◽  
Bacillus Subtilis Wb800n ◽  
Cellulose Binding

Traditional methods of recombinant protein purification are uneconomic and inconvenient to the secreted proteins at large-volume. CBM3a, a module from cellulosome’s scaffoldin of Clostridium thermocellum, directs the binding of the cellulase complex on the cheap cellulose substrate. Most of previous studies about CBM3a fused with cellulases as the purification tag were conducted in intracellular Escherichia coli system. In this research, we used the extracellular Bacillus subtilis WB800N expression system to investigate the CBM3a-tag fused with endoglucanase CelA into plasmid pHT. The results indicated that protein CelA was secrected and purified by CBM3a-tag binding on the Regenerated Amorphous Cellulose (RAC) subtrate. This can be used for further improvement in protein purification tag designing.

Deciphering characteristics of the designer cellulosome from Bacillus subtilis WB800N via enzymatic analysis

2017 ◽  
Vol 117 ◽  
pp. 147-155 ◽  
Author(s):  
Chia-Chi Lin ◽  
Cally Joe San Yap ◽  
Shu-Chen Kan ◽  
Nai-Chi Hsueh ◽  
Liang-Yu Yang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Enzymatic Analysis ◽  
Bacillus Subtilis Wb800n ◽  
Designer Cellulosome

scholarly journals Functional Analysis of the Bacillus subtilis Zur Regulon

2002 ◽  
Vol 184 (23) ◽  
pp. 6508-6514 ◽  
Author(s):  
Ahmed Gaballa ◽  
Tao Wang ◽  
Rick W. Ye ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Metal Ion ◽  
Dna Microarrays ◽  
Optimal Growth ◽  
Zinc Uptake ◽  
Uptake System ◽  
Mobility Shift ◽  
Transport Pathway ◽  
Dnase I Footprinting ◽  
Transporter Family

ABSTRACT The Bacillus subtilis zinc uptake repressor (Zur) regulates genes involved in zinc uptake. We have used DNA microarrays to identify genes that are derepressed in a zur mutant. In addition to members of the two previously identified Zur-regulated operons (yciC and ycdHI-yceA), we identified two other genes, yciA and yciB, as targets of Zur regulation. Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation. Zur binds to an ∼28-bp operator upstream of the yciA gene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons, yciC and ycdHI-yceA. Analysis of a yciA-lacZ fusion indicates that this operon is induced under zinc starvation conditions and derepressed in the zur mutant. Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway. Mutation of yciA or yciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of the ycdH mutant under conditions of zinc limitation. Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system. Mutant strains lacking all three identified zinc uptake systems (yciABC, ycdHI-yceA, and zosA) are dependent on micromolar levels of added zinc for optimal growth.

scholarly journals Analysis of the Binding of Expansin Exl1, from Pectobacterium carotovorum, to Plant Xylem and Comparison to EXLX1 from Bacillus subtilis

ACS Omega ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. 7008-7018 ◽  
Author(s):  
Omar E. Tovar-Herrera ◽  
Mabel Rodríguez ◽  
Miguel Olarte-Lozano ◽  
Jimmy Andrés Sampedro-Guerrero ◽  
Adán Guerrero ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pectobacterium Carotovorum

scholarly journals Comparative Evaluation of Agroindustrial Byproducts for the Production of Alkaline Protease by Wild and Mutant Strains ofBacillus subtilisin Submerged and Solid State Fermentation

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Hamid Mukhtar ◽  
Ikramul Haq
Keyword(s):  
Bacillus Subtilis ◽  
Solid State ◽  
Wheat Bran ◽  
Solid State Fermentation ◽  
Soybean Meal ◽  
Alkaline Protease ◽  
Enzyme Production ◽  
Enhanced Production ◽  
Industrial Byproducts ◽  
Agroindustrial Byproducts

The present study describes the screening of different agroindustrial byproducts for enhanced production of alkaline protease by a wild and EMS induced mutant strain ofBacillus subtilisIH-72EMS8. During submerged fermentation, different agro-industrial byproducts were tested which include defatted seed meals of rape, guar, sunflower, gluten, cotton, soybean, and gram. In addition to these meals, rice bran, wheat bran, and wheat flour were also evaluated for protease production. Of all the byproducts tested, soybean meal at a concentration of 20 g/L gave maximum production of the enzyme, that is, 5.74  ±  0.26 U/mL from wild and 11.28  ±  0.45 U/mL from mutant strain, during submerged fermentation. Different mesh sizes (coarse, medium, and fine) of the soybean meal were also evaluated, and a finely ground soybean meal (fine mesh) was found to be the best. In addition to the defatted seed meals, their alkali extracts were also tested for the production of alkaline protease byBacillus subtilis, but these were proved nonsignificant for enhanced production of the enzyme. The production of the enzyme was also studied in solid state fermentation, and different agro-industrial byproducts were also evaluated for enzyme production. Wheat bran partially replaced with guar meal was found as the best substrate for maximum enzyme production under solid state fermentation conditions.

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