scholarly journals Isolation, Extraction, Purification, and Molecular Characterization for Thermostable α-Amylase from Locally Isolated Bacillus Species in Sudan

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Maha A. Rakaz ◽  
Mohammed O. Hussien ◽  
Hanan M. Ibrahim
Keyword(s):  
Bacillus Cereus ◽  
Bacillus Licheniformis ◽  
Soil Bacteria ◽  
Biochemical Characterization ◽  
Amylase Activity ◽  
Industrial Applications ◽  
Soil Samples ◽  
Bacillus Species ◽  
Good Source ◽  
Extraction And Purification

The aim of this study was to isolate some soil bacteria strain that produced α-amylase and subsequent extraction and purification. One hundred soil samples were collected from different geographical areas in Khartoum State such as north Omdurman, Toti Island, and Soba. Samples were analyzed for starch hydrolyzing bacteria. Among several bacteria isolated, Bacillus cereus and Bacillus licheniformis were identified as active α-amylase producers. Both bacteria showed a large zone of clearance of 20 mm when grown on starch-agar plates. The identity was conducted using biochemical characterization and confirmed by sequencing their 16S-rDNA. The constitutive nature of amylase was proved by amplification of the amylase gene from the genome of B. licheniformis. The α-amylase activity from the spent medium of B. cereus and B. licheniformis was optimized at pH 8.0 and temperature of 45°C and 65°C, respectively. The α-amylase produced by both bacteria is alkalophilic and thermophilic. The experiments confirmed that B. licheniformis can be a good source of amylase for industrial applications in Sudan.

scholarly journals Biochemical Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. HS Isolated in the Odiel Marshlands

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 337
Author(s):  
Patricia Gómez-Villegas ◽  
Javier Vigara ◽  
Luis Romero ◽  
Cecilia Gotor ◽  
Sara Raposo ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Amylase Activity ◽  
Extreme Environments ◽  
Three Dimensional ◽  
Large Family ◽  
Industrial Applications ◽  
Dimensional Structure ◽  
Glycosyl Hydrolases ◽  
Calcium Dependent ◽  
Alpha Amylases

Alpha-amylases are a large family of α,1-4-endo-glycosyl hydrolases distributed in all kingdoms of life. The need for poly-extremotolerant amylases encouraged their search in extreme environments, where archaea become ideal candidates to provide new enzymes that are able to work in the harsh conditions demanded in many industrial applications. In this study, a collection of haloarchaea isolated from Odiel saltern ponds in the southwest of Spain was screened for their amylase activity. The strain that exhibited the highest activity was selected and identified as Haloarcula sp. HS. We demonstrated the existence in both, cellular and extracellular extracts of the new strain, of functional α-amylase activities, which showed to be moderately thermotolerant (optimum around 60 °C), extremely halotolerant (optimum over 25% NaCl), and calcium-dependent. The tryptic digestion followed by HPLC-MS/MS analysis of the partially purified cellular and extracellular extracts allowed to identify the sequence of three alpha-amylases, which despite sharing a low sequence identity, exhibited high three-dimensional structure homology, conserving the typical domains and most of the key consensus residues of α-amylases. Moreover, we proved the potential of the extracellular α-amylase from Haloarcula sp. HS to treat bakery wastes under high salinity conditions.

scholarly journals RECONSTITUTION OF DIFFERENT SIGNAL PEPTIDES FOR ENHANCED THERMOSTABLE ALPHA AMYLASE SECRETION IN Bacillus subtilis

2018 ◽  
Vol 56 (1) ◽  
pp. 7 ◽  
Author(s):  
Nguyen Thị Da ◽  
Tran Dinh Man ◽  
Nguyen Kim Thoa
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Bacillus Cereus ◽  
Bacillus Licheniformis ◽  
Signal Peptide ◽  
Amylase Activity ◽  
Alpha Amylase ◽  
Amylase Secretion ◽  
Signal Peptides ◽  
Extracellular Amylase

Three signal peptides of alpha amylase genes of three isolated strains that were Bacillus licheniformis DA23, Bacillus subtilis D5-2, Bacillus cereus CN1-5 were successfully sequenced. Three predicted “Sec – type” signal peptides have a length varying from 27 (CN1-5) to 33 residues (D5-2).  The secretion of alpha amylase of the recombination B. subtilis 168MPgrac strain (pHV33–PgracAmy3BT2) with 71.4U/ml was larger than that of 168MPamy with 53.2U/ml. Base on analyzed rerults of PAGE and zymogram about molecular weight, alpha amylases in both strains were the same size, nearly 58kDa. The extracellular amylase activity of signal peptide SsubtilisD5.2 in 168M was highest with 76.4±4.3 U/ml in four signal peptide targets.  

scholarly journals Identification and Characterization of Arsenic Transforming Bacillus Species from Abandoned Mining Regions of Madhya Pradesh and Jharkhand

2021 ◽  
Vol 15 (1) ◽  
pp. 175-185
Author(s):  
Ankur Bhardwaj ◽  
Rakesh Kumar Sharma ◽  
Gajendra Bahadur Singh
Keyword(s):  
Biochemical Characterization ◽  
Arsenic Concentration ◽  
Luria Bertani ◽  
Soil Samples ◽  
Madhya Pradesh ◽  
Bacillus Species ◽  
Pcr Analysis ◽  
Bacterial Strains ◽  
Reducing Ability

The arsenic (As) comprehensiveness in nature has aggravated the expansion of arsenic fortification and detoxification components in microorganisms. Many microorganisms discovered today with ability to oxidize arsenite (As3+) into arsenate (As5+) or reduce As5+ to As3+. In this study, two bacterial strains designated 3AB3 and 5AB2 was isolated from the soil samples collected from abandoned mining region of Madhya Pradesh and Jharkhand, India and arsenic concentration has been determined in both water and soil samples. Enrichment culturing method was employed for isolating bacteria and further they are screened for their redox ability. The isolated strains exhibited maximum growth at 30°C, at pH 7.0 in arsenic stressed Luria Bertani broth, checked through UV-Vis spectrophotometer at OD-620nm. Biochemical characterization of isolated strains was performed with various confirmation tests. Phylogenetic analysis of selected bacterial strains through MEGA-X confirmed their relationship to the genus Bacillus. Further, they are tested for transformation ability of arsenic (MSA method) and gene identification was done in selected isolated strains (PCR method). The result of this study shows that, even after abandoning the mining activities, concentration of arsenic increases in ground water by reducing ability of bacterial strains. PCR analysis depicted the presence of genes arsR, arsB and arsC in the strain 3AB3 and gene aoxB in 5AB2 respectively.

scholarly journals Thermo-resistant enzyme-producing microorganisms isolated from composting

2023 ◽  
Vol 83 ◽  
Author(s):  
S. P. M. Cotta ◽  
M. S. Marins ◽  
I.E. Marriel ◽  
U.G.P Lana ◽  
E.A. Gomes ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Bacillus Licheniformis ◽  
Cellulase Activity ◽  
Ribosomal Gene ◽  
Thermomyces Lanuginosus ◽  
Universal Primers ◽  
Bacillus Species ◽  
Mineral Fertilizers ◽  
Biological Additive

Abstract Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

scholarly journals Purification and Properties of an Esterase from Bacillus licheniformis and it’s Application in Synthesis of Octyl Acetate

2020 ◽  
Vol 14 (1) ◽  
pp. 113-121
Author(s):  
Kamal K. Bhardwaj ◽  
Adarsh Dogra ◽  
Smita Kapoor ◽  
Akshita Mehta ◽  
Reena Gupta
Keyword(s):  
Bacillus Licheniformis ◽  
Column Chromatography ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Maximum Yield ◽  
Industrial Applications ◽  
Molar Ratio ◽  
Simple Method ◽  
Octyl Acetate ◽  
Short Period

Background: Esterase plays a major role in the degradation of natural materials, industrial pollutants and also provides an immense contribution to the eco-friendly approaches in various industrial applications. Objective: In the present study, extracellular esterase from bacterial isolate Bacillus licheniformis was purified, characterized and used in the synthesis of octyl acetate. Methods: Purification of esterase from Bacillus licheniformis was achieved using Sephadex G-75 column chromatography. Gas chromatography was used to analyze the octyl acetate synthesis. Results: The enzyme was salted out using ammonium sulphate precipitation and 60-70% saturation gave maximum specific activity of the enzyme during precipitation. A purification fold of 6.46 and yield of 9.69% was achieved when esterase from Bacillus licheniformis was purified using Sephadex G-75 column chromatography. Native as well as SDS-PAGE analysis gave a single band of 42 kDa. This showed that the enzyme was purified to homogeneity and it was a monomer with molecular weight of 42 kDa. Biochemical characterization of the enzyme revealed that it had optimum temperature of 45°C in 0.1 M Tris-HCl buffer of pH 8.0. On optimizing different parameters, such as molar ratio of reactants, incubation time, temperature, and amount of protein, the % yield of octyl acetate was found to be 77.3%. Conclusion: In this work, simple method was used to purify esterase and the enzyme was further used in producing esters/products of commercial value within a reasonably short period of 12 h with a maximum yield of 77.3%.

Sidrophore Production and Phosphate Solubilization by Bacillus cereus and Pseudomonas fluorescens Isolated from Iraqi Soils and Soil Characterization

2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Zaid Raad Abbas ◽  
Aqeel Mohammed Majeed Al-Ezee ◽  
Sawsan H
Keyword(s):  
Bacillus Cereus ◽  
Pseudomonas Fluorescens ◽  
Phosphate Solubilization ◽  
Bacterial Species ◽  
Nutrient Status ◽  
Bacterial Count ◽  
Soil Samples ◽  
Phosphate Solubilizing Bacteria ◽  
Clear Zone ◽  
Zone Formation

This study was conducted to explore the ability of Pseudomonas fluorescens and Bacillus cereus to solubilizing a phosphate in soil for enhancing the planting growth and, its relation with soill characterization. The isolates were identified as P.fluorescens and B. cereus using convential analysis and, its phosphate solubilization ability and sidrophore was shown by the clear zone formation on National Botanical Research Institute���s Phosphate medium. Moreover, Pseudomonas fluorescens isolates (n = 9) and three of B. cereus isolated from agricultural area in Baghdad university, Mustansiriyah university and Diyala bridge. Results displayed that bacterial count were varied in soil samples according to their region, and ranging from 30 to 60 *10 2 CFU/g in Baghdad university soil to 10���20 *10 2 CFU/g in Mustansiriyah university soil, the Baghdad soil macronutrient which included: NH4, NO3, P, and K were, 8.42, 20.53, 19.09, 218.73 respectively, While the physio analysis revealed that the mean of pH was 7.3 and EC was 8.63. on the other hand the micronutrient analysis indicated that the soil samples were included Ca, Fe, Mn, Zn and Cu which gave their mean 5025.9, 8.9, 4.9, 0.5 and 1.5 respectevily. Results revealed that all isolated bacteria (9 isolates of P.fluorescens and three isolates of B. cereus gave ahalo zone which mean their ability to be phosphate solubilizing bacteria at 100%. Results revealed that all isolated bacteria were detected a ability to produce high levels from chelating agents (siderophores)) by P.fluorescens and. B cereus at 100%, when appeared ahalo clear zone. Furthermore, the high levels of phosphate solubilization and siderophore production were grouped in bacterial species isolated from Iraqi soils. might be attributed to many soil factors such as soil nutrient status, soil acidity, water content, organic matter and soil enzyme activities.

Purification and Characterization of Natural Solid-Substrate Degrading and Alcohol Producing Hyperthermostable Alkaline Amylase from Bacillus cereus (sm-sr14)

2020 ◽  
Vol 21 (9) ◽  
pp. 872-881
Author(s):  
Sumit Sahoo ◽  
Sudipta Roy ◽  
Dipannita Santra ◽  
Sayantani Maiti ◽  
Sonali Roul ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Main Property ◽  
Solid Substrate ◽  
Industrial Applications ◽  
Starch Degradation ◽  
Significant Similarity ◽  
Alcohol Production ◽  
Cazy Database ◽  
Tim Barrel ◽  
Alkaline Amylase

Objective: Amylases enzymes hydrolyze starch molecules to produce diverse products including dextrins, and progressively smaller polymers. These include glucose units linked through α-1- 1, α-1-4, α-1-6, glycosidic bonds. Methods: This enzyme carrying an (α /β) 8 or TIM barrel structure is also produced containing the catalytic site residues. These groups of enzymes possess four conserved regions in their primary sequence. In the Carbohydrate-Degrading Enzyme (CAZy) database, α-amylases are classified into different Glycoside Hydrolase Families (GHF) based on their amino acid sequence. The present objective was to study one such enzyme based on its molecular characterization after purification in our laboratory. Its main property of solid-natural starch degradation was extensively investigated for its pharmaceutical/ industrial applications. Results: Amylase producing bacteria Bacillus cereus sm-sr14 (Accession no. KM251578.1) was purified to homogeneity on a Seralose 6B-150 gel-matrix and gave a single peak during HPLC. MALDITOF mass-spectrometry with bioinformatics studies revealed its significant similarity to α/β hydrolase family. The enzyme showed an efficient application; favourable Km, Vmax and Kcat during the catalysis of different natural solid starch materials. Analysis for hydrolytic product showed that this enzyme can be classified as the exo-amylase asit produced a significant amount of glucose. Conclusion: Besides the purified enzyme, the present organism Bacillus cereus sm-sr14 could degrade natural solid starch materials like potato and rice up to the application level in the pharmaceutical/ industrial field for alcohol production.

Isolation, identification and in silico study of native cellulase producing bacteria

2019 ◽  
Vol 17 ◽  
Author(s):  
Farzane Kargar ◽  
Mojtaba Mortazavi ◽  
Mahmood Maleki ◽  
Masoud Torkzadeh Mahani ◽  
Younes Ghasemi ◽  
...  
Keyword(s):  
Growth Rate ◽  
Bacillus Cereus ◽  
16S Rdna ◽  
Enzyme Production ◽  
Rational Design ◽  
Cellulase Production ◽  
Bacillus Species ◽  
Bacillus Sp ◽  
Chain Polymer ◽  
Bacillus Toyonensis

Aims: The purpose of this study was to screen the bacteria producing cellulase enzymes and their bioinformatics studies. Background: Cellulose is a long-chain polymer of glucose that hydrolyzes by cellulases to glucose molecules. In order to design the new biotechnological applications, some strategies have been used as increasing the efficiency of enzyme production, generating cost-effective enzymes, producing stable enzymes and identification of new strains. Objective: On the other hand, some bacteria special features have made them suitable candidates for the identification of the new source of enzymes. In this regard, some native strains of bacteria were screened. Method: These bacteria were grown on a culture containing the liquid M9 media containing CMC to ensure the synthesis of cellulase. The formation of a clear area in the culture medium indicated decomposition of cellulose. In the following, the DNA of these bacteria were extracted and their 16S rDNA genes were amplified. Result: The results show that nine samples were able to synthesize cellulase. In following, these strains were identified using 16S rDNA. The results show that these screened bacteria belonged to the Bacillus sp., Alcaligenes sp., Alcaligenes sp., and Enterobacter sp.conclusionThe enzyme activity analysis shows that the Bacillus toyonensis, Bacillus sp. strain XA15-411 Bacillus cereus have produced the maximum yield of cellulases. However, these amounts of enzyme production in these samples are not proportional to their growth rate. As the bacterial growth chart within 4 consecutive days shows that the Alcaligenes sp. Bacillus cereus, Bacillus toyonensis, Bacillus sp. strain XA15-411 have a maximum growth rate. The study of the phylogenetic tree also shows that Bacillus species are more abundant in the production of cellulase enzyme. These bioinformatics analyses show that the Bacillus species have different evolutionary relationships and evolved in different evolutionary time. Other: However, for maximum cellulase production by this bacteria, some information as optimum temperature, optimum pH, carbon and nitrogen sources are needed for the ideal formulation of media composition. The cellulase production is closely controlled in microorganisms and the cellulase yields appear to depend on a variety of factors. However, the further studies are needed for cloning, purification and application of these new microbial cellulases in the different commercial fields as in food, detergent, and pharmaceutical, paper, textile industries and also various chemical industries. However, these novel enzymes can be further engineered through rational design or using random mutagenesis techniques.

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