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.

scholarly journals Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant

2017 ◽  
Vol 7 (4) ◽  
pp. 1 ◽  
Author(s):  
Sreedevi Basavaraju ◽  
Chandrasekhar Kathera ◽  
Pramoda Kumari Jasti
Keyword(s):  
Bacillus Cereus ◽  
Ammonium Sulphate ◽  
Alkaline Protease ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Tween 20 ◽  
Original Activity ◽  
Protease Enzyme

The alkaline protease produced by Bacillus cereus UV-15 mutant was purified by precipitation with ammonium sulphate and gel filtration through sephadex G-100. The enzyme has shown to have a molecular weight of 29kDa by SDS polyacrylamide gel electrophoresis. The extracted protease enzyme was purified by 16.64 fold through ammonium sulphate precipitation and chromatography separation in Sephadex G-100. The purified protease had a specific activity of 2915 (U/mg). The zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which coincided with the band obtained with SDS–PAGE. The enzyme was remained active and stable at pH 8-11, with an optimum at pH 10.0. The protease was stable in the temperature ranging from 40°C to 60°C, but gradually decreased at temperature 70°C. The optimum temperature for protease activity was determined at 60°C. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. At 1% concentration of Tween-20 and Tween-80, the enzyme retained 78% and 94% relative activity respectively. Alkaline protease retained 95% activity toward 0.5% concentration of the anionic detergent SDS. The enzyme showed compatibility at 50°C with commercial detergents such as Ariel, Surf excel, Rin, wheel, Tide and Nirma. In the presence of Ariel and Rin the enzyme retained about 72 and 75% of the original activity respectively. The supplementation of the enzyme in detergents could improve the cleansing performance towards the blood stains and suggested to be used as a detergent additive. The enzyme also removed goat hide hairs completely after 15 hr of incubation. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.

scholarly journals The N-Terminal β-Sheet of the Hyperthermophilic Endoglucanase from Pyrococcus horikoshii Is Critical for Thermostability

2012 ◽  
Vol 78 (9) ◽  
pp. 3059-3067 ◽  
Author(s):  
Trent C. Yang ◽  
Steve Legault ◽  
Emery A. Kayiranga ◽  
Jyothi Kumaran ◽  
Kazuhiko Ishikawa ◽  
...  
Keyword(s):  
Triosephosphate Isomerase ◽  
Industrial Applications ◽  
Crystalline Cellulose ◽  
Bioinformatics Analyses ◽  
Pyrococcus Horikoshii ◽  
Content Type ◽  
Enzyme Thermostability ◽  
Tim Barrel ◽  
Key Residues ◽  
Β Sheet

ABSTRACTThe β-1,4-endoglucanase (EC 3.2.1.4) from the hyperthermophilic archaeonPyrococcus horikoshii(EGPh) has strong hydrolyzing activity toward crystalline cellulose. When EGPh is used in combination with β-glucosidase (EC 3.2.1.21), cellulose is completely hydrolyzed to glucose at high temperature, suggesting great potential for EGPh in bioethanol industrial applications. The crystal structure of EGPh shows a triosephosphate isomerase (TIM) (β/α)8-barrel fold with an N-terminal antiparallel β-sheet at the opposite side of the active site and a very short C-terminal sequence outside of the barrel structure. We describe here the function of the peripheral sequences outside of the TIM barrel core structure. Sequential deletions were performed from both N and C termini. The activity, thermostability, and pH stability of the expressed mutants were assessed and compared to the wild-type EGPh enzyme. Our results demonstrate that the TIM barrel core is essential for enzyme activity and that the N-terminal β-sheet is critical for enzyme thermostability. Bioinformatics analyses identified potential key residues which may contribute to enzyme hyperthermostability.

scholarly journals Biochemical and structural investigation of rutinosidase from Aspergillus oryzae

2020 ◽  
Author(s):  
Koki Makabe ◽  
Ruka Hirota ◽  
Yoshihito Shiono ◽  
Yoshikazu Tanaka ◽  
Takuya Koseki
Keyword(s):  
Crystal Structure ◽  
Aspergillus Oryzae ◽  
Structural Investigation ◽  
Signal Sequence ◽  
Structural Similarity ◽  
Industrial Applications ◽  
Flavonoid Glycoside ◽  
Flavonoid Glycosides ◽  
High Catalytic Activity ◽  
Tim Barrel

The rutinosidase-encoding gene Aorut has been expressed in Pichia pastoris with its native signal sequence from Aspergillus oryzae. Biochemical and structural investigation of the purified recombinant mature AoRut, designated as rAoRutM, was performed in this study. A 1.7 Å resolution crystal structure of rAoRutM was determined, which is an essential step forward in the utilization of AoRut as a potential catalyst. The crystal structure of rAoRutM was represented by a (β/α)8 TIM barrel fold with structural similarity to rutinosidase from Aspergillus niger (AnRut) and an exo-β (1, 3)-glucanase from Candida albicans. The crystal structure revealed that the catalytic site was located in a deep cleft, similar to AnRut, and internal cavities and water molecules were also present. Purified rAoRutM hydrolyzed not only 7-O-linked and 3-O-linked flavonoid rutinosides, but also 7-O-linked and 3-O-linked flavonoid glucosides. rAoRutM displayed high catalytic activity toward quercetin 3-O-linked substrates such as rutin and isoquercitrin, rather than the 7-O-linked substrate, quercetin-7-O-glucoside. Unexpectedly, purified rAoRutM exhibited increased thermostability after treatment with endo-β-N-acetylglucosaminidase H. Circular dichroism (CD) spectra of purified intact rAoRutM and the enzyme after N-deglycosylation showed a typical α-helical CD profile, however, the molar ellipticity values of the peaks at 208 nm and 212 nm varied. The Km and kcat values for the substrates modified by rutinose were higher than those for substrates modified by β-D-glucose. Importance Flavonoid glycosides constitute a class of secondary metabolites widely distributed in nature. These compounds are involved in the bitter taste or clouding in plant-based foods or beverages, respectively. Flavonoid glycoside degradation can proceed through two alternative enzymatic pathways: one that is mediated by monoglycosidases, and the other catalyzed by a diglycosidase. The present study on the biochemical and structural investigation of A. oryzae rutinosidase provides a potential biocatalyst for industrial applications of flavonoids.

scholarly journals Genomic analysis of Bacillus cereus NWUAB01 and its heavy metal removal from polluted soil

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ayansina Segun Ayangbenro ◽  
Olubukola Oluranti Babalola
Keyword(s):  
Heavy Metal ◽  
Surface Tension ◽  
Bacillus Cereus ◽  
Metal Removal ◽  
Heavy Metal Removal ◽  
Gene Clusters ◽  
Industrial Applications ◽  
Luria Bertani ◽  
Biosurfactant Production ◽  
Screening Methods

AbstractMicroorganisms that display unique biotechnological characteristics are usually selected for industrial applications. Bacillus cereus NWUAB01 was isolated from a mining soil and its heavy metal resistance was determined on Luria–Bertani agar. The biosurfactant production was determined by screening methods such as drop collapse, emulsification and surface tension measurement. The biosurfactant produced was evaluated for metal removal (100 mg/L of each metal) from contaminated soil. The genome of the organism was sequenced using Illumina Miseq platform. Strain NWUAB01 tolerated 200 mg/L of Cd and Cr, and was also tolerant to 1000 mg/L of Pb. The biosurfactant was characterised as a lipopeptide with a metal-complexing property. The biosurfactant had a surface tension of 39.5 mN/m with metal removal efficiency of 69%, 54% and 43% for Pb, Cd and Cr respectively. The genome revealed genes responsible for metal transport/resistance and biosynthetic gene clusters involved in the synthesis of various secondary metabolites. Putative genes for transport/resistance to cadmium, chromium, copper, arsenic, lead and zinc were present in the genome. Genes responsible for biopolymer synthesis were also present in the genome. This study highlights biosurfactant production and heavy metal removal of strain NWUAB01 that can be harnessed for biotechnological applications.

scholarly journals Biosynthesis of Fatty Alcohols in Engineered Microbial Cell Factories: Advances and Limitations

2020 ◽  
Vol 8 ◽  
Author(s):  
Anagha Krishnan ◽  
Bonnie A. McNeil ◽  
David T. Stuart
Keyword(s):  
Fatty Acid ◽  
Fatty Alcohol ◽  
Industrial Applications ◽  
Microbial Cell ◽  
Fatty Alcohols ◽  
Scale Production ◽  
Endogenous Fatty Acid ◽  
Alcohol Production ◽  
Microbial Cell Factories ◽  
Cell Factories

Concerns about climate change and environmental destruction have led to interest in technologies that can replace fossil fuels and petrochemicals with compounds derived from sustainable sources that have lower environmental impact. Fatty alcohols produced by chemical synthesis from ethylene or by chemical conversion of plant oils have a large range of industrial applications. These chemicals can be synthesized through biological routes but their free forms are produced in trace amounts naturally. This review focuses on how genetic engineering of endogenous fatty acid metabolism and heterologous expression of fatty alcohol producing enzymes have come together resulting in the current state of the field for production of fatty alcohols by microbial cell factories. We provide an overview of endogenous fatty acid synthesis, enzymatic methods of conversion to fatty alcohols and review the research to date on microbial fatty alcohol production. The primary focus is on work performed in the model microorganisms, Escherichia coli and Saccharomyces cerevisiae but advances made with cyanobacteria and oleaginous yeasts are also considered. The limitations to production of fatty alcohols by microbial cell factories are detailed along with consideration to potential research directions that may aid in achieving viable commercial scale production of fatty alcohols from renewable feedstock.

scholarly journals Characterizing Activity and Thermostability of GH5 Cellulase Chimeras from Mesophilic and Thermophilic Parents

2018 ◽  
Author(s):  
Fei Zheng ◽  
Josh V. Vermaas ◽  
Jie Zheng ◽  
Yuan Wang ◽  
Tao Tu ◽  
...  
Keyword(s):  
Thermal Properties ◽  
Rational Design ◽  
Enzyme Stability ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Essential Property ◽  
Tim Barrel ◽  
Dynamics Simulations ◽  
Temperature Optima ◽  
Hybrid Enzymes

ABSTRACTCellulases from glycoside hydrolase (GH) family 5 are key enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme stability and activity. Despite their shared topology, the thermostability of GH5 enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on a previously characterized thermophilic GH5 cellulase from Talaromyces emersonii (TeEgl5A, with an optimal temperature of 90°C), we created ten hybrid enzymes with the mesophilic cellulase from Prosthecium opalus (PoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optima (10 and 20°C), T50 (15 and 19°C), Tm (16.5 and 22.9°C), and extended half life, t1/2 (~240- and 650-fold at 55°C) relative to the mesophilic parent enzyme, and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 cellulase from Aspergillus nidulans (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics simulations (MD) of both PoCel5 and TeEgl5A parent enzymes as well as their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2 and α3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to the mesophilic parent PoCel5.IMPORTANCEThermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM-barrel fragments and even fragments from unrelated folds, to generate new structures. However, there are not many research on GH5 cellulases. In this study, two GH5 cellulases, which showed TIM-barrel structure, PoCel5 and TeEgl5A with different thermal properties were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

scholarly journals Bio-Delipidation of Dissolved Air Flotation Pre-Treated Poultry Slaughterhouse Wastewater

2018 ◽  
Author(s):  
Siyasanga Mbulawa ◽  
Seteno Karabo Obed Ntwampe ◽  
Moses Basitere ◽  
Yolanda Mpentshu ◽  
Cynthia Dlangamandla ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Oxygen Demand ◽  
Industrial Applications ◽  
Quality Parameters ◽  
Bacterial Strains ◽  
Oil And Grease ◽  
Dissolved Air Flotation ◽  
Slaughterhouse Wastewater ◽  
Air Flotation ◽  
Dissolved Air

Delipidation is a method of defatting that is generally associated with the removal of residual lipids or lipid groups from matrices in which they are present in minute quantities. The bio-delipidation of protein-rich poultry slaughterhouse wastewater (PSW) pre-treated with a dissolved air flotation (DAF) system was developed using microbial lipases from bacterial strains isolated from the PSW. The efficacy of the bio-delipidation system was quantitatively characterised by comparing the quality parameters i.e., fats, oil and grease (FOGs), turbidity, total suspended solutes (TSS), total chemical oxygen demand (tCOD) and protein concentration of the DAF pre-treated PSW and bio-lipidized samples. As hypothesised, the bio-delipidation system was able to effectively reduce the levels of these quality parameters when crude lipases of Bacillus cereus AB1 (BF3) and Bacillus cereus CC-1 (B30) strains were used. Strain-dependent quality characteristics were also observed in bio-delipidized samples. The study successfully managed to complement physical reduction techniques (DAF) with biological strategies (bio-delipidation) for improved PSW quality, with potential industrial applications.

scholarly journals Purification and characterisation of thermostable α-amylases from microbial sources

BioResources ◽  
2019 ◽  
Vol 15 (1) ◽  
pp. 2005-2029
Author(s):  
Si J. Lim ◽  
Siti Nur Hazwani-Oslan ◽  
Siti N. Oslan
Keyword(s):  
Column Chromatography ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Promising Result ◽  
Potato Starch ◽  
Industrial Applications ◽  
Wheat Starch ◽  
Starch Degradation ◽  
Affinity Column ◽  
Microbial Sources

α-Amylases (E.C 3.2.1.1) hydrolyse starch into smaller moieties such as maltose and glucose by breaking α-1,4-glycosidic linkages. The application of α-amylases in various industries has made the large-scale productions of these enzymes crucial. Thermostable α-amylase that catalyses starch degradation at the temperatures higher than 50 °C is favourable in harsh industrial applications. Due to ease in genetic manipulation and bulk production, this enzyme is most preferably produced by microorganisms. Bacillus sp. and Escherichia coli are commonly used microbial expression hosts for α-amylases (30 to 205 kDa in molecular weight). These amylases can be purified using ultrafiltration, salt precipitation, dialysis, and column chromatography. Recently, affinity column chromatography has shown the most promising result where the recovery rate was 38 to 60% and purification up to 13.2-fold. Microbial thermostable α-amylases have the optimum temperature and pH ranging from 50 °C to 100 °C and 5.0 to 10.5, respectively. These enzymes have high specificity towards potato starch, wheat starch, amylose, and amylopectin. EDTA (1 mM) gave the highest inhibitory effect (79%), but Ca2+ (5 mM) was the most effective co-factor with 155%. This review provides insight regarding thermostable α-amylases obtained from microbial sources for industrial applications.

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