Production of Alkaline Protease from Bacillus licheniformis through Statistical Optimization of Growth Media by Response Surface Methodology

Proteases are the hydrolytic enzymes which hydrolyzes peptide bond between proteins with paramount applications in pharmaceutical and industrial sector. Therefore production of proteases with efficient characteristics of biotechnological interest from novel strain is significant. Hence, in this study, an alkaline serine protease produced by Bacillus cereus strain S8 (MTCC NO 11901) was purified and characterized. The alkaline protease was purified by ammonium sulfate precipitation (50%), ion exchange (DEAE-Cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. As a result of this purification, a protein with specific activity of 300U/mg protein was obtained with purification fold 17.04 and recovery percentage of 34.6%. The molecular weight of the purified protease was determined using SDS-PAGE under non-reducing (71 kDa) and reducing conditions (35 kDa and 22 kDa). Zymogram analysis revealed that proteolytic activity was only associated with 22 kDa. These results indicate that existence of the enzyme as dimer in its native state. The molecular weight of the protease (22 kDa) was also determined by gel filtration (Sephadex G-200) chromatography and it was calculated as 21.8 kDa. The optimum activity of the protease was observed at pH 10.0 and temperature 70 °C with great stability towards pH and temperature with casein as a specific substrate. The enzyme was completely inhibited by PMSF and TLCK indicating that it is a serine protease of trypsin type. The enzyme exhibits a great stability towards organic solvents, oxidizing and bleaching agents and it is negatively influenced by Li2+ and Co2+ metal ions. The purified protein was further characterized by Matrix Assisted Laser Desorption Ionization/Mass Spectroscopy (MALDI/MS) analysis which reveals that total number of amino acids is 208 with isoelectric point 9.52.

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Section: Methodsmentioning

confidence: 99%

Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8

Lakshmi

Kumar

Hemalatha

2018

Journal of Genetic Engineering and Biotechnology

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“…It is worth noting that the present study was carried out at the optimal growth temperature of the genus Bacillus and Staphylococcus (Lakshmi & Hemalatha, ; Meira, Barbosa, Athayde, Siqueira‐Junior, & Souza, ; Simões, Simões, & Vieira, 2005). It is known that temperature is one of the parameters that most influence the formation of bacterial biofilms.…”

Section: Discussionmentioning

confidence: 99%

Bacteria isolated from the bovine gelatin production line: biofilm formation and use of different sanitation procedures to eliminate the biofilms

Férnandes

Oliveira

Cheriegate³

et al. 2018

Journal of Food Safety

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The objective of this study was to evaluate the biofilm formation by Staphylococcus aureus, Bacillus cereus, and Bacillus licheniformis, as well to verify the efficiency of the sanitation procedures (cleaning, disinfection, and cleaning + disinfection) on the biofilm removal. Biofilms were formed after immersion of stainless steel and PVC coupons into different culture media (semi‐finished bovine gelatin, final gelatin, and hydrolyzed collagen) at 35°C for 5, 32, and 48 hr. After 32 hr of contact, all microorganisms were capable to form biofilms on the different surfaces in contact with all culture media tested. The semi‐finished gelatin and the hydrolyzed collagen provided a higher biofilm formation (counts between 3.54 and 7.87 log CFU/cm2) when compared to the final gelatin (counts between 3.05 and 6.70 log CFU/cm2). The cleaning step complemented with the disinfection with peracetic acid was the only procedure capable of removing all biofilms (counts <3 log CFU/cm2). Practical applications In this study, we investigated the biofilm formation by different isolates from bovine gelatin, simulating the processing conditions of gelatin and hydrolyzed collagen. The present study showed a peculiar result since the gelatin was the culture medium that provided a lower biofilm formation, while the hydrolyzed collagen and the semi‐finished gelatin provided the best conditions for the formation of biofilms. In addition, we verified that the use of detergent or disinfectant alone was not efficient in the removal of the biofilms formed. In view of this, we suggest the requirements to prevent the formation of microbial biofilms of different bacterial species in the gelatin processing industry. In addition, we suggest measures to remove the biofilms from food processing surfaces.

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“…Production of crude alkaline protease from Bacillus cereus strain S8 (MTCC NO 11901) was carried out by statistically optimized media supplemented with molasses, 1% (w/v); potassium nitrate, 0.75% (w/v); salt solution, 5% (v/v) -{MgSO4.7H2O, 0.5% (w/v); KH2PO4, 0.5% (w/v)}; FeSO4.7H2O, 0.01% (w/v) and CaCO3, 0.5% formulated by (Lakshmi and Hemalatha, 2016).The protease is optically active at pH 10.0 and temperature 70 o C with great stability towards pH and temperature with casein as a specific substrate. The enzyme is a serine protease of trypsin type as it is completely inhibited by PMSF and TLCK.…”

Section: Production Of Crude Alkaline Proteasementioning

confidence: 99%

Industrial Applications of Alkaline Protease with Novel Properties from Bacillus Cereus Strain S8

B.K.M,

Sowjanya

et al. 2023

J. Adv. Zool.

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Proteases are the enzymes which catalyze the hydrolysis of peptide bonds in proteins and they are widely used industrial enzymes. The present paper reports the applications of alkaline protease from Bacillus cereus strain S8 in different industrial sectors. The protease was optimally active at pH 10. 0 and temperature 700C. This enzyme has been used as an alternative for conventional chemical treatments for dehairing of goat skins, chitin recovery from shrimp waste, silk degumming agent and as well as in detergent formulations. Thus, this protease is ecofriendly by reducing the pollution load by replacing conventional chemical methods. The deproteinization activity of Bacillus cereus strain S8 protease was better than many proteases reported due to its thermostability and its activity at alkaline pH. The percentage of protein removal from natural shrimp waste was 87% after 3 h of incubation at 70°C. The protease is effective as silk degumming agent as it promotes sericin hydrolysis with 19% weight loss of treated silk threads. Silk degumming is a very essential step for the removal of sericin protein from silk fibers for giving it shine and softness. The protease is also effective and profitable in dehairing of goat skins by promoting fine leather without affecting its quality as evidenced by Scanning electron micrograph (SEM) micrographs. Due to its unique properties it also compatible with various detergent formulations in industrial sector. These results suggest that the alkaline protease from Bacillus cereus strain S8 is significant in the industry from the prospects of its ability to withstand harsh conditions in various industrial applications.

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Production of Alkaline Protease from Bacillus licheniformis through Statistical Optimization of Growth Media by Response Surface Methodology

Cited by 12 publications

References 17 publications

Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8

Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8

Bacteria isolated from the bovine gelatin production line: biofilm formation and use of different sanitation procedures to eliminate the biofilms

Industrial Applications of Alkaline Protease with Novel Properties from Bacillus Cereus Strain S8

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