Cold-active extracellular alkaline protease from an alkaliphilicStenotrophomonas maltophilia: production of enzyme and its industrial applications

2009 ◽  
Vol 55 (11) ◽  
pp. 1294-1301 ◽  
Author(s):  
Mohammed Kuddus ◽  
Pramod W. Ramteke
Keyword(s):  
High Efficiency ◽  
Western Himalaya ◽  
Industrial Applications ◽  
Culture Conditions ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Protease Production ◽  
Cold Active ◽  
Metalloprotease Inhibitors ◽  
Potential Component

A novel psychro-tolerant bacterium Stenotrophomonas maltophilia (MTCC 7528) with an ability to produce extracellular, cold-active, alkaline, and detergent-stable protease was isolated from soil samples obtained from Gangotri glacier, Western Himalaya, India. The culture conditions for higher protease production were optimized with respect to incubation time, agitation, substrate, pH, and temperature. Maximum protease production of 56.2 U·mL–1was achieved in the medium at 20 °C and pH 9.0 after 120 h incubation. The protease was partially purified by ion-exchange chromatography and approximately 55-fold purification was achieved. The purified enzyme was a 75 kDa protease with maximum activity and stability at pH 10 and 20 °C. The activity of enzyme is stimulated by Mn2+and inhibited completely by metalloprotease inhibitors, indicating that it is a metalloprotease. The protease showed excellent stability and compatibility with commercial detergents and exhibited high efficiency for the removal of different types of protein-containing stains at low temperature. The wash performance analysis of blood and grass stains on cotton fabric showed an increase in reflectance by 26% and 23%, respectively, after treatment with enzyme in comparison to detergent only. These results indicate that it may be a potential component to use as a detergent additive for cold washing and in environmental bioremediation in cold regions.

scholarly journals Effect of Culture Conditions on the Production of an Extracellular Protease by Bacillus sp. Isolated from Soil Sample of Lavizan Jungle Park

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Abbas Akhavan Sepahy ◽  
Leila Jabalameli
Keyword(s):  
Enzyme Production ◽  
Corn Steep Liquor ◽  
Experimental Studies ◽  
Environmental Parameters ◽  
Nitrogen Sources ◽  
Culture Conditions ◽  
Maximum Activity ◽  
Protease Production ◽  
North East ◽  
Steep Liquor

Soil samples of Tehran jungle parks were screened for proteolytic Bacilli. Among eighteen protease producers one of the isolates obtained from Lavizan park, in north east of Tehran, was selected for further experimental studies. This isolate was identified as Bacillus sp. strain CR-179 based on partial sequencing of 16S rRNA. Various nutritional and environmental parameters affected protease production by Bacillus sp. strain CR-179. Protease production by this Bacillus cultivated in liquid cultures reached a maximum at 24 h, with levels of 340.908 U/mL. Starch and maltose were the best substrates for enzyme production while some pure sugars such as fructose, glucose, and sucrose could not influence production of protease. Among various organic nitrogen sources corn steep liquor, which is commercial, was found as the best substrate followed by yeast extract, whey protein, and beef extract. The optimal pH and optimal temperature of enzyme production were 8.0 and 45°C, respectively. Studies on enzymatic characterization revealed that crude protease showed maximum activity at pH 9.0 and 60°C, which is indicating the enzyme to be thermoalkaline protease.

New Insight into Old Bacillus Lipase: Solvent Stable Mesophilic Lipase Demonstrating Enzyme Activity towards Cold

2015 ◽  
Vol 25 (5) ◽  
pp. 340-348 ◽  
Author(s):  
Jyoti Khurana ◽  
Rakesh Kumar ◽  
Arbind Kumar ◽  
Kashmir Singh ◽  
Ranvir Singh ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Lipase Gene ◽  
Reading Frame ◽  
Wide Range ◽  
Cold Active ◽  
Solvent Stable ◽  
Insight Into

<b><i>Background:</i></b><i>Bacillus</i> lipases are grouped in subfamily 1.4, which are the smallest known lipases having a molecular mass of 19.6 kDa. Lipases active in a wide range of temperatures, specifically at low temperatures, have an advantage under low water conditions for industrial application. <b><i>Methods:</i></b> The lipase gene was cloned and expressed in <i>Escherichia coli</i>. The protein was purified and biochemically characterized in detail. <b><i>Results:</i></b> A lipase gene was cloned from a mesophilic <i>Bacillus</i> isolate. Sequence analysis revealed an open reading frame of 633 bp in length. The predicted molecular mass of protein was 22.6 kDa. The purified enzyme displayed optimal activity at 35°C and pH 8.0. Interestingly, this mesophilic enzyme was also cold active showing retention of 75 and 55% relative enzyme activity at 20 and 10°C, respectively. The purified lipase was stable in various organic solvents (50% v/v) and ionic liquids (5% v/v). The enzyme displayed maximum activity with paranitrophenyl laurate (C<sub>12</sub>). k<sub>cat</sub>/K<sub>m</sub> values for the purified lipase were calculated to be 5.8 ± 0.6 × 10<sup>-6</sup>. <b><i>Conclusions:</i></b> The lipase showed tolerance to various solvents as well as activity at low temperature. Therefore, this lipase might be of great potential to be employed in various industrial applications.

Cytotoxic Effect of Pseudomonas aurogenosa Protease on Cancer Cells

2019 ◽  
Vol 10 (03) ◽  
Author(s):  
Ghanyia J. Shanyoor ◽  
Fatima R. Abdul ◽  
Nehad A. Taher ◽  
Ihsan A. Raheem
Keyword(s):  
Cell Line ◽  
Normal Cell ◽  
Cytotoxic Effect ◽  
Human Cancer ◽  
Skim Milk ◽  
Exchange Chromatography ◽  
Protease Production ◽  
Normal Cell Line ◽  
Protease Enzyme ◽  
Embryonic Fibroblast

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) and the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method and it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column and the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , and one normal cell line Ref ( Rat embryonic fibroblast ) . The cancer and normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8and 0.16 mg/ml) then incubated for additional 48h at 37C0 and the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andslight toxicity ( 37.12% ) on normal cell line (Ref) in a concentration (0.8mg/ml).

Constraints in Adoption ofModern Farm Machines by Tribal Farmers in Ramgarh District of Jharkhand

2019 ◽  
Vol 6 (03) ◽  
Author(s):  
PK SUNDARAM ◽  
BIKASH SARKAR ◽  
UJJWAL KUMAR ◽  
AP ANURAG ◽  
DK RAGHAV ◽  
...  
Keyword(s):  
Cell Line ◽  
Normal Cell ◽  
Human Cancer ◽  
Skim Milk ◽  
Exchange Chromatography ◽  
Protease Production ◽  
Normal Cell Line ◽  
Protease Enzyme ◽  
Tribal Farmers ◽  
Embryonic Fibroblast

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) andamp; the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method andamp; it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column andamp; the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , andamp; one normal cell line Ref ( Rat embryonic fibroblast ). The cancer andamp; normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8andamp; 0.16 mg/ml) then incubated for additional 48h at 37C 0 andamp; the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andamp;slight toxicity ( 37.12% )on normal cell line (Ref) in a concentration (0.8mg/ml).

scholarly journals Insights into the Functionality of the Putative Residues Involved in Enterocin AS-48 Maturation

2010 ◽  
Vol 76 (21) ◽  
pp. 7268-7276 ◽  
Author(s):  
Rubén Cebrián ◽  
Mercedes Maqueda ◽  
José Luis Neira ◽  
Eva Valdivia ◽  
Manuel Martínez-Bueno ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
High Efficiency ◽  
Specific Protein ◽  
Culture Conditions ◽  
Site Directed Mutagenesis ◽  
Circular Structure ◽  
Cleavage Enzyme ◽  
Site Recognition ◽  
Biosynthetic Machinery

ABSTRACT AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein consists of an N-terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His-1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2-2 strain containing the necessary native biosynthetic machinery for enterocin production. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81 His-1Ile ) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

scholarly journals Red Mud-Based Polyaluminum Ferric Chloride Flocculant: Preparation, Characterization and Flocculation Performance

2021 ◽  
Author(s):  
Yong Cheng ◽  
Longjun Xu ◽  
Chenglun Liu ◽  
Zao Jiang ◽  
Qiyuan Zhang ◽  
...  
Keyword(s):  
Red Mud ◽  
High Efficiency ◽  
Low Cost ◽  
Oxygen Demand ◽  
Industrial Applications ◽  
Iron Chloride ◽  
Raw Material ◽  
Molar Ratio ◽  
Polymerization Temperature ◽  
Solution Ph

Abstract In this work, red mud was used as raw material to extract Al and Fe with hydrochloric acid. The high-efficiency polyaluminum iron chloride (PAFC) flocculant was prepared via adjusting the pH of the leaching solution, the molar ratio of aluminum and iron, and the polymerization temperature. The effect of synthesis and flocculation conditions on the flocculation performance of aged landfill leachate was investigated. The results confirmed that the PAFC prepared at the polymerization pH of 2.5, the Al/Fe molar ratio of 8, and the polymerization temperature of 70 °C had the optimum flocculation effect. The flocculation consequences of PAFC and commercial polyaluminum iron chloride flocculant (CPAFC) under different flocculation conditions were compared. The chemical oxygen demand (COD), UV254, chroma and settlement height of PAFC at flocculant concentration of 60 g/L and solution pH of 6 were 72.2%, 79.2%, 82.9% and 9.5 cm (within 90 min), respectively. PAFC has excellent flocculation performance and can be used as a simple, potentially low-cost wastewater treatment agent in industrial applications.

scholarly journals STUDY ON THE IN VITRO MORPHOGENETIC RESPONSE FROM SHOOT AND CALLUS CULTURES OF Blepharis maderaspatensis (L.) Heyne ex. Roth

2020 ◽  
Vol 1 (40) ◽  
pp. 28-38
Author(s):  
Trang Phuong Nguyen Thi ◽  
Quang Minh Bui ◽  
Hai Duc Le ◽  
Linh Quoc Nguyen
Keyword(s):  
High Efficiency ◽  
Raw Materials ◽  
Callus Cultures ◽  
Culture Conditions ◽  
Ms Medium ◽  
Axillary Shoots ◽  
Scientific Database ◽  
Medicinal Value ◽  
Shoot Growth Rate

Blepharis maderaspatensis (L.) Heyne ex. Roth is a short-term plant which contains many important secondarycompounds with high medicinal value. Currently, most of the researches focus on chemical composition and pharmacological activity, but the source of raw materials is very limited. In this study, the first step is transferring the samples from nature into in vitro culture conditions to understand the effects of the factors related to shooting and callus morphogenesis was performed, the first node from shoots apical meristem was isolated and sterilized with 1.5% NaOCl for 20 minutes to achieve high efficiency with 86.11% sterile samples and 85.56% shoot growth rate after 2 weeks of culture on MS medium. The shoot generation from axillary shoots was continued to be investigated with the highest number of shoots formed on MS medium supplemented with BA (1 mg / l) showed 1.53 shoots/implant which the height and the number of leavesare 3.65cm and 6.67, respectively. Besides, the formation of callus from leaves of MS medium supplemented with 2.4 - D (0.25 mg / l) achieved the rate of 66.67% of cultured samples, forming good callus after 4 weeks of culture. The results of the study not only contribute importantly to understanding morphogenesis for micropropagation purposes but also serve as the scientific database for further studies at the cellular and molecular levels of this plant.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

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