Casein Agar Plate Research Articles

Neurotoxic Acrylamide Mitigation By L-Asparaginase Enzyme Extracted From Actinomycetes Species.

Acrylamide is a chemical that is generated in starchy foods, coffee, and bread, etc during high-temperature cooking processes, such as frying, roasting, and baking and is a major food safety concern. Acrylamide toxicity targets the nervous and reproductive systems, causing symptoms like muscle weakness and numbness therefore there is a need to solve this problem by biological method. Actinomycetes are an excellent source of L-asparaginase, an enzyme which specifically catalyses the breakdown of amino acid L-asparagine to aspartic acid and ammonia. If starchy food products are not treated with L- asparginase enzyme then at high temperature L-asparagine present in food converts to acrylamide. Hence, water and soil samples were obtained from Uran, Sai, and Panvel regions and Actinomycetes were isolated onto Starch Casein Agar plates. Biochemical analyses revealed negative results for TSI, nitrate reduction, and citrate but positive for catalase and starch hydrolysis. Noteworthy enzymatic activity at 2.66µg/ml/min was observed. Optimization was performed with various parameters. Production and purification of L-asparaginase by submerged fermentation, followed by ammonium precipitation, dialysis, and subsequent application to pretreat potatoes. After incubation and frying, high-performance liquid chromatography analysis exhibited a reduction in acrylamide levels. The study's meticulous approach, from isolation to application, addresses critical food safety issues, marking a significant contribution.

Endophytes Producing Proteases from Custard Apple (Annona squamosa L.) Leaves

The cost of the proteolytic enzymes and their inability to remove “fine” hairs from the animal hide has been the main obstacles to the commercialization of an enzymatic dehairing procedure. Endophytic microbes that grow inside various plant tissues without causing tissue damage in the host plant are the important sources of bioactive compounds. They are rich in secondary metabolites and known to produce wide range of enzymes. Several endophytes showing different properties have been reported from Annona leaves and hence this study was performed to isolate endophytes having proteolytic properties. Endophytes from custard apple leaves were isolated and screened for proteolytic activity on casein agar plates. The protease enzyme activity of the promising isolates was determined using casein substrate and tyrosine release assay. Present study conducted on the bacterial endophytes from the Custard apple leaves showed that the plant-associated bacterial endophytes are a good reservoir of proteases. Two promising isolates were obtained on the casein agar plates. These two isolates C and D possessed 113U/ml and 102 U/ml enzyme activity respectively. The isolate C showed more dehairing capacity than isolate D. Purified forms of these enzymes can prove to be potential candidates in biotechnological applications.

Effects of Temperature and pH on the Stability of Protease Produced by Alcaligenes faecalis P2

Background: Bacterial proteases represent a group of very important industrial enzymes. They are involved in the hydrolysis of peptide bond found in protein. Industrial application of bacterial proteases has been limited by low yield and instability at biotechnological process conditions. This study was design to investigate the effect of temperature and pH on the stability of protease produced by Alcaligene faecalis strain P2.
 Methodology: Protease-producing bacteria were isolated from beans effluent-impacted soil and screened for protease production on Casein agar plate. Protease assay was carried out following standard method for protease determination and the stability of the protease produced was investigated over temperature range of 20 to 90 oC and pH range of 3 to 12. The protease-producing bacterium was identified using its molecular characteristics.
 Results: Protease assay result showed that the amount of tyrosine released by one unit of the crude enzyme was 0.176 µmol/mL resulting in 0.19 U/mL protease activity. Stability studies showed that the protease had wide spectrum of pH and temperature stability. The protease was most stable at pH 9 and temperature of 40 oC after 30 min. Maximum protease activity of 1.259 U/mL was recorded with pH 9 after 30 min. The protease-producing bacterium was classified as Alcaligene faecalis P2 based on phylogenetic analysis of its 16S gene analysis. The sequences have been submitted to GenBank under the accession number MZ477004.
 Conclusion: This study therefore has demonstrated that beans effluent-impacted soil harbours protease producing bacteria with high industrial potentials. In addition, the study revealed that the protease produced in this study can retain its activity over wide temperature and pH.

Increment in protease activity of Lysobacter enzymogenes strain by ultra violet radiation.

Background and Objectives:Increasing the amount of protease from microbial sources is in the focus of attention. Random mutagenesis by physical methods like ultraviolet (UV) radiation is a cost effective and convenient procedure for strain improvement. Therefore, in the present study attempts were made to investigate the effect of UV radiation on Lysobacter enzymogenes in order to increase its protease activity.Materials and Methods:UV mutagenesis was induced in L. enzymogenes fresh culture at the distance of 20 cm from light source for different exposure times of 70, 90, 150 and 200 seconds. The mutated isolates were randomly cultured from the nutrient agar medium to casein agar plate, as a selective medium. The primary screening was performed by observing hydrolysis of casein in the plate and the secondary screening was carried out on skim milk agar on the basis of zone of hydrolysis using bacterial supernatants. Quantification of protease activity was done by Anson’s method using tyrosine as standard.Results:UV radiation resulted in obtaining 12 mutants out of 100 examined L. enzymogenes strains with increased protease activity. The mutant M2, at 90s exposure time was selected as the best mutant bacterium which produced 1.96 fold more protease over the parent strain.Conclusion:Random mutation by UV radiation is a simple and convenient method to increase the protease activity of Lysobacter enzymogenes. Furthermore, it seems that the middle time of exposure to UV, 90 s, was the best time because it can induce mutagenesis but did not hamper the bacteria growth and viability.

Degradation of complex casein polymer: Production and optimization of a novel serine metalloprotease from Aspergillus niger KIBGE-IB36

Abstract Serine metalloproteases have substantial scientific interest due to their unique physicochemical properties and capability to degrade complex casein polymer. In this contemporary study, the production of protease was enhanced by optimizing various physicochemical parameters and one-variable at a time approach used under submerged fermentation conditions. Among four different previously isolated Aspergillus species, Aspergillus niger KIBGE-IB36 represented high protease titer along with casein hydrolyzing zone on casein agar plate. Maximum protease production was achieved at 30 °C after 5 days of cultivation period at pH-6.0. Glucose (2.5 g L-1) and casein (2.5 g L-1) augmented protease production within the growth medium. Further increment in protease production was attained when different combinations of nitrogen and mineral salts concentrations used. Peptone (20.0 g L-1), yeast extract (0.50 g L-1), CaCl2 (0.10 g L-1), MgSO4 (0.10 g L-1) and K2HPO4 (0.30 g L-1) boosted the protease secretion. The current study is imperative for the large scale production of serine metalloprotease that also possess extraordinary prominence in food processing industries.

Screening of Amylase and Cellulase Enzymes from Marine Actinomycetes

Actinomycetes have an important role in the Industrial enzymes, Antibiotics, vitamins and organic acids. We have isolated 114 actinomycetes strains from Chennai coastal region by using starch casein agar plate method. Out of these 114 actinomycetes strains, 13 actinomycetes strains showed amylase activity by starch agar plate method and 12 strains showed cellulase activity by cellulase agar plate method, which was indicated by clear zone of dark blue color formation for amylase activity and dark yellow color formation for cellulase activity. Based on the maximum amylase and cellulase producing strains MB23 and BN08 was found to be a potential strains for further investigation.

Selection of Marine Actinomycetes with Bioactive Potential Isolated from Sediments of Bay of Bengal and Characterization of Promising Isolate, ABT-103

Aim: Marine actinomycetes are a potential and untapped source for the production of novel bioactive compounds. The aim of the present work is to isolate actinomycetes strains from the marine sediments of Bay of Bengal for the production of bioactive compounds and characterization of the potential actinomycetes. Place and Duration of Study: Department of Chemical Engineering and Biotechnology, ANITS, Visakhapatnam, between March, 2014 and April, 2015. Materials and Methods: Marine sediment samples were collected along the coast of Bay of Bengal, Visakhapatnam, India. Actinomycetes were isolated on Starch Casein Agar plates by pour plate and spread plate methods. Morphologically distinct pure isolates were tested for antimicrobial activity by Cross-streak method in preliminary screening and by Cup-plate method in secondary Short Research Article Sridevi et al.; BMRJ, 10(2): 1-9, 2015; Article no.BMRJ.20132 2 screening. The marine isolates were also screened for enzyme activities of amylase, lipase, protease and L-asparaginase. The most potential isolate was characterized up to genus level based on morphological, chemotaxonomic, biochemical and physiological characteristics. Results: A total of 74 bacterial strains were isolated from marine sediment samples of Bay of Bengal. Among them, 13 morphologically distinct pure isolates were screened for antimicrobial activity and also for enzyme activities. Five isolates exhibited antimicrobial activity among which ABT-205 isolate showed broad spectrum antimicrobial activity against both bacteria and fungi, and ABT-103 exhibited maximum antifungal activity. Screening for enzyme activities revealed that nine isolates exhibited enzyme activities of which, ABT-101 and ABT-201 are highly potential for the production of Protease, whereas ABT-103 and ABT-206 were best producers of Amylase and Lipase enzymes, respectively. Among all the isolates screened, ABT-103 was found to be a promising isolate as it produced red pigment, exopolysaccharide, amylase and exhibited antifungal activity. Hence, the isolate ABT-103 was characterized and identified as Streptomyces species. Conclusion: The selected marine actinomycetes isolates, ABT-101, ABT-103, ABT-201, ABT-205 and ABT-206 could be useful or the production of novel antibiotics, enzymes with different physical and physiological properties, red pigment, exopolysaccharide and other compounds for various Biotechnological applications.

English

Protease-producing bacterium Bacillus cereus SU12 was isolated from oysterSaccostrea cucullata. Fifteen strains of bacteria were isolated from oyster S. cucullata and screened for secretion of protease on casein agar plates. Among them, SU12 isolate was selected due to its high enzyme production capacity and was identified as B. cereus SU12 on the basis of its morphological, biochemical and 16S rDNA properties. Media and cultivation conditions were studied to optimize bacterial growth and protease production which includes different carbon and nitrogen sources, in addition to different factors such as incubation time, pH, temperature, NaCl concentrations. At pH 7, temperature 40°C and 2.5% NaCl concentration, carbon source such as starch and beef extract as nitrogen source, the protease activity was maximum. Extracellular protease was isolated, purified to 8.73 fold by diethyl aminoethyl (DEAE) ion-exchange chromatography and its specific activity was determined to be 886.56 U/mg and the sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) showed a single band for the purified enzyme, with an apparent molecular weight of 66 kDa. These findings suggest that the scope for the use of B. cereus SU12 strain as suited organism for the industrial production of the extracellular protease enzyme.   Key words: Protease, Bacillus cereus SU12, Oyster, diethyl aminoethyl (DEAE) ion-exchange chromatography, optimization.

Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: A human Lymphatic Filarial parasite

Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.

Biosynthesis of Gold Nanoparticles Using Therapeutic Enzyme: <I>In-Vitro</I> and<I> In-Vivo</I> Efficacy Study

The present contribution deals with the synthesis of gold nanoparticles using a therapeutic enzyme serratiopeptidase followed by the in-vitro and in-vivo evaluation to demonstrate the retention of therapeutic activity of the enzyme. Here, gold nanoparticles were synthesized at 25 degrees C and physiological pH 7. The formation of serratiopeptidase reduced gold nanoparticles was confirmed by UV visible spectroscopy, transmission electron microscopy, X-ray diffractometer and fourier transform infrared spectroscopy. Proteolytic enzyme activity assay revealed that the composite contained 64% enzyme capped on AuNps while the remaining 36% enzyme remained in the supernatant. Further, retention of enzymatic activity of these nanoparticles was confirmed by in-vitro casein agar plate method as well as by in-vivo anti-inflammatory activity performed in experimental animals. Six month stability study at ambient temperature (25 degrees C) revealed that gold nanoparticles were stable as indicated by no shift in the surface plasmon band. In conclusion, physiological condition is an important process condition for controlled synthesis of highly stable gold nanoparticle with respect to retention of enzymatic activity. Also use of gold nanoparticles as a carrier for serratiopeptidase led to an improved anti-inflammatory response.

Screening of Extracellular Keratinase Producing Bacteria from Feather Processing Areas in Vellore, Tamil Nadu, India

The aim of the current study was to isolate keratinolytic bacteria from the soil samples collected from different feather processing areas in Vellore, TN, India. The isolation was performed by serial dilution and plating method. Total eight bacteria were isolated from the collected soil samples. All isolates were screened for keratinolytic activity by Casein agar plate method, among eight bacterial isolates only one (H5) isolate showed the keratinolytic activity in Casein agar medium. H5 isolate (potential strain) was identified as Bacillus sp. by microscopic and biochemical experiments. The best enzyme activity was observed at pH 7 and temperature 30ºC. Keywords: Keratinolytic; Spread plate method; Bacillus sp.; Bacteria. © 2010 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. DOI: 10.3329/jsr.v2i3.4567 J. Sci. Res. 2 (3), 559-565 (2010)

Sodium azide induced mutation of Actinomycetes I

Sodium azide was employed to generate mutants of actinomycetes from Mount Everest region, viz. Sagarmatha National Park, Jorsale, Kalapatthar and Namche. Individual isolates of actinomycetes were inoculated on Starch Casein Agar plates containing 10, 50 and 100 ppm of sodium azide. Depending on complete inhibition of their growth , actinomycete isolates were categorized as highly sensitive , moderately sensitive and tolerant, respectively. Out of a total of 38 actinomycetes isolates, two (5.3%) were highly sensitive, 23 (60.5%) moderately sensitive and 13 (34.2%) tolerant. A total of 21 mutants were generated by treating 8 of the 23 moderately sensitive actinomycetes with 20, 30 and 40 ppm (sublethal concentrations ) of sodium azide and analyzed morphologically and biochemically. All mutants like wild strains were able to hydrolyse starch, Tween 20 and casein and utilize fructose and lactose. All were urease and catalase positive , but were different from wild strains in colony color or texture or both. Altogether nine (43 % ) mutants having negative mutation in loci of utilization of galactose or mannose (loss-of-function LOF, single mutants ) , two LOF double mutants in utilization of both galactose and arabinose , one gain-of-function (GOF) single mutant having positive mutation in the loci of utilization of galactose, one GOF double mutant in utilization of mannose and galactose and five double mutants (25%) having GOF ( arabinose or sucrose ) as well as LOF (mannose or galactose or arabinose) were obtained. Key words : Mt. Everest, Sagarmatha, actinomycetes, sodium azide, mutation, LOF, GOF DOI: 10.3126/ijls.v3i0.2385

Variation in extracellular protease production among clinical isolates of Staphylococcus aureus due to different levels of expression of the protease repressor sarA.

Staphylococcus aureus produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92 S. aureus strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 strains appeared to be protease negative. The major protease genes were present in all protease-positive (n = 5) and protease-negative (n = 12) strains analyzed. Northern blotting showed that transcription of the protease genes was suppressed due to increased sigma factor B (SigB)-dependent expression of the protease repressor SarA. Other SigB-dependent traits such as pigmentation and expression of asp 23 were also increased in protease-negative compared to protease-positive strains. Inactivation of sarA in three protease-negative strains resulted in increased transcription of all protease genes and increased protease production, while overexpression of sarA in a strain producing protease at high levels repressed protease production. Our results suggest that the protease genes are conserved among clinical S. aureus strains and that the level of SigB-dependent expression of the protease repressor sarA determines the level of protease production in each strain.

Effect of isolation temperature on the characteristics of extracellular proteases produced by Antarctic bacteria

Protease-producing psychrotolerant bacteria were isolated from Antarctic biotopes on casein agar plates using different incubation temperatures. Most of the isolates were non-spore-forming Gram-negative motile rods with catalase activity, 30% were pigmented and none of them were glucose fermenters. All the strains were grown in liquid cultures at 20°C and protease secretion was tested using the azocasein method. Despite their capacity for production of a clear zone of hydrolysis in agar plates, some strains did not produce detectable levels of proteolytic activity in liquid cultures. The lowest apparent optimum temperature for protease activity found in culture supernatants was 40°C. Almost all the strains showed activation energy values about 10-20 kJ-mol?1 lower than that observed for a mesophilic Subtilisin. Most of the proteases showed optimal activity at neutral or alkaline pH values and developed a multiple-band profile on gelatine-SDS-PAGE. It was observed that the lower the strain isolation temperature was, the more stongly cold-adapted–in terms of optimal temperature and activation energy–were the proteases produced by them. This dependence of the characteristics of the proteases on the isolation temperature is an important factor to take into account in the design of screening programmes directed towards the isolation of psychrotolerant bacteria able to produce proteases strongly or weakly adapted to work in the cold. The Antarctic area explored proved to be a promising source of proteolytic bacteria with potential use in industrial processes to be carried out at low or moderate temperatures.

Application of casein agar plate method for the determination of protease activity.

Casein agar plate method for protease assay was developed by the method of Cheeseman with a slight modification. With this modified method, 1μg-1 mg/10μl or 10μg-1 mg/0.1 ml of trypsin was successfully determined and the sensitivity of the method was about 10 times higher than that of the original method. This is one of the simple method for the determination of neutral and alkaline protease activity and was found to be applicable to the field of biochemical study. This method is shown to be very suitable for the measurement of activity change observed when protease is treated with various inhibitors and antiserum, respectively. Especially, the results of inhibition test with protease inhibitors by this method agreed well with those obtained by the conventional method determining the remaining protease activity after preincubation of the inhibitor with protease solution. In the clinical analysis with experimental animals, the method was successfully applied for the detection of trypsin in fecal specimen and duodenum Juice. Furthermore, it was also useful for check of routine protease assay of each fraction isolated by column chromatography.

Bioautography of proteinase inhibitors of microbial origin : A simple enzymatic detection procedure on casein agar plates

Bioautography of proteinase inhibitors of microbial origin : A simple enzymatic detection procedure on casein agar plates

A modification for increasing the sensitivity of the casein-agar plate assay: a simple semiquantitative assay for thermophilic and mesophilic proteases

A casein-agar plate assay was used for the quantitative determination of both mesophilic and thermophilic proteases. Because many proteases are thermostable, assay at higher temperatures is possible. The sensitivity of the plate assay increased with temperature, the optimum assay temperature depending on the thermostability of the enzyme (e.g. Thermus protease, 75°C; thermolysin, 65°C; trypsin, 65°C; α-chymotrypsin, 45°C). A positive correlation was observed between incubation temperature and the density of the para-casein precipitate, increasing the accuracy of diameter measurement. Using this modified, thermostable proteases could be assayed at levels well below the limits of detection of other methods (e.g. 40 pg of thermolysin and 300 pg of trypsin detectable at 65°C, a 16-fold increase in the sensitivity for trypsin compared with a conventional plate assay (Fossum, K. (1970) Acta Pathol. Microbiol. Scand. Sect. B 78, 350–361)). The sensitivity of the plate assay could be further increased by the inclusion of some detergents and chaotropic agents in the gel.

Fibrinolytic Activity of Lung and Spleen Extracts Observed in Conventional but not in Germ-Free Rats

Protease-like activity which split plasminogen-free fibrin was demonstrated in 2 M KSCN extracts of the lung and spleen of conventional rats. The activity was virtually undetectable in tissue extracts from germ-free rats. The extracts from the conventional rat tissues split fibrin and fibrinogen remarkably at neutral pH, but not casein, when examined using fibrin, fibrinogen-agar and casein-agar plates. The fibrinolytic activity was inhibited by STI and DFP, indicating a serine protease nature. The activity was not inhibited by TLCK, t-AMCHA or dansyl-L-arginine-methylpiperidine amide (a selective synthetic thrombin inhibitor, OM189). It was neither activated nor inhibited by cysteine, KCN or iodoacetic acid. The results obtained indicate that the protease-like activity of the lung and spleen extracted with 2 M KSCN from conventional rats has properties which differ from those of trypsin, plasmin, plasminogen-activator, thrombin, and cathepsin A, B and C.

Studies on the Effect of Food Additives on the Activity of Trypsin, using Cup and Disc Methods

For the purpose of studying the effect of food additives on the activity of trypsin, the cup and disc methods were applied. Eight kinds of food additives, i.e., sodium benzoate, sodium dehydroacetate, dulcin, sodium cyclamate, sodium glutamate, glycine, Phloxine and Rose Bengale were tested. In each experimental run, one of these additives was dissolved in 20 ml of 1.5% agar solution (pH 7.6) which contained 0.5% of casein. The casein-agar medium which contained no food additives, was used as the control. These casein-agar solutions were poured into petri-dishes and solidified. Then, diluted trypsinsolutions were added through stainless cup or paper disc to the casein-agar plates in petri-dishes. The dishes were incubated at 37°for 24 hr. After incubation, transparent circles were formed in the plates by the action of trypsin on casein. The outlines of the digested casein circles became distinct by adding 9% trichloroacetic acid. After removal of trichloroacetic acid, the diameters of the circles were measured. The plot of the mean diameters of circles versus logarithm of enzyme concentration became linear. Thus the ratio of the enzyme activity in the presence of each food additive to that of control was calculated. The ratios were very small when dulcin, Phloxine and Rose Bengale were added to the reaction medium. These results showed that these three additives were inhibitory for the tryptic digestion of casein. It was also shown that other five additives had no effect on the activity of trypsin.

Differential Action of Animal, Vegetable, and Microbial Rennets on Caseins as Revealed by Casein Agar Plate Assay Method

The action of rennets prepared from animal (cow and buffalo), vegetable, and microbial sources was studied on caseins from cow and buffalo milks, using the casein-agar gel diffusion technique. Both cow and buffalo rennets were observed to form two concentric precipitated zones while diffusing through an agar plate containing 1% whole casein (cow or buffalo) and 10–20mM calcium chloride. A subsequent clearance zone was also visible following the second zone of precipitation.Rates of increase in zone diameters for both precipitated zones were dependent on the nature of rennet and also on the type of casein, cow rennet being faster on cow casein than on buffalo casein. Buffalo rennet was equally reactive with both caseins.Rennet preparations from either vegetable or microorganisms exhibited a different action on casein-agar plate, forming only one precipitated zone followed by a clear zone.