isolation of l-asparaginase from coliform

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Slide 1: 

Screening, optimization and production of L-Asparaginase from water organism GUIDE: PRESENTED BY: SHRI R.V.KARADI Arpan J. Shah Dept. of Pharmaceutical Biotechnology K.L.E.S’s College of Pharmacy Belgaum-10,karnataka.

Contents : 

Introduction Objectives Methodology Results and Discussions Summary References Contents

Introduction : 

L-Asparaginase (L-asparagine amino hydrolase, LA) catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. L-asparagine is an essential amino acid for the growth of tumor cells where as the growth of normal cells is independent of its requirement. Introduction

Introduction : 

LA is present in many animal tissues, bacteria, plants and in the serum of certain rodents but not in mankind. Whereas microbial LA has attracted considerable attention since the demonstration that LA from E. coli has antitumour activity (Mashburn and Wriston, 1964; Broom, 1961) Coliform bacteria are a commonly used indicator of sanitary quantity of foods and water, which defined as rod-shaped, gram-Ve, oxidase negative and lactose fermenting. They have a diverse rang of enzymatic activity and are capable of catalyzing various biochemical reactions. Thus, there is enoumorous scope for investigation to explore the possibilities of deriving new products of economic importance. The reported work deals with isolation of coliform bacteria and optimization of various parameters for the maximum L-asparaginase production. Introduction

Objectives : 

The main objectives in the present work are: Screening of coliform bacteria for the isolation of L-asparaginase from the water. Identification and characterization of isolated micro-organism. Optimization of medium to obtain a maximum yield. Purification and molecular weight determination of enzyme. Objectives

Review of Literature : 

Review of Literature Dhevangi P and Poorami E collected marine actinomycete from Parangipettai and Cochin coastal areas of South India (PDK7 & PDK2). The partially purified L-asparaginase showed a specific activity of 64.07 IU/mg protein, 83-fold pure and yielded 2.18 % of protein. The enzyme was stable at range of temperature 60°C to 80°C. And have a molecular weight of 140 kDa. Fengsheng Zhao and Jiaye Yu developed a method to release L-asparaginase (EC 3.5.1.1) from ATCC Escherichia coli 11303 cells by chemical permeabilization was studied. aleria J Harwood, isolated Total and Fecal coliform bacteria from the cloaca and feces of the estuarine diamondback terrapin.

Review of Literature : 

Review of Literature Peterson RE and Ciegler A, developed a medium enrichment technique coupled with aeration optimization for increasing yield of L-asparaginase from Erwinia aroideae NRRL B-138 Peterson RE, studied on 123 species of bacteria for L-asparaginase production, Erwinia aroideae NRRL B-138 provided the highest yields.

Methodology : 

Screening of L-Asparaginase producing organism Characterization of isolates Determination of enzyme activity Chromatographic study Optimization of fermentation parameters Production and Purification of L-asparaginase Quantitative estimation of protein by Lowry’s method Molecular weight determination by SDS-PAGE Methodology

Screening of LA producing organism : 

Collection of water samples Isolation of coliform bacteria Two methods were used. Serial tube method (Multiple Tube Fermentation) Presences of coliforms were further confirm by streaking Eosin-Methylene Blue agar from positive MacConkey broth tubes. 2. Modified ISP-5 medium (Rapid Screening Method) Content of Modified ISP-5 medium (g/l): L-asparagine (1.0), Lactose (20.0),Dipotassium phosphate (1.0),Trace salt solution (1.0 ml), Phenol red (1.0 ml), Agar (20.0),Final pH (at 25°C) 7.0 ±0.2. Screening of LA producing organism

Characterization of isolates : 

The 12 screened organisms, E.coli and Pseudomonas sp. (positive control) was tested for various biochemical reaction. Lactose fermentation and Gas production Hydrogen sulfide 7. Arginine decarboxylase Methyl red 8. Citrate utilization Voges-Proskauer 9. Urease production Phenylalanine deaminase 10. Gas from glucose Indole production 11. Gram staining 12. Motility Characterization of isolates

Determination of enzyme activity : 

The enzyme activity was determined by Nesslerization method. Standard curve of L-asparaginase: Taken by using a marketed product of VHB life science i.e. Leucoginase (5000 IU). Dissolve 5000 IU of Leucoginase in 5 ml of 0.2M Tris-HCL Buffer, pH 8.6 Procedure: For each different enzyme dilution pipette into test tubes as follows: Test Blank 1) 0.05M Tris-HCl Buffer, pH 8.6 0.2ml 0.2ml 2) 0.01M L-asparagine 1.7ml 1.7ml 3) L-asparaginase 0.2,0.4,0.6,0.8,1ml ------ Incubate at 37°C for exactly 15 minutes 4) 1.5M trichloroacetic acid 2ml 2ml Determination of enzyme activity

Determination of enzyme activity : 

Clarify by centrifugation and add 0.5 ml of clear supernatant to 7.0 ml reagent grade water. Add 1.0 ml of Nessler's reagent and incubate at room temperature for 10 minutes. Yellow colour developed, absorbance was taken at 480 nm in UV spectroscopy. 2. Standard curve of ammonium sulphate: Dissolve 1.179 gm ammonium sulfate to a final volume of 100 ml. Dilute 1.4 ml of this solution to 100 ml to give 1 umole NH3 per ml. 3. Determination of enzymatic activity form the isolates : TGY broth(50 ml) was inoculated with the selected isolates AS-2; incubated on a rotary shaker incubator at 37°C for 24 hr. Cells were harvested by centrifugation and were washed twice with of phosphate buffer (Na2HPO4, 4.757 g; KH2PO4, 4.539 g; Triton X-100, 0.125 ml; water to 1.0 liter). Determination of enzyme activity

Determination of enzyme activity : 

The washed cells were suspended in 3 ml of 0.05M Tris-HCL Buffer, pH 8.6. In test tubes 1 ml of the suspension was placed with 9 ml of 0.05M Tris-HCL Buffer and 10 ml of 0.01 M L-asparagine. Incubated at 37°C for 15 min, and the reaction was stopped by the addition of 0.5 ml of 1.5M Trichloroacetic acid. Precipitated proteins were removed by centrifugation Take 0.5 ml of supernatant and diluted with 7.0 ml of distilled water. Add 1 ml of Nessler’s reagent to the above solution. Yellow colour developed, absorbance was taken at 480 nm in UV spectroscopy. Determination of enzyme activity

Chromatographic study : 

The basic principle involved in TLC is adsorption process; the solute competes with the solvent for the surface sites of the adsorbent. In this study thin layer chromatography was use to find out the conversion of L-asparagine into L-aspartic acid. Mobile phase: n-butanol: acetic acid: water (5:4:1) Visualizing agent: Ninhydrine Chromatographic study

Optimization of fermentation parameter : 

Environmental parameters: pH and Temperature Nutritional parameters: Carbon source and Nitrogen TGY broth as a Basal medium. (Glucose 1.0; Dipotassium hydrogen phosphate 1.0; Yeast extract 5.0; Tryptone 5.0; pH adjusted to 7.0) During the each optimization L-asparaginase production was monitored at every 12 hours intervals over a 48 hours fermentation period through nesslerization method. Optimization of fermentation parameter

Optimization of Environmental parameters : 

1. Optimization of pH: The effect of pH on L-asparaginase production was studied by growing AS-2 strain in basal medium of different pH i.e. 5 to 10. pH was maintained by using phosphate buffer. 2. Optimization of Temperature: To observed the effect of temperature on LA production basal medium was inoculated with AS-2 strain and incubated at different temperatures 27°C, 30°C, 34°C, 37°C separately for 48hours. Optimization of Environmental parameters

Optimization of Nutritional parameters : 

Optimization of Nutritional parameters 1. Carbon source: Basal medium contain 0.1% glucose. Glucose, Sucrose and Mannitol was used for optimization. 2. Nitrogen source: Basal medium contain 0.5% yeast extracts. Peptone, Casein and Yeast extract was used for optimization. Basal medium with these carbon/nitrogen sources in a conc. of 0.1%, 0.5%, 1.0% and 1.5%, was inoculated with AS-2 strain; incubated at 37 °C for 48 hrs.

Optimal duration and Biomass measurement : 

AS-2 strain was inoculated in optimized TGY broth and incubated for 40 hours. Enzymatic activity was measured at every 1 hour interval by measuring the librated ammonia by direct nesslerization method. Biomass production was measure by dry weight method. Optimal duration and Biomass measurement

Production and Purification of L-asparaginase : 

Production of L-Asparaginase: Employing the optimized medium and other optimized conditions LA production was done by growing AS-2 strain in a lab. Scale fermentor for 30 hrs. 2. Partial purification of L-asparaginase: At the end of fermentation period the fermentor medium pooled together and centrifuged to separate the broth and cells. The enzyme was extracted from cells by grinding it with an alumina. The extract was centrifuged. The pooled supernatant fluids were treated to remove the nucleic acid by adding 1M MnCl2 drop wise. After stirring for 1 hour, the solution was centrifuged at 18,000 rpm for 20 min and the residue was discarded. Production and Purification of L-asparaginase

Production and Purification of L-asparaginase : 

Solid (NH4)2SO4 was added to the supernatant fluid to achieved 20% saturation. The suspension was further centrifuged and solid (NH4)2SO4 was added to supernatant to reach 40% and 60% saturation. The precipitates (0-20%, 20-40% and 40-60%) were collected by centrifugation, the collected precipitates were dissolved in Tris-HCL buffer, pH 8.6 and dialyzed overnight against the same buffer. After the exclusion of ammonium sulphate, enzymatic activity in each dialyzed solution was determined; the fraction that showing the highest enzyme activity was designate as partially purified extract (PPE). Production and Purification of L-asparaginase

Production and purification of L-asparaginase : 

Production and purification of L-asparaginase Sephadex G-200 gel filtration Proteins obtained after 20-40%saturation (PPE) were dissolved in 0.05 M Tris-HCl (pH 8.6) buffer and loaded onto prequilibrated column with 0.05 M Tris-HCl Sephadex G-200. It was eluted with same buffer. Fractions were collected at flow rate 5ml/30mins. Total protein was monitored at 280 nm and the collected fractions were assayed for LA activity by procedure described earlier.

Quantitative estimation of protein by Lowry’s method : 

Reagents Required: 1. 4% sodium carbonate in 0.2 N Sodium Hydroxide (Reagent A). 2. 1.0% copper sulphate in 2% sodium potassium tartarate (Reagent B). 3. Alkaline copper solution: Mix 50 ml of A and 1 ml of B, prior to use (Reagent C). 4. Folin - Ciocalteau reagent solution Working standard solution: Weigh accurately 50 mg of Bovine Serum Albumin (BSA) and dissolve in 5 ml of distilled water. Dilute 1 ml of stock solution to 10 ml of distilled water to get 1 mg/ ml concentration. Quantitative estimation of protein by Lowry’s method

Quantitative estimation of protein by Lowry’s method : 

Procedure: 1. Pipette out 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 ml of the working standard into a series of test tubes. 2. Make up the volume to 1 ml in all the test tubes. A tube with 1 ml of water serves as the blank. 3. Add 2.5 ml of reagent C to each tube including the blank. Mix well and allow standing for 10 min. 4. Then add 0.2 ml of reagent D, mix well and incubate at room temperature in the dark for 30 min. Blue Colour is developed. 5. Take the reading at 660 nm. 6. Draw a standard graph. Quantitative estimation of protein by Lowry’s method

Molecular weight determination by SDS-PAGE : 

Molecular weight determination by SDS-PAGE Molecular weight of L-asparaginase was determined by SDS-PAGE with respect to the various markers like Pepsin 35kDa, Egg albumin 45kDa, Lipase 56 kDa, Bovin albumin 66kDa, Phosphorylase 97kDa and β-Galactosidase 116 kDa.

Results and Discussion : 

Isolation of coliform bacteria After collection of water samples isolation of coliforms were done by using the two methods 1. Serial tube method (Multiple Tube Fermentation) After the incubation period, we observed the change in MacConkey broth colour initially red to yellow indicates the fermentation of lactose into the acidic metabolites. This was due to the presence of coliform bacteria which were differentiated from non-coliform as well as other gram negative bacteria by streaking EMB agar from previously positive MacConkey broth tubes. Results and Discussion

Screening of LA producing organism : 

After the incubation period, two colonies were found on EMB agar, one was in pale colour; coded as AS-1 and another was in pink colour; coded as AS-2. Screening of LA producing organism

Slide 27: 

2. Modified ISP-5 medium (Rapid Screening Method) Modified ISP-5 medium was used with and without carbon source (lactose) where medium without lactose was served as control to observed that weather the medium colour changes from yellow to pink due to the lactose fermentation or the conversion of L-asparagine into the L-aspartic acid and ammonia. After the overnight incubation Colonies were observed with the pink surrounding on the ISP-5 with Lactose. There were no colonies observed on ISP-5 medium without carbon source (Lactose).

Screening of LA producing organism : 

After 7 days incubation ISP-5 medium with lactose became pinkish in colour. colonies were observed on the ISP-5 medium without Lactose with the pinkish zone surrounding From this we concluded that pinkish colour surrounding the bacterial colonies was due to the liberation of ammonia which makes the pH of the medium alkaline. Colonies with the pinkish surrounding were coded as AS-3, AS-4, AS-5, AS-6, AS-7, AS-8, AS-9, AS-10, AS-11 and AS-12. Screening of LA producing organism

Screening of LA producing organism : 

we concluded that in the presence of carbon source i.e. lactose bacteria required short incubation time. Two organisms isolated by Serial tube method and Ten organisms isolated by modified ISP-5 medium were sub-cultured on Nutrient agar for further use. Screening of LA producing organism ISP-5 Medium without Lactose after 7 days of incubation ISP-5 Medium with Lactose after 7 days of incubation

Characterization of isolates : 

All isolates were firstly subjected to the lactose fermentation and gas production to satisfy the definition of coliform bacteria. Table-1 lists the result of lactose fermentation and gas production and for the further characterization we selected isolates which were either lactose fermenting or gas producing or both. Characterization of isolates Table-1 lactose fermentation and gas production

Characterization of isolates : 

Strain AS-1, AS-2, AS-7, AS-9, AS-10 and AS-11 were subjected to various biooxidation pathways (biochemical tests). Known bacterial strains (E. coli, Pseudomonas sp.) were tested as positive control. AS-2 strain was found to be lactose fermenting, Gram –Ve, rod shaped, producing H2S, utilized citrate as carbon source and VP –Ve. All mention characters of AS-2 strain, were compared with the characters of Entrobacteriaceae family described by Sleigh and Duguid (2005) where we concluded that the AS-2 was closely related to Citrobacter sp. AS-1 strain was found as non-lactose fermenting, Gram –Ve, rod shaped and urease positive. Which are the characters of genus Providencia Characterization of isolates

Characterization of isolates : 

Characterization of isolates Table-2 lists the results of various biochemical tests +, positive reaction; -, negative reaction; ++, strong positive reaction; (-): Gram negative.

Characterization of isolates : 

Characterization of isolates Acid from lactose Hydrogen sulfide production test Methyl red test Voges-Proskauer test

Characterization of isolates : 

Characterization of isolates Citrate utilization test Urease production test Arginine decarboxylase test Indole production test

Characterization of isolates : 

Characterization of isolates Gas production from glucose Gram staining of AS-2

Determination of enzyme activity : 

Standard curve of L-asparaginase: Taken by using a marketed product of VHB life science i.e. Leucoginase (5000 IU). Determination of enzyme activity

Determination of enzyme activity : 

2. Standard curve of ammonium sulphate: Determination of enzyme activity AS-2 strain was checked for enzyme production by growing it into TGY broth, which showed positive result.

Chromatographic study : 

TLC was performed to check the conversion of L-asparagine into L-aspartic acid. Rf values of sample and standard L-aspartic acid was 0.68 and 0.70, respectively. Chromatographic study TLC (From left; Aspartic acid, Asparagine, Mixture, Treated solution)

Optimization of fermentation parameter : 

Optimization of Environmental parameters: Studies indicate that pH-8 is an optimum extarcellular pH with the enzymatic activity (810 IU/ml) at 24 hours. 37°C found an optimal temperature for the L-asparaginase production with a enzymatic activity (658 IU/ml) at 24 hours. Optimization of fermentation parameter

Optimization of fermentation parameter : 

Optimization of fermentation parameter Optimization of nutritional parameters: Carbon source: The present studies stated that 1.0% glucose in basal medium is required for an optimal production of L-asparaginase with a yield of 650 IU/ml at 24 hours . Whereas 1.0% Sucrose having a low yield (361 IU/ml) at 24 hours 1.0% Mannitol having a low yield (498 IU/ml) at 24 hours 2. Nitrogen source: Yeast extract with a 1.5% concentration in Basal medium gives a maximum enzyme production with a yield of 371 IU/ml at 24 hours. 1.0% peptone having a lower yield of 315 IU/ml at 24 hours. 1.0% casein having a lower yield of 298 IU/ml at 24 hours

Optimization of carbon source : 

Optimization of carbon source

Optimization of nitrogen source : 

Optimization of nitrogen source

Optimal duration and Biomass measurement : 

Enzymatic activity was at lowest values in the log phase and increasing in the eaxpontial phase; at 26 hours it reached to the maximum values and in the early stationary phase (upto 30 hours) of the growth cycle the activity was stable and continue in decreasing at late stationary phase. Optimal duration and Biomass measurement

Slide 44: 

On the based of above study the optimized fermentation parameters are described below: Optimized TGY broth (gm/100ml): Glucose 1.0 Yeast extract 1.5 K2HPO4 0.1 Tryptone 0.5 pH - 8.0 Temperature - 37C Incubation period – 30 hours Optimal duration- 26 hours

Production and Purification of L-asparaginase : 

Production and Purification of L-asparaginase As the L-asparaginase is intracellular product, in the purification studies cells were separated by centrifugation and supernatant was discarded. A summary of purification steps is given in Table-3.

Production and purification of L-asparaginase : 

Production and purification of L-asparaginase Sephadex G-200 gel filtration Two peaks were obtained one at fraction number 16 (high λmax) and the at fraction number 22 (low λmax), indicates two different proteins molecules. All the fractions were assayed for LA activity, but only fraction number 16 found to be maximum activity, was pooled and characterized by SDS-PAGE.

Quantitative estimation of protein by Lowry’s method : 

Quantitative estimation of protein by Lowry’s method

Molecular weight determination by SDS-PAGE : 

Molecular weight determination by SDS-PAGE SDS-PAGE showed that the enzyme is one band with electrophoretic mobility of 0.25 which is same as phosphorylase, indicates that molecular weight of isolated protein is 97 kDa.

Summary and conclusion : 

Two colonies (AS-1 and AS-2) and ten colonies (AS-3 to AS-12) were isolated form the MacConkey broth/EMB agar and ISP-5 medium respectively. 12 screened bacteria were tested for lactose fermentation which is the characteristic for the coliform bacteria. Based on the result non-lactose fermenting bacteria were sorted out and lactose fermenting/ gas producing bacteria were tested for other biochemical reaction. Based on the biochemical test AS-1 strain and AS-2 strain was probably classified under the genus of Providencia and Citrobacter. As the Citrobacter is a coliform bacterium, was taken for the L-asparaginase production. Summary and conclusion

Summary and conclusion : 

L- asparaginase convert the L-asparagine into L-aspartic acid and ammonia, was conformed by TLC and Nessler’s reagent respectively. Optimization of fermentation parameters were carried out for increasing the yield of L-asparaginase from the Citrobacter. Results showed that pH-8 and 37°C was the optimum Temperature. Glucose and Yeast extract was selected as optimal and nitrogen source in basal medium in concentration of 1.0% and 1.5% respectively. By using the optimized fermentation parameters optimal duration and biomass production was measured. Result indicates L-asparaginase production was growth associated and the maximum production was obtained at 26 hours. Summary and conclusion

Summary and conclusion : 

L-asparaginase was purified by ammonium sulphate precipitation method. Precipitate which was collected from the fraction of 20-40% saturation showed maximum activity. Two peaks were obtained one at fraction number 16 (high λmax) and the at fraction number 22 (low λmax), indicates two different proteins molecules. Molecular weight of the LA was determined by SDS-PAGE and with standard marker, which was found 97 kDa. Summary and conclusion

References : 

Kidd JG. Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum. J Exp Med 1953;98:583-91. Broome JD. Evidence that the L-asparaginase activity of guinea pig serum is responsible for its antilymphoma effects. Nature (London) 1961;191:1114-5. Yelline TY, Wriston JC. Antagonism of purified L-asparaginase from Guinea pig serum toward lymphoma. Science 1966;151:998-1004. Mashburn LT, Wriston JC. Tumour inhibitory effect of L-asparaginase from E.coli. Arch Biochem 1964;105:450-8. Information on coliform bacteria is available on URL: http://www.doh.wa.gov/ehp/dw. 12/7/07 References

References : 

Collee JA, Frases AG, Marmion BP. Practical medical microbiology, 13th ed. Churchill livingstones, vol-2 of microbiology. 2005 Dubey RC, Maheshwari DK. Practical microbiology. 1st ed. New Delhi: S.Chand and Company LTD; 2005. p. 286-89. Biochemical test for the bacteria are available on URL: http://whitewolf.newcastle.edu.au/techinfo/proc_bacto_biochem.html 20/7/07 Cappuccino JG, Shearman N. Microbiology - a laboratory manual. 6th ed. Delhi: Pearson education; 2006. Ananthnarayan and Paniker. Text book of microbiology. 7th ed. Hyderabad: Orient Longmun Private Limited; 2005. p. 274. References

References : 

Bilimoria MH. Conditions for the production of L-asparaginase 2 by coliform bacteria. Appl Microbiol 1969;12: 1025-30. Peterson RE, Ciegler A. L-asparaginase production by various bacteria. Appl Microbiol 1969;929-30. Lowry OH, Rosebrough NJ, Fare AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. Collee JA, Frases AG, Marmion BP. Practical medical microbiology, 13th ed. Churchill livingstones, vol-2 of microbiology. 2005 ammonia detection by nesslerization method is available on URL:http://www.worthington-biochem.com/ASPR/assay.html. 12/4/2007 References