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1371

Alkhassaki et al. World Journal of Pharmaceutical Research

OPTIMUM CONDITIONS FOR L-ASPARGINASE PRODUCTION BY

LOCAL ISOLATE OF PSEUDOMONAS AERUGINOSA P4

Elaf I. Alkhassaki* and Asmaa A. Hussein

Department of Biotechnology, College of Science, University of Al-Nahrain.

ABSTRACT

In this study, a total of forty six samples were collected for isolation of

P. aeruginosa. These include 14 samples taken from wounds, 20

samples of burns and 12 samples of ear infections collected from Al-

yarmouk hospital. From these samples, a total of 29 bacterial isolates

were obtained .when subjected to morphological and microscopical

tests. L-asparginase enzyme activity was determined by quantitative

methods using L-aspargine as substrate for these isolates, results

indicated that 6 isolates of them are l-asparginase producer with

different specific activity ranged between (0.02-0.15) U/mg and the

isolate Pseudomonas aeruginosa P5 was the most efficient in the

production of l-asparginase with specific activity of 0.15 U/mg protein therefore, it was

chosen to determine the optimum conditions for l-asparginase production. Maximum l-

asparginase production was achieved after supplementation of the minimal salt medium

(pH7) with 0.1% glycerol, 0.1% tryptone and incubated at 37oC in shaker incubator (150

rpm) for 24h. Under these conditions, the specific activity of l-asparginase produced in

culture supernatant was sharply increased to 0.6 U/mg protein.

KEYWORDS: L-asparginase, Pseudomonas aeruginosa, l-asparginase activity,

optimization.

INTRODUCTION

L-asparaginase (L-asparagine aminohydrolase EC 3.5.1.1), The enzyme L-asparginase

belongs to a group of hydrolysis enzymes, it works in the presence of water on the L-

asparagine amino acid analysis to L-aspartic acid and ammonia.[28]

L-asparaginase has been

produced throughout the world by both submerged and solid-state fermentations hydrolysis

process through an attack nucleophile.[7]

L-asparginase has two types, L-asparaginase I and

World Journal of Pharmaceutical Research SJIF Impact Factor 6.805

Volume 5, Issue 11, 1371-1384. Research Article ISSN 2277– 7105

*Corresponding Author

Dr. Elaf I. Alkhassaki

Department of

Biotechnology, College of

Science, University of Al-

Nahrain.

Article Received on

16 Sept. 2016,

Revised on 06 Oct. 2016,

Accepted on 26 Oct. 2016

DOI: 10.20959/wjpr201611-7311


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L-asparaginase II. Whereas L-asparaginase I is a constitutive cytoplasmic enzyme and its

synthesis is almost unaffected by the growth conditions, L-asparaginase II is an inducible

periplasmic enzyme, and its amount in the bacterial cell varies greatly depending on the

growth conditions.[16]

The L-asparginases are enzymes expressed and produced by different

microorganisms.[7]

The production of L-asparaginase has been studied in Serratia marcescens

.[13]

, Erwinia carotovora.[17]

Escherichia coli.[31]

Enterobacter aerogenes.[20]

Pseudomonas

aeruginosa.[1]

Bacillus subtilis.[6]

Aspergillus tamari, Aspergillus niger, and Aspergillus

terreus.[19]

with various carbon and nitrogen sources under both aerobic and fermentative

conditions. L-asparginase can be used in food processing by reduce the level of acrylamide

up to 90% in a range of starchy foods without changing the taste and appearance of the end

product(10) and can be consider as one of the major important enzyme come from the fact

that acute lymphoblastic leukemia cells and some other suspected tumor cells are unable to

synthesize the non-essential amino acid asparagine, whereas normal cells are able to make

their own asparagine; thus leukemic cells require high amount of asparagine. These leukemic

cells depend on circulating asparagine. Asparginase, however, catalyzes the conversion of L-

asparagine to aspartic acid and ammonia. This deprives the leukemic cell of circulating

asparagine, which leads to cell death.[23]

The enzyme has also been studied for application in

L-asparagine biosensor for leukemia.[31]

It can also be associated with a coagulopathy as it

decreases protein synthesis, including synthesis of coagulation factors (e.g. progressive

isolated decrease of fibrinogen) and anticoagulant factor (generally antithrombin III;

sometimes protein C&S as well), leading to bleeding or thrombotic events such as stroke.[22]

Ruzzo et al.,[26]

mentioned that Asparagine is required for development and function of the

brain, Patterson and Marc,[25]

noted that it is important to protein function.

According to those mentioned above this study was aimed to isolate a higher l-asparginase

from Pseudomonas aeruginosa and studying the optimum condition for its production.

MATERIALS AND METHODS

Samples collection

A total of 46 specimens were collected from the Burns, ear infections and wounds from al-

yarmouk hospital in Baghdad. Swab specimens were aseptically transferred under cooling

conditions to the laboratory for analysis. Each specimen was inoculated on Pseudomonas

isolation agar. All plates were incubated aerobically in incubator at 37ºC for 24 hrs.

https://en.wikipedia.org/wiki/Microorganisms

https://en.wikipedia.org/wiki/Non-essential_amino_acid

https://en.wikipedia.org/wiki/Ammonia

https://en.wikipedia.org/wiki/Cell_death

https://en.wikipedia.org/wiki/Coagulopathy

https://en.wikipedia.org/wiki/Fibrinogen

https://en.wikipedia.org/wiki/Antithrombin

https://en.wikipedia.org/wiki/Protein_C

https://en.wikipedia.org/wiki/Protein_S

https://en.wikipedia.org/wiki/Bleeding

https://en.wikipedia.org/wiki/Blood_clotting


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Determination of specific L-asparginase activity for Pseudomonas aeruginosa

To screen the ability of local P. aeruginosa isolates for extracellular L-asparginase

production. Enzyme activity was determined by qualitative methods using ninhydrine reagent

as indicator to screen for the isolate that produce L-asparginase, Fig (1) showed the that

colonies producing an enzyme were detected which gave a purple spots color directly

proportional to the quantity of enzyme produced and can be easily distinguished from the

brown background of the filter paper which represents all positions do not contain any

bacterial growth . Also, Enzyme activity was determined by quantitative methods using L-

aspargine as substrate. A volume of 100 μl of fresh culture of each bacterial isolates was used

to inoculate the Brain heart infusion broth in a conical flask and incubated in a shaker

incubator (150 rpm) at 37ºC for 24hrs. After incubation, the culture was centrifuged, pellets

were discarded, and supernatants were taken and assayed for L-asparginase activity by

measuring ammonia concentration at 436nm.The assay mixture contained: (0.1) ml of L-

aspargine was added to 2.5 ml of tris buffer (pH8.6) then incubated for 30 minutes at 37

oC.The reaction was stopped by adding 0.5 ml of the 5 % TCA and the solution was

centrifuged at 6000 rpm for 20 minutes. The assay mixture contained: 4.4ml of distilled

water, 0.5ml of nissler reagent and 0.1ml of l-asparginase. The blank sample was prepared by

mixing 4.5 ml of distilled water and 0.5 ml of nissler reagent.

Ammonia concentration was determined in the sample as mentioned above and the enzymatic

activity was calculated according to the following equation.

Concentration of ammonia liberated (Ϻg / ml)

Enzymatic activity (U / ml) = ——————————————————

Reaction time (30 minutes) × 14

Absorbance at wavelength 436 nm

Concentration of ammonia liberated = ————————————

Slop (Ϻg / ml)

Enzyme activity was expressed in units; 1U being defined as the amount of enzyme causing

the formation of 1μmol of product per minute under the assay conditions used.[27]


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Fig(1): L-asparginase qualitative assay in P. Aeruginosa P4 placed on ninhydrin socked

filter paper

Determination of protein concentration

Protein concentration was determined according to the method described by Bradford, (4)

and as follow:

Standerd curve of Bovine serum albumin (BSA) was plotted by using different

concentrations from the BSA stock solution according to the following volumes.

Table (1): Bovine serum albumin standard curve composition.

Then 2.5 ml of Coomassi brilliant blue G-250 dye was added, mixed and left to stand for

2 min at room temperature.

The absorbance at 595 nm was measured; the blank was prepared from 0.5 ml of Tris-

HCl buffer and 2.5 ml of the dye reagent.

A standard curve was plotted between the BSA concentrations against the corresponding

absorbance of bovine serum albumin.


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Protein concentration was estimated by mixing 0.1ml of the tast sample, 0.4 ml of Tris-

HCl and 2.5 ml of Coomasi brilliant blue G-250, left to stand for 2 min at room

temperature then measuring the absorbance at 595 nm.

Fig. (2) Bovine serum albumin standard curve.

Identification of the isolate

The selected isolate with high l-asparginase activity was identified based on its

morphological and biochemical characteristics. The morphological characterization involved

culturing the isolate on cetramide agar plates for studying the appearance of the colonies,

following that, cells shape and Gram reaction were studied. Biochemical characteristics of

the isolate was based on catalase, oxidase indole and citrate utilization, vitek test was used to

ensure the biochemical results according to method described by Logan and Turnbull.[16]

Optimum carbon and nitrogen source for l-asparginase production

Various carbon (glucose, mannitol, sucrose, glycerol, and maltose) and nitrogen sources

(peptone, casein, tryptone, minitol and yeast extract) at initial concentration of 0.1% (w/v)

were screened individually in minimal salt medium(28) which compost of g/L {Yeast extract

(8) , Na2HPO4 (10.75), K 2HPO 4 (3.55), MgSO4 (0.025), MnCl2.4H2O (0.0025),

FeSO4.7H2O (0.0027), CaCl2.6H2O (0.015)} the initial pH of the medium was set at 7.5. L-

asparginase activity was determined in the supernatants after inoculation of the medium with

the locally isolated Pseudomonas aeruginosa culture, and incubated at 37°C under shaking

(150 rpm).

Optimum pH for l-asparginase production

Optimal pH for production of l-asparginase was determined by preparing the medium with

different pH values (4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9). L-asparginase activity was measured


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in the supernatant after inoculation of the production medium with Pseudomonas aeruginosa

log phase culture, and incubation at 37°C under shaking (150 rpm).

Optimum temperature for laccase production

Pseudomonas aeruginosa was grown in the production medium and incubated at different

temperatures (20, 25, 30, 37, 40, 45, 50, 60 ) °C. The l-asparginase activity was determined in

supernatants after centrifugation at 6000 rpm for 20min.

Optimum inoculums size for l-asparginase production

Effect of different inoculum size of the selected isolate for the over producer Pseudomonas

aeruginosa on l-asparginase production was studied by inoculating the production medium,

individually with a serial dilution ranging between (103 and 10

9cell/ml).

RESULTS AND DISCUSSION

Isolation of Pseudomonas aeruginosa.

Forty six burns, ear infections and wounds samples were collected from Al-Yarmouk hospital

in Bagdad. 29 bacterial isolates were obtained,6 isolates were identified as Pseudomonas

aeruginosa. When subjected to morphological and microscopical tests in which growing

colonies on the cetrmide agar had a pale yellow colonies with a grape odor. After cells were

stained with Gram stain, results showed that they were Gram negative.

Screening ability of Pseudomonas aeruginosa for l-asparginase production

To screen the ability of local Pseudomonas aeruginosa isolates for l-asparginase production.

Enzyme activity was determined by quantitative methods using nisller reagent. Results

indicated that 6 of 29 isolates are l-asparginase producing with different specific activities

Table (2). Depending on these results, the isolate named P4was found to be the most efficient

in the production of l-asparginase with specific activity about 0.144 U/mg protein, therefore it

was chosen for further study.

Table (2): Specific activity of l-asparginase produced by 6 local isolates of Pseudomonas

aeruginosa.

Isolate number Specific activity

(U\mg)

P4 0.144

P5 0.092

P9 0.104


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P15 0.084

P25 0.119

P37 0.128

Identification of the isolate

To identify selected Pseudomonas aeruginosa P4 isolate with high l-asparginase activity. It

was subjected to the biochemical test and vitek. Results in Tables (3) and (4) shows that this

isolate was Pseudomonas aeruginosa according to the criteria of Bergys Manual of

systematic bacteriology and vitek test (3,16).

Table (3) Biochemical characterization of Pseudomonas aeruginosa P4.

Test

Growth under

aerobic

condition

β-

haemolysis Catalase Oxidase Indole Citrate

Lactose

fermentation

P4 + + + + - + -

(+) positive results (-) negative results.

Table(4)Vitek test result for identification of Pseudomonas aeruginosa P4.

Test Res

ult Test

Resul

t Test

Resul

t Test

Resul

t Test Result

APPA - ADO - PyrA - IARL - Dcel -

H2S - BNAG - AGLT

p - dGLU + GGT +

BGLU - dMAL - dMAN + dMNE + BXYL -

ProA + LIP + PLE - TyrA - URE -

SAC - dTAG - dTRE + CIT + MNT +

ILATK + AGLU - SUCT + NAGA - AGAL -

GlyA - ODC - LDC - IHISa - CMT +

O129R + GGAA - IML

Ta + ELLM - ILATa -

BGAL - OFF - BAIap + dSOR - 5KG -

PHOS - BGUR -

(+) positive results (-) negative results.

Optimum conditions for production of l-asparginase production

The over producer Pseudomonas aeruginosa characterized with its high ability in l-

asparginase production was used to determine the optimum conditions for l-asparginase

production.


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Optimum carbon and nitrogen sources

Optimum carbon and nitrogen source was investigated by using sucrose, glycerol, glucose

and minitol (as carbon source) and yeast extract, tryptone, peptone and casein hydrolysate (as

nitrogen source). Pseudomonas aeruginosa P4 was cultivated in a minimal salt media

containing 0.1% from each of these various carbon and nitrogen sources. Results in Figure

(3) and Figure (4) shows that, this isolate was capable of utilizing different carbon sources as

a sole source for carbon and energy, while production of l-asparginase was varied according

to the type of the carbon source. Since, glycerol was the best carbon source for l-asparginase

production, while glucose, sucrose and minitol were the less effectives. The type of nitrogen

source also affected enzyme production, among the various nitrogen sources, maximum l-

asparginase specific activity was obtained when tryptone was added to the medium. Also,

good level of enzyme activity was obtained with other nitrogen sources.

Mokrane,[20]

mentioned that best carbon source of culture medium for L-asparginase

production from P. aeruginosa was glycerol in a concentration of 0.1%. Repression of L-

asparaginase synthesis by glucose has been shown in some bacteria like Escherichia coli and

Enterobacter aerogenes.[7]

Figure (3) Effect of carbon source on l-asparginase production by Pseudomonas

aeruginosa P4.


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Figure (4) Effect of carbon source on l-asparginase production by Pseudomonas

aeruginosa P4.

Optimum inoculum size

Different inoculums sizes were used to determine the optimum for l-asparginase production

by P4. These inoculum sizes were ranged between (103-10

9) cell/ml. results illustrated in Fig.

(5) shows that production of l-asparginase by P5 was affected by the inoculums size. L-

asparginase production was increased slightly with increasing inoculums size to 108 cell/ml,

and then it was decreased above this size. However, maximum specific activity (0.28 U/mg)

was obtaind using 108CFU/ml.

Figure (5): Effect of inoculum size on L-asparginase production by P.aeruginosaP4after

incubation at 37oC for 24 h.

Production of enzyme in sufficient amount required optimum inoculums size of cells;

lowering inoculum size required longer time for cells to multiply for sufficient number and

produce enzyme. On the other hand, an increase in the number of the inoculum would ensure

a rapid proliferation and biomass synthesis, after a certain time, enzyme production could be


1380


decreased because of the depletion in the nutrients which may result in decreased in

metabolic activity (12). Niladevi et al. (24) recorded that a very low inoculum size was found

to be inadequate for enzyme production, while the inoculum level above the optimum

reduced the yield probably due to the competition for nutrients.In another report, both

Bacillus subtilis and Bacillus cereus exhibited highest enzyme activity at 300 μl inoculum

size (9).Also, an inoculum size of 1x108 spores/ml showed the highest yield (11.0 IU) of L-

asparaginase by Streptomyces albidoflavus (2).

Optimum pH

In order to investigate the effect of the initial medium pH on l-asparginase production by the

P. aeruginosa P4, the production medium was adjusted to different pH values ranged

between pH 4.5 and pH 9. Fig.(6) shows that maximum l-asparginase production was

obtained when the pH value of the production medium was adjusted to 7, at this value the

enzyme specific activity in culture filtrate was 0.4 U/mg protein.

A decrease or increase in hydrogen ions (H+) concentration causes pH changes in the culture

medium which may lead to drastic changes in the three-dimensional structure of proteins

because H+ and/or OH- compete with hydrogen bonds and ionic bonds in an enzyme,

resulting in enzymes denaturation (30). Khamna et al. (13) reported that the optimum pH for

L-asparaginase production from actinomycetes was observed at 7.0.

Figure (6): Effect of medium pH on L-asparginase production by P. aeruginosaP4after

incubation at 37oC for 24 hrs.

Optimum incubation temperatures

In this study, different incubation temperatures (20, 25, 30, 37, 40, 50 and 60) oC were used

to determine the optimum temperature for l-asparginase production by P. aeruginosa P4. Fig.

https://www.google.iq/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwjwmZbysvvOAhURkRQKHWF3CQUQFggaMAA&url=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FStreptomyces_albidoflavus&usg=AFQjCNG_Hx1oWUtXYsfh9uewlTdijwCixQ&bvm=bv.131783435,d.bGg


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(7) shows that l-asparginase specific activity is increased with increasing temperature from

30-37℃, since the specific activity was 5800 u/mg protein at 30℃ has increased to 0.4U/mg

protein at 37℃, but decreased at higher temperatures.

Generally, for any enzymatic reaction, temperature below or above the optimal temperature

will drastically reduce the rate of reaction. This may be due to the enzyme denaturation, or to

losing its characteristics of three-dimensional structure. Denaturation of a protein involves the

breakage of hydrogen bonds and other non-covalent bonds (30). According to Kushwaha et

al.(15) the bacterial L-asparginase showed maximum activity at 37°C under optimized

conditions.Also, the maximum enzyme activity from endophytic Bacteria appeared as 0.65

IU/ml was at 37°C; the enzyme activity increases up to 37°C and then decreases at 40°and

45°C (11).

Figure (7): Effect of incubation temperature on L-asparginase production by P.

aeruginosaP4

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