Introduction

Lasparaginase belong to group of homologous amidohydrolases family[2].L-asparaginase

L-asparagine + H2O → aspartic acid + Ammonia (NH3)[12,13].

In 1963 it was revealed that antineoplastic activity of guinea pig sera was due to the presence of L-Asparaginase. Broome’s statement was confirmed by partially purifying L-asparaginase from the sera of guinea pig [3].They are naturally occurring enzymes expressed and produce by animal tissue, bacteria,plant, and in serum of certain rodent, but not in mankind . Different types of asparaginase can be used for different industrial and pharmaceutical purposes. Asparaginase are used to reduce the formation of acryl amide, a suspected carcinogen, in starchy food product such as snacks and biscuits. By adding asparaginase is converted into another common amino acid, aspartic acid, and ammonia.

L-Asparaginase has received increased attention in recent years for its anticarcinogenic potential and is used as a chemotherapeutic agent for acute lymphocytic leukemia and less frequently for acute myeloblastic leukemia,chronic lymphocytic leukemia, hodgking’s disease, melonosarcoma and non hodgking’s lymphoma.The reason it is preferred for this purpose is that, it is biodegradable, non-toxic and can be administered at the local site quite easily [2].

Material and Mathod

Sample collection

Following sample were collected from the various places with view to isolate potent L-asparagine producing microorganism. The different soil sample are

Table1: sampling sites for samples

Serial no Sample code Collection site
1 DI Marine soil from Dumas
2 GI Lake garden soil from Motavarachha
3 VI Vegetable field soil from sardar market
4 HI Hilly area soil from Mahabaleshwer Maharastra
5 CF Coconut soil from Mini goa
6 COM Compost soil from utran
7 CON Constructed area soil from utran
8 FI Forest soil from silvassa
9 LI Lake area soil from puna gam
10 MI Muddy soil from utran

All the sample were collected in polythene bag from the depth of 10 cm from different location and characteristics [2,14].

Screening and isolation of microorganisms

Isolation of bacteria

Different soil samples treated bacterial isolation by serial dilution technique using nutrient agar medium. Antifungal agents (Fluconazole 75ug/ ml, ketocanazole 75 ug/ml) were added to control the fungal contamination. The inoculated agar plates were incubated at 370 c for 24hr [2].

Primary Screening of L-asparaginase producing microorganisms

Initially isolated organisms streak on the modified nutrient plate containing 1% L-asparginase and phenol red indicator (2.5%).L-asparaginase producing colonies were selected on the bases of formation of pink color around the colonies .That isolated colony maintained on the nutrient agar slant [3]. And L- asparaginase producing colony confirmed by streaking on the glucose asparagine agar plate incubate at 370 c for 24hr [9].

Phenotypic Identification

The morphological and colony characteristics were studied using nutrient agar plate. The physiological characteristics of all the obtained isolates were studied the biochemical characteristics( Indole, catalase , oxidase, triple sugar iron test, methyl red , voges-proskauer, citrate , hydrogen sulphide production ,nitrate reduction , gelatin hydrolysis , urease ,casein hydrolysis) and sugar fermentation were also carried out using standard refrence biochemical tests for identification of medical bacteria by jean E. Mac Faddin[5].

Secondary screening of potential isolates

Agar cup method was carried out to quantify the enzyme. Sterile nutrient agar was modified by adding 1% L- asparagine and (2.5%) phenol red indicator. The isolates were grown in 10 ml sterile nutrient broth for 24 hr and add in respective wells. Plates were incubated for 24 hr at 370c. Diameter of pink zones around the well were measured to quantify the amount of enzyme produced [8,6]. Maximum zone given organism take for enzyme production by nesslerization.

Quantity assay for enzyme production

Broth based assays were performed as an effort to screen the isolate that would produce highest yield of extracellular L-asparaginase was examined for enzyme activity by submerged fermentation in Tryptone glucose yeast extract (TGY-Broth with 1% asparagine at pH7.0) broth. Inocula were prepared in Erlenmeyer flask containing 50 ml (TGY) broth After 24 hr incubation on rotary shaker at (180 rpm /min) at 370 c, then transferred for enzyme production, a 250 ml volume of TGY broth in 500-ml Erlenmeyer flask was inoculated 5ml of inoculums (2.304 OD at 600nm) of the bacterium to be tested. The broth is incubated in a shaker incubator at (180 rpm/min) at 370 c. For different time interval 24hr, 48 hr, 72 hr, 96hr, 144hr. After Incubation the culture broth was centrifuged at 10,000 ×g for 10 min, supernatants were collected and enzyme assay done was by Nesselerisation[15,11].

Assay

The enzyme L-asparaginase was assayed colorimetrically and for the purpose standard curve was prepared using ammonium sulphate concentration gradient [1]. Assay of enzyme was carried by taking 0.5ml of 0.04M asparagine in a test tube, to which 0.5ml of 0.5 M buffer (acetate buffer pH-5.4). 0.5ml of enzyme and 0.5ml of distilled water was added to make up the volume up to 2.0ml and incubate the reaction mixture for 30min. After the incubation period the reaction was stopped by adding 0.5ml of 1.5M TCA (Trichloracetic acid). After that take 0.1ml supernatant then add 3.7ml distilled water and then add 0.2 ml Nessler’s reagent. Then incubate at room temperature for 15 to 20 min Mixed well and centrifuged at 4000 rpm at room temperature.then after take OD at 450nm. The blank was run by adding enzyme preparation after the addition of TCA. The enzyme activity was expressed in International unit [4,7].

International Unit (IU)

One IU of L-Asparaginase is the amount of enzyme which liberates 1μmol of ammonia per minute per ml [μmole/ml/min][10].

Calculation:[2]

Micromoles ammonia releasedUnits/ml = 10 minutes x ml enzyme in reaction

Result

Based on the primary screening by serial dilution technique on Nutrient agar plate 49 isolates were found. Among them 30 isolates gave pink color around the colony on modified nutrient agar plate and L-asparaginase producing colony confirmed by observe on glucose asparagine agar plate. Pink color producing colony shown in fig 1

Image 1

Figure 1: modified nutrient agar plate [A] showing un-inoculated control PR+, LASP+ plate [B] and [C] showing production of L-asparaginase by microorganisms. Plate [D] showing growth on glucose asparagine agar plate.

The total number of isolates proceeded for their morphological character and motility test. The colony characteristics of the obtained isolates were studied on nutrient agar plate, which give circular/rhizoid, white/pink, small/large colonies with entire margin after 24hr incubation. In them 10 were gram positive, among them 9 were cocci and 1 was large rod and 20 were gram negative generally short rods and some were long rods. Following Table.2 shows results of various biochemical characteristics of the isolates.

Table 2: Biochemical characterization of the isolates.

ISOLATES Biochemical
Test Carbohydrate Probable identity
Oxidase Catalase Indole Methyl red V-P test Citrate H2S production Urease TSI Nitrate reduction Gelatin hydrolysis Casein hydrolysis Glucose Mannitol Sucrose Lactose
DI2 + + + + + + Alk/alk + + + + + Staphylococcus spp.
DI3 + + + + + + Alk/ A + + + + Vibrio spp.
DI5 + + Alk/alk + + + + + Staphlococcus spp.
GI2 + + + + + Alk/alk + + + + Vibrio spp.
GI3 + + + + Alk/ A + + + + + Vibrio spp.
GI4 + + + + + + A/A + + + + + Xanthomonas spp.
GI6 + + + + Alk/alk + + + + S.epidermidis
VI1 + + + + + Alk/alk + + + + + Aeromonas spp.
VI3 + + + + + A/A + + + + Xanthomonas spp.
VI4 + + + + + + Alk/alk + + + + + S. aureus
VI8 + + + A/A + + + Klebsiella mobilis
HI1 + + + + + A/A + + + + + + Aeromonas spp.
HI4 + + + + Alk/alk + + + + S .epidermidis
HI5 + + Alk/alk + + + + Klebsiella mobilis
CF2 + + + + Alk/alk + + + + Aeromonas spp.
CF3 + + + Alk/ A + + + + + + Serratia spp.
CF4 + + Alk/alk + + + + + Bacillus spp.
COM1 + + + + + Alk/alk + + + + + Aeromonas spp.
COM3 + + + + + A/ alk + + + + + Vibrio spp.
COM5 + Alk/alk + + + Vibrio spp.
CON1 + + + + + + Alk/alk + + + + + Staphylococcus spp.
CON3 + + Alk/alk + + + + + Staphylococcus spp.
CON5 + + + + Alk/alk + + + + Staphylococcus spp.
FI1 + + + + + + Alk/alk + + + + + S. aureus
FI2 + + + + + A/ A + + + + Pseudomonas spp.
FI4 + + + + + A/ alk + + + + Pseudomonas spp.
LI1 + + + + Alk/alk + + + + + Pseudomonas spp.
MI1 + + + Alk/ A + + + + Aeromonas spp.
MI2 + + + Alk/ A + + + + + + Serratia spp.
MI3 + Alk/alk + + + + Haemophilus spp.

(‘+’: positive, ‘-’: negative, ‘ A’ : for acidic gave yellow coli and ‘Alk’: for basic gave pink color on TSI slant)

By studying morphological and biochemical characteristics L-asparginase producing various species were obtained such as, Staphylococcus spp.: 9, Bacillus spp.: 1, Psuedomaonas spp.: 3, Aeromonas spp.: 5, Vibrio spp : 5, Serratia spp.: 2, Enterobacter spp.;2, Haemophillus spp.:1,Xanthomonas spp.: 2. The total number of isolates are shown in Fig 2.

Image 2

Figure 2: Isolated species of L-asparaginase producers

Secondary screening, by agar cup method L-asparaginase producing 30 isolates gave different zone of diameter after 24 hrs incubation. Out of this garden area soil sample isolate GI3 was found good L-asparginase producer as it exhibited 52mm pink zone. (Shown in table no: 3) So the GI3 isolate were selected for enzyme assay. However there are many reports that justify the selection of potential isolate depending on the diameter of pink zone around the L-asparaginase producing colonies. The zone obtaining isolates showing in below fig 3.

Image 3

Figure 3: modified nutrient agar plate [A] showing un-inoculated control PR+, LASP+ plate and [B] & [C] showing pink zone obtain by isolates.

Table: 3 Showing the zone of diameter in mm on modified nutrient agar plate

Isolates Diameter of zone in mm
DI2 44mm
DI3 20mm
DI5 29mm
GI2 8.5mm
GI3 17mm
GI4 35mm
GI6 32mm
VI1 35mm
VI3 25mm
VI4 18mm
VI8 23mm
HI1 30mm
HI4 23mm
HI5 40mm
CF2 40mm
CF3 34mm
CF4 20mm
COM1 42mm
COM3 36mm
COM5 25mm
CON1 21mm
CON3 40mm
CON5 15mm
FI1 17mm
FI2 23mm
FI4 30mm
LI1 40mm
MI1 24mm
MI2 50mm
MI3 25mm

Enzyme activity was done by using ammonium sulfate as a standard (Table :4 and Fig: 4 shown the optical density and standard graph). The enyme activity and enzyme activity graph of selected isolate GI3 shown in below (Table:5 and fig :5)u

Table: 4 Optical density of standard graph

(NH4)2SO4conc.(μM/ml) Optical Density (nm)
0.011 0.072
0.023 0.287
0.047 0.404
0.095 0.708
0.142 1.011
0.19 1.174
0.237 1.582
0.285 2.054
0.333 2.211
0.383 2.442
0.428 2.637
0.4758 2.892

Image 4

Figure 4: Standard graph

Table 5: Activity of GI3 isolate

isolate Time interval L-asparaginase activity IU/ml
0hr 0.666
24hr 1.333
GI3 48hr 6
72hr 8
96hr 18.66
144hr 18.66

Image 5

Figure 5: Enzyme activity graph

In the enzyme production at 48 hr ,72 hr,96 hr the log phase gave increasing amount of activity after 96 hr the production of enzyme being constant and enzyme activity is 18.66 IU/ml.

Discussion

The study highlights the screening , Identification and Production of L-asparaginase producing microorganisms. A total 30 isolates were obtained from different soil sample. Isolates were identify based on their morphological and biochemical characteristics by macfaddin and bergey’s manual. By zone assay GI3 isolate give highest zone 52mm . M. R. Bhat et al.,(2015) found maximum zone giving organisms from forest soil. From the all isolates maximum zone giving isolate GI3 shows 18.66 IU/ml enzyme activity . Sahira Nsayef Muslim et al., (2015) found 13.53 IU/ml enzyme activity.

Conclusion

The preliminary work of Screening L-asparaginase producing isolates from chosen sampling site and biochemical identification of isolates under study has been successfully carried out. By agar well diffusion method GI3 isolate give maximum zone 2.6 cm. and the enzyme activity after 96hr is 18.66IU/ml. This isolate is further carried out for optimization and therapeutic agent.