pharmaceutically significant enzymes- tyrosinase …

www.wjpr.net Vol 7, Issue 04, 2018.

89

Majumder et al. World Journal of Pharmaceutical Research

PHARMACEUTICALLY SIGNIFICANT ENZYMES- TYROSINASE

AND ASPERGINASE FROM MESOPHILIC STREPTOMYCES-A3DR2S

Dr. D. R. Majumder*, Sara Lambate1, Aqsa Firfire

2, Rahat Palekar

3, Asfiya Shaikh

4,

Ashlesha Bhandari5 and Reshma Sayyed

6

*Head, Department of Microbiology, Abeda Inamdar Senior College, 2390-B, K. B

Hidayatullah Road, Azam Campus Pune-411001. Maharashtra, India.

1-6Department Microbiology, Abeda Inamdar Sr. College, Pune.

ABSTRACT

In the present study, Actinomycete strain producing Tyrosinase,

Asparginase, along with Amylase, Protease and Lipase was isolated

from garden soil. Tyrosinase and Asparginase were chosen for further

study because of their pharmaceutical and commercial importance.

Tyrosinase catalyzes the bioconversion of an amino acid L-Tyrosine to

L-DOPA (3,4-dihydroxyphenylalanine). L-DOPA has therapeutic

importance in the treatment of Parkinson’s disease. L-asparginase also

known as L-asparagine amidohydrolase, is the enzyme with anti-tumor

activity and used as a chemotherapeutic agent against acute

lymphoblastic leukemia and lymphosarcoma. From microscopic

observation the isolate was identified as Streptomyce sp A3DR2S. The

optimization result revealed that the maximum Tyrosinase and L-DOPA

production was at pH-8 and room temperature 370C with ideal substrate

concentration of 0.1%. Similarly, maximum production of Asparginase was recorded at pH 6.5

and temperature 370C with substrate concentration of 1%. Specific activity of tyrosinase was

calculated to be 232.75U/mg and that of asparginase it was 4.30 U/mg. Purity of both the

enzymes was checked by SDS-PAGE revealing the presence of a single band with a molecular

weight of 38kDa for Tyrosinase and 150kDa for Asparginase approximately. Km and Vm of

tyrosinase was calculated as 2.7mM and 5.4mM/ml/min and that of asparginase the values

are 3.75mM and 7.5mM/ml/min.

World Journal of Pharmaceutical Research SJIF Impact Factor 8.074

Volume 7, Issue 04, 89-109. Conference Article ISSN 2277–7105

Article Received on

02 Jan. 2018,

Revised on 23 Jan. 2018,

Accepted on 12 Feb. 2018

DOI: 10.20959/wjpr20184-10766

*Corresponding Author

Dr. D. R. Majumder

Head, Department of

Microbiology, Abeda

Inamdar Senior College,

2390-B, K. B Hidayatullah

Road, Azam Campus

Pune-411001. Maharashtra,

India.


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KEYWORDS: Streptomyce sp A3DR2S, Amylase, Protease, Lipase, Tyrosinase,

Asparginase, L-DOPA.

INTRODUCTION

Soil Microorganisms have important role in degradation of complex organic matter. One of

the important soil microbes is Actinomycete which has more than 100 Genera. They are

special group of microorganism which has both characters of Bacteria and fungi.[1]

Taxonomically they are placed with bacteria in class Schizomycetes and order

Actinomycetales. They are unicellular like bacteria but also produce mycelium like fungi.

Colonies of Actinomycetes grow slowly, show powdery consistency and stick to agar surface.

They are heterotrophic, aerobic and mesophilic (25-30◦C) organisms. Some are thermophilic

growing at 55-65◦C, e.g. Streptomyces and Thermoactinomycetes. The actinomycetes are

found in various habitats in nature.[2,3]

Actinomycetes can synthesize different bioactive compounds like enzymes, antibiotics, etc.

which are industrially and pharmaceutically important.[4]

Tyrosinase and asparginase are two

such enzymes having pharmaceutical importance.

1) Tyrosinase (monophenol, o-diphenol: oxygen oxidoreductase, EC 1.14.18.1), often also

called polyphenol oxidases are copper containing metalloproteins. It is an oxidase that is the

rate-limiting enzyme for controlling the production of melanin. It is mainly involved in two

distinct reactions of melanin synthesis in presence of molecular oxygen: firstly, the

hydroxylation of a monophenol and secondly, the conversion of an O-diphenol to the

corresponding O-Quinone. O-Quinone undergoes several reactions to eventually form

melanin.[5]

Figure 1: Structure of Tyrosinase. Coppers: blue, histidines: green, dioxygen molecule:

red active site.[6-8]


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Biotransformation of Tyrosine to L-DOPA

Conversion of L-Tyrosine to L-DOPA is a one-step oxidation reaction, catalyzed by enzyme

tyrosinase in presence of oxygen. Two oxygen molecules reacts with the two copper atoms

present in the active site of tyrosinase and forms a highly reactive intermediate which then

further oxidizes the substrate.[9]

Fig. 2: Bioconversion of L-tyrosine to L-DOPA by Tyrosinase.[6-8]

Application of Tyrosinase

1) L-DOPA Production

L-DOPA has a high economic value because it is the main drug for the treatment of

Parkinson’s disease.[10]

L-DOPA is a chemical precursor of dopamine and is a derivative of

amino acid L-tyrosine. Tyrosinase oxidizes tyrosine to melanin. The first two intermediates in

the process are L-DOPA and Dopaquinone. L-DOPA is metabolically converted into

dopamine in the body by an enzyme called aromatic L-amino acid decarboxylase (AADC).


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Dopamine is a neurotransmitter. Its deficiency in human results in degenerative disorder

called Parkinson's disease which affects central nervous system. But, Dopamine cannot be

administered because it cannot cross the blood brain barrier whereas L-DOPA which is a

precursor of Dopamine can cross the barrier. For this reason, L-DOPA can be used in the

treatment of Parkinson's disease. L-DOPA is marketed under the brand names like Sinemet,

Atamet, Parcopa and Stelova. Chemical synthesis of L-DOPA is time consuming process and

involves byproducts which lack applications. Therefore, a cost effective, time saving and

economical process has to be developed. Enzymatic bioconversion is an alternative to

chemical process. For the first time, Shiet et al reported the production of L-DOPA from

fungi. Tyrosinase also has various other applications like in dye industry. Bacterial

tyrosinases have been tested in the detoxification of wastewater by removal of phenolic

compounds and decolourisation.[3,11]

Medical applications of tyrosinase include the

production of melanin as natural antibacterial compounds for the treatment of wounds, i.e. the

local application of melanin precursor and tyrosinase in the form of a cream or ointment.

Tyrosinases are also suggested to be potential tools in treating melanoma.[12,13]

It is also used

as a target for the activation of prodrugs in food industries for modification of food proteins

via crosslinking.[14,15]

Synthetic melanin is also used for protection against radiation (UV,

X-ray, and gamma ray) and as cation exchangers, drug carriers, antioxidants, antiviral agents

or immunogens. There is considerable information of the application of this potential enzyme

in food, medicine, and agricultural industries as well as for analytical and environmental

purposes.[11]

2) L-asparaginase

L-asparaginase (L-asparagine amidohydrolase, (EC3.5.1.1) is an enzyme used as a

chemotherapeutic agent for the treatment of acute lymphoblastic leukemia, acute

myeloblastic leukemia, chronic lymphocytic leukemia[16]

, Hodgkin′s disease, melanosarcoma

and also as food processing aid to reduce the formation of acrylamide during frying of

starchy food at high temperature.[17,18]

The enzymes isolated from E.coli and Erwinia

carotovora are now used in the treatment of acute lymphoblastic leukemia. Among the

actinomyces Streptomyces species such as S.karnatakensis S.venezualae, S.

longsporusflavus etc have been explored for L-asparaginase production.[19]


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Mechanism of action

Asparaginase is used as an antitumor Agent because leukemic cells cannot produce the

non-essential amino acid asparagine on their own, which is very essential for the growth of

the tumor cells, where as normal cells can reduce their own asparagine. The products of

Asparaginase reaction are aspartic acid and ammonia.[20]

This deprives the leukemic cell of

circulating asparagine and prevents them from the rapid malignant growth. Therefore, the

function of L-asparaginase in treatment of cancer is to reduce the concentration of

L-asparagine from serum by catalyzing the breakdown thus controlling the tumor growth

effectively. Protein and RNA synthesis is inhibited in the absence of asparagine and as a

consequence cell cycle arrest and apoptosis is induced in leukemic cell lines.[17, 21, 22]

Fig. 3: Bio-conversion of L-asparagine to L-aspartate by Asparginase.[23]

MATERIAL AND METHOD

Isolation of Actinomycete

1.0 g of soil sample collected from Azam campus, Camp, Pune was suspended in 9ml sterile

saline and serially diluted upto10-6

and 0.1 ml was plated on CSA Agar medium (Hi Media).

The plates were incubated for 4-5 days at 37◦C.

Identification of Actinomycete

Identification of Actinomycete was done by slide culture technique.

Screening of Actinomycetes for extracellular enzymes production

The isolate was subjected to screening for different enzymatic activities (Amylase,

Gelatinase, Protease, asparaginase and Tyrosinase) by spot inoculation on respective

medium. The plates were incubated at 37◦C for respective time and results were recorded.

The details are given in Table 1.


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Table 1: Screening of Actinomycetes for Extracellular Enzymes Production.

Sr.

No. Enzyme Medium Incubation Reagent used

Criteria for positive

enzyme activity

1. Amylase Starch Agar (Hi media)

37◦C for 2

days Iodine solution

Zone of hydrolysis

around the spot after

flooding the plate with

Iodine solution

2. Protease Gelatin

Agar 37

◦C for 2

days

Frazier's reagent : Hgcl2 15g, conc.

HCL 20ml,

Distilled

Water 100ml

zone of hydrolysis

around the spot by

flooding the plate with

Frazier's reagent

3. Lipase Tween

80-agar 37

◦C for 2-3

days -

Precipitation around

the spot

4. Tyrosinase Tyrosine

agar

37◦C for 7

days -

Development of brown

color

5. Asparaginase Asparagine

dextrose

salt agar

37◦C for 4

days -

Development of pink

color

Composition of the Medium used for Detection of Enzyme Production

Gelatin Agar- Nutrient Agar with 1% gelatin powder

Tween 80 Agar- Peptone-10g, NaCl-5g, CaCl2.2H2O- 0.1g, Tween 80-10g, Agar-20g,

Distilled water-1000ml, pH-6.

Tyrosine agar- Peptone-0.5g, Beef Extract-0.3g, L-Tyrosine-0.1g, Agar-2g, pH-7, DW 100ml

Modified M9 Agar- KH2PO4-0.1g, L-asparagine-1g, MgSO4-0.05g, Dextrose-0.2g, Agar-20g,

Distilled water-100ml, Phenol Red- 0.00, pH-6.5.

Tyrosinase and Asparaginase were chosen for further studies because of its pharmaceutical

applications.

The isolate was inoculated in Tyrosine and Asparagine dextrose salt broth and incubated at

37◦C for respective days. After development of brown color in Tyrosine broth, tyrosinase assay

and detection of L-DOPA was done. Similarly, after development of pink color in Asparagine

dextrose salt broth, Asparginase assay and detection of aspartate was done.

Quantitative Estimation of Tyrosinase and Asparginase

1) Tyrosinase Assay

Solutions were pipette into test tube in the following order:


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Table 2: Protocol for Tyrosinase Assay.

Solution to be added Amount (ml)

0.5M phosphate buffer 1.0

0.001M L-tyrosine 1.0

Distilled water 1.0

The reaction mixture was oxygenated by bubbling oxygen through a capillary tube for 4-5

minutes. Absorbance was recorded at 280 nm for 4-5 minutes to establish blank. 0.1 ml of the

supernatant of the enriched culture expected to be producing tyrosinase was added to the

reaction mixture and absorbance was recorded for 10-12 minutes. Calculation of enzyme

activity was done using the following formula:

Units of enzyme/ ml = (∆A280nm/min test - ∆ A280nm/min Blank) x DF

(0.001)(0.1)

DF = Dilution factor

0.01= The change in A280nm/minute per unit of Tyrosinase at pH 6.5 at 25°C in a 3 ml

reaction mix

0.1ml = Volume of enzyme used

2) Asparginase Assay

Solutions were pipette into test tube in the following order:

Table 3: Protocol for Asparginase Assay.

Solution to be added Amount (ml)

0.04 M L-Asparagine 0.5

50mM of tris buffer 0.5

enzyme solution 0.5

distilled water 2

The above mixture was incubated at 37°C for 30 minutes. The reaction was stopped by

addition of 0.5ml of 1.5M of TCA. Take 3.7 ml of distilled water + 0.1 ml of above mixture +

0.2ml of Nessler’s reagent and immediately recorded the absorbance at 450nm and the

amount of liberated ammonia was determined. Enzyme activity was calculated by using the

formula.

Units/mL enzyme = (μmole of NH3 liberated) (2.5)

(0.1) (30) (0.5)

2.5ml= Initial volume of enzyme mixture

0.1ml=Volume of enzyme mixture used in final reaction


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30min=Incubation time

0.5ml=Volume of enzyme used

Quantitative Detection of L-DOPA

Chemical detection of L-DOPA was done by Arnow’ Method

Requirements

1) 0.5N Hydrochloric acid

2) Nitrite-molybdate reagent. Dissolve 10 g of sodium nitrite and 10 g of sodium

molybdate in 100 ml.of distilled water

3) 1N sodium hydroxide solution

4) Standard curve of L-DOPA (Sigma Aldrich) (Range: 0.001-0.01mg/ml)

The method has the following steps.

Filter enriched broth using Whatman filter paper No.1. 1ml of cell free broth was taken and

the reagents were added in the following order, mixing well after each addition.

Table 4: Protocol for Arnow's Method.

The absorbance recorded is directly proportional to amount of L-DOPA present in the

reaction mixture.

Qualitative Detection of L-DOPA Production by HPLC.

The isolate was inoculated in Tyrosine broth. After 48 hours sample was withdrawn and

centrifuged. The supernatant was collected and was subjected to HPLC analysis for the

detection of L-DOPA.

Qualitative Detection of L-Aspartate Production by TLC

Cell free broth was subjected to thin layer chromatography (TLC) for the confirmation of

L-aspartate production, using butanol acetic acid and water as a mobile phase and silica gel as a

Reagent Amount (ml)

0.5 N hydrochloric acid 1

Nitrite-molybdate reagent 1

Yellow color results at this point

1 1N sodium hydroxide 1

Red color develops at this point. Make Up volume with distilled

water to 5 ml

Record the absorbance at 530nm


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stationary phase. Ninhydrin solution was used as spraying agent for identification. Rf was

value of aspartate was calculated by the following formula,

Rf = Distance travel by solute

Distance travel by solvent

Optimization of Different Parameters for Enhanced Enzyme Production

1) pH

In order to check the enhanced production of Tyrosinase and Asparginase, different buffer

systems of a specific pH value ranging from pH 4-8 for tyrosinase and pH 5.5, 6.5 and 7.5 for

asparginase were used. Cells were harvested from appropriate broth and inoculated into buffers

containing 0.1% Tyrosine and 1mg/ml Asparagine. The flasks were incubated on a shaker at

370C for 4-5 days. The broth was then centrifuged and the supernatant was checked for enzyme

units and concentration of L-DOPA.

2) Temperature

Cells were harvested and inoculated in buffer of optimum pH containing 0.1% tyrosine and

1mg/ml aspargine and incubated at different temperatures 50C, 37

0C and 55

0C for Tyrosinase

and 270C, 37

0C and 55

0C for Asparaginase. After incubation of 4-5 days, the broth was

centrifuged and the supernatant was assayed for tyrosinase and asparginase activity as well as

for L-DOPA production.

3) Substrate Concentration (L-Tyrosine and L-Asparagine)

Cells were harvested and inoculated in buffer of optimum pH containing different

concentration of substrate and incubated at optimum temperature for 4-5days. For tyrosinase

production- 0.05, 0.1 and 1 g% substrate concentration were taken and for asparaginase

production- 0.5, 1 and 1.5 mg/ml substrate concentration were taken. After incubation, the

broth was centrifuged and the supernatant was assayed for enzyme activity and L-DOPA

production.

Purification of Tyrosinase and Asparginase from Actinomycetes

Enriched culture was centrifuged and both the enzyme were further purified from the filtrate,

following the steps given below.


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Ammonium Sulphate Precipitation

Ammonium sulphate powder was added to the 10ml of cell free broth slowly with continuous

stirring in an ice bath, until 60% saturation is achieved. The mixture was refrigerated overnight,

followed by centrifugation at 6000-10,000g in a refrigerated centrifuge. The precipitate was

dissolved in 0.2 M phosphate buffer (pH 7.0). The suspended precipitate was then checked for

enzyme activity and total protein content.

Dialysis

Dialysis membrane was pre-treated in EDTA for 10 min and washed three times with distilled

water and boiled for 10 min after every wash. The membrane was filled up with the suspended

precipitate and sealed at both ends. The dialysis bag was then suspended overnight at 4ºC in a

glass beaker containing 0.2 M phosphate buffer (pH 7.0) with continuous mixing using a

magnetic stirrer. The dialyzed extract was checked for enzyme activity and total protein

content.

Protein Content: Folin-Lowry Method

Protein content was measured with Bovine Serum Albumin (BSA) as standard protein by

Folin-Lowry method. 1 ml of sample was mixed with 5 ml of Alkaline solution and incubated

for 10 min. 0.5ml of Folin-Ciocalteu was added and incubated for 30min. Absorbance was

measured at 530 nm. Protein content was expressed as milligram of protein per milliliter of

sample. Following enzyme assay and protein determination, specific activity of Tyrosinase and

Asparginase was calculated using the following formula-

Specific Activity = enzyme Units/ml /min

mg of protein/ml

SDS-PAGE

To check the purity of dialyzed sample SDS-PAGE was carried out in a 30% polyacrylamide

gel using Tris-glycine buffer (pH 8.3) according to protocol of Laemmeli. Dialyzed sample

was loaded on to polyacrylamide gel. Silver staining was performed in order to visualize the

protein bands. The gel results were documented in Gel Doc EZ imager.


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Determination of Km and Vm for Asparaginase and Tyrosinase

For determining the Km and Vm values Enzyme activity was calculated (velocity) using

different substrate concentration. Velocity was plotted against substrate concentration and

Michaelis-Menton constant was determined.

RESULT

Identification of Actinomycetes

From soil sample one isolate A3DR2S was isolated of which colony morphology and

microscopic observation is shown in Figure-4.

Screening of Actinomycetes for Extracellular Enzymes Production

Screening of extracellular enzyme activities was done and the isolate gave positive results for

enzymes Amylase, Protease, Lipase, Tyrosinase and Asparginase. The results for the

enzymes production are given below in Fig 5 and table 5.

(a) (b) (c)

Figure-4: (a) Growth of Isolate A3DR2S on CSA (b) Growth of Isolate A3DR2S Slide

culture technique (c) Microscopic view of Isolate A3DR2S


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(A) (B)

(C)

(D) (E)

Figure-5: Different Enzyme Activities of Isolate A3DR2S (A) Amylase (B) Protease (C)

Lipase (D) Tyrosinase (E) Asparginase.

Table 5: Positive Enzyme Activities of The Isolate.

Enzymes Observation Result

Amylase Zone of hydrolysis was observed around the spot after flooding the

plate Iodine solution +

Protease Zone of hydrolysis was observed around the spot by flooding the plate

with Frazier's reagent +

Lipase Precipitation was seen around the spot +

Asparginase Development of pink color +

Tyrosinase Development of brown color +

Qualitative detection of L-DOPA production by HPLC.

After 48 hr. sample was harvested from tyrosine broth by centrifugation and subjected to

HPLC.


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The comparison of retention time of standard L-DOPA with that produced by isolate is given

below in table 6.

Table 6: HPLC analysis for standard L-DOPA and sample.

Retention time of standard L-DOPA Retention time of test samples (215nm)

5.1 to 6.4 min 5.2 to 6.3

Fig 6: HPLC graph of std L-DOPA and test sample.

Qualitative detection of L-Aspartate by TLC

After development of pink color in Aspargine dextrose salt broth sample was harvested and

subjected to TLC. The Rf value of the Aspartate was found to be (0.46cm) and that of the

standard run Aspartic acid under the same condition was found to be (0.49cm).

Fig 7: TLC of Aspartate


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Optimization of different parameters for Tyrosinase production and enhanced L-DOPA

production -

Different parameters for enhanced enzyme and L-DOPA production were optimized and the

results are as follows.

1) pH Optimization

The results in fig 8 indicate that optimum pH was pH 8.0 for tyrosinase production, 287 U/ml

and that for L-DOPA production, 0.028 mg/ml was pH 7.5.

Fig 8: Tyrosinase and L-DOPA production at different pH.

2) Temperature Optimization

After incubation, respective broths were centrifuged and supernatants were assayed for

enzymes and L-DOPA production. The results are summarized in the table given below.

Table 6: Units of tyrosinase and amount of L-DOPA obtained per ml, at different

temperature.

Temperature Enzyme U/ml L-DOPA (mg/ml)

5ºC 0 0

37 ºC 285 0.026

55 ºC 0 0

3) Substrate Concentration

After incubation, the broth was centrifuged and the supernatant was assayed for enzyme

activity and L-DOPA production. The results are summarized in the table given below.


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Table 7: Units of tyrosinase and amount of L-DOPA obtained per ml at different tyrosine

concentration.

L-tyrosine (g/100ml) Enzyme U/ml L-DOPA (mg/ml)

0.05 200 0.015

0.1 285 0.02

1 80 0.013

Optimization of different parameters for Asparaginase production

1) pH Optimization.

Table 8: Asparginase production at different pH values.

2) Temperature Optimization

Table 9: Units of Asparginase obtained per ml, at different temperature.

Temperature(◦c) Enzyme units (U/ml)

27 0.306

37 0.66

55 0.36

3) Substrate Concentration

Table 10: Units of Asparaginase obtained per ml at Asparagine concentration.

Purification of tyrosinase and Asparginase

Enriched culture was filtered and was partially purified by 60% Ammonium sulphate

precipitation and by Dialysis. The comparison of Specific activity and fold purification of

crude and purified extract is depicted in table 11 and table 12.

The purity of dialyzed fraction was checked by SDS-PAGE according to protocol of

Laemmli,1970.The result summarized in Fig 3.5, revealed the presence of a single band with a

molecular weight of 30kDa for Tyrosinase and 150kDa for Asparginase approximately.

pH Enzyme units (U/ml)

5.5 0.66

6.5 0.76

7.5 0

Asparagine (mg/100ml) Enzyme units (U/ml)

0.5 0.46

1 0.66

1.5 0.49


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Table 11: Purification chart of pure and crude extract of tyrosinase.

Sample Total volume

(ml)

Enzyme activity

(U/ml)

Protein content

(mg/ml)

Specific activity

(U/mg)

Fold

purification

%

yield

Crude CFB 95 285 4.56 62.5 1 100

Dialysed extract 50 186.2 0.8 232.75 3.72 65.3

Table 12: Purification chart of pure and crude extract of Asparginase.

Total volume

(ml)

Enzyme activity

(U/ml)

Protein content

(mg/ml)

Specific activity

(U/mg)

Fold

purification

%

yield

Crude CFB 10 0.66 0.3 2.22 1 100

Dialysed extract 10 0.56 0.13 4.30 1.93 84

Figure-9: SDS-PAGE Gel Doc image of partially purified tyrosinase stained by silver

staining technique. (1) Ladder (2) Dialysed extract of tyrosinase (3) Dialysed extract of

asparginase. Circle indicates 38 kDa of approximate molecular weight of tyrosinase in

dialysed extract and 150kDa of approximate molecular weight of asparginase in

dialysed extract.

Determination of Km and Vm for Tyrosinase

Substrate concentration (L-tyrosine)-10mM.


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Fig 10: Michaelis-Menton graph for Tyrosinase.

Determination of Km and Vm for Asparginase

0

2

4

6

8

0 2 4 6 8 10

velo

city

U/m

l/m

in

substrate conc mM

Michealis-Menton

Fig 11: Michaelis-Menton graph for Asparginase.

Table 13: Determination of Km and Vm for Asparginase and Tyrosinase.

Enzyme Km (mM) Vmax (mM/ml/min)

Tyrosinase 2.7 5.4

Asparginase 3.75 7.5

DISCUSSION

Actinomycetes are predominantly screened for its biological significance in the industries.

They have a wide range of bioactive compounds. Streptomyces spp. produce one third of

bioactive metabolites such as antibiotics, enzymes, anti-tumor compounds etc. which are

essential for the treatment of dreadful human diseases. In the present study, the


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Actinomycetes strain A3DR2S isolated from soil was screened for different industrially

important enzymes. The result revealed that the isolate produce five enzymes viz, Amylase,

Protease, Lipase, Tyrosinase and Asparginase. The study conducted by previous workers

reported the production of enzyme Amylase, Protease, Xylanase and Lipase from different

Actinomycetes isolated from soil.[24,25]

Melanin biosynthesis (charcoal blackish

pigmentation) is a two-step biocatalytic process mediated by tyrosinase enzyme using

tyrosine as the substrate material. Biochemically, tyrosinase enzyme possesses both

monophenolase activity (hydroxylation of monophenols to o-diphenols) and diphenolase

activity (oxidation of o-diphenols to o-quinones).In the first reaction, the tyrosine is

converted to L-Dopa (3, 4-dihydroxyphenyl alanine), which upon subsequent catalysis step

results in production of melanin.[26, 27]

L-DOPA is pivotal in the treatment of Parkinson’s

disease. Asparaginase is a pharmacologically active anticancer therapeutic agent. Therefore,

the present study is focused on Tyrosinase and Asparginase production. Tyrosinase was

partially purified by Ammonium Sulphate precipitation and dialysis. Total protein was

assayed by Folin-Lowry method. Specific activity of Tyrosinase was calculated for purified

extract and it was found to be 232.75 U/mg and reported is 0.38 U/mg in purified extract.[28]

Specific activity of asparaginase was calculated for purified extract and it was found to be

4.30 U/mg. and reported is 17 U/mg in purified extract[29]

The production of Tyrosinase and

L-DOPA was optimized at different pH, temperature and tyrosine concentration. In present

study tyrosinase and L-DOPA production was maximum at pH-8 and pH-7.8 respectively and

temperature-37ºC. 0.1%.Tyrosine concentration is best at which tyrosinase and L-DOPA

production was maximum. In the earlier study[30]

maximum production was at pH 6.5 and

temperature 30ºC. The purity of dialyzed sample was checked by SDS-PAGE and the

purified fraction showed the presence of single band corresponding to approximately 38 kDA

of tyrosinase and that of Asparaginase was 150 kDA . In the previous study [28]

tyrosinase was

purified by SDS-PAGE and molecular weight was approximately 30 kDA. Asparginase

activity of the isolate was determined qualitatively (based on colour intensity) and

quantitatively (amount of ammonia liberated). Optimization studies were further carried out

which revealed maximum production at pH 6.5, temperature 37˚C, substrate concentration

1%. TLC was carried out to check production of L-aspartate as L-asparginase converts L-

aspargine to L-aspartate and Rf value was calculated (0.46cm) and compared with standard.

Whereas in the study conducted by Noura El-Ahmady et al[31]

showed the maximum

production of L-Asparginase at 40°C and pH 8.5 with substrate concentration of

0.01M.Further enzyme kinetic parameters such as Michaelis-Menton constants Km and Vm


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for both enzymes were studied. Km and Vm for tyrosinase are (Km-2.7mM,Vm-5.4

mM/ml/min) and for asparaginase (Km-3.75M, Vm 7.5 U/ml/min) which are comparable

with reported Km and Vmax of Tyrosinase and asparginase respectively.[28,31]

ACKNOWLEDGEMENT

We would like to thank Dr. E.M.Khan, Principal of Abeda Inamdar Senior College for

providing us with the necessary infrastructure conducive for research.

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pharmaceutically significant enzymes- tyrosinase …

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