55

CHAPTER 3

PRODUCTION AND PURIFICATION OF TYROSINASE

3.1. PREFACE

Tyrosinases are a group of copper containing metalloproteins and are normally

referred as polyphenol oxidases, which belongs to a major class of oxidoreductase

enzymes. It is a bifunctional enzyme and catalyzes two reactions in the presence of

molecular oxygen, exhibiting monophenolase or cresolase activity and diphenolase or

catecholase activity. It plays a significant catalytic properties led the tyrosinase for

several applications. Catalytic properties of enzymes are generally varied from the

sources of their origin aiming at particular applications.

Biological sources including plants, animals and microorganisms are

predominant sources for the synthesis of tyrosinase. Mushroom tyrosinase is

commercial source of tyrosinase available in the world market. Although submerged

and solid state processes were explored for the production of tyrosinase at large scale,

the submerged bioprocess is leading. Several methods mainly including

immobilization, mutations and recombinant DNA techniques have been explored for

the enhanced production of enzymes at higher scale. However, Response Surface

Methodology (RSM) with suitable statistical design is very attractive and most

preferred one for the enhanced production of enzymes. The purification of enzyme is an

important criterion before subjecting for any major applications. Ammonium sulpahte

precipitation, dialysis, gel filtration and column chromatography are the basic methods

normally employed for the purification of enzymes.

This chapter covers the production of tyrosinase by an efficient isolate of

Streptomyces tuirus DBZ39. A submerged bioprocess was optimized with major

physicochemical and nutritional conditions. Response Surface Methodology with

Central Composite Design (CCD) was employed to optimized critical process variables

for the enhanced production of tyrosinase. The enzyme was purified by ammonium

sulpahte precipitation, dialysis, gel filtration and column chromatography.

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3.2. REVIEW OF LITERATURE

3.2.1. Tyrosinase and bioprocess

Tyrosinase: The browning of fruits and vegetables has been interest of research for

years (Almeida and Nogueira, 1995). The process of browning involves the enzyme-

catalyzed synthesis of a dark pigment named melanin (Friedman, 1997). The enzyme

that plays a crucial role in the formation of melanin is tyrosinase. In addition to plants

and animals, a wide range of microorganisms such as bacteria, fungi, yeast and

actinomycetes, have proven to be proficient sources for tyrosinase.

Tyrosinase is an important multicopper containing enzyme having EC No.

1.14.18.1 and belongs to oxidoreductase family (Lerch, 1983). It is mainly referred as

monophenol monooxygenase, dihydroxyphenylalanine: oxygen oxidoreductase,

polyphenol oxidase; catechol oxidase and oxygen oxidoreductase. Tyrosinases are

nearly ubiquitously distributed in nature. It is found in both prokaryotes and eukaryotic

microbes, invertebrates, plants and mammals. The most extensively investigated

tyrosinases are from mushroom (Sung et al., 2003). It has been suggested that there is

no common tyrosinase protein structure occurring across all species. However, all

tyrosinases have a common binuclear type 3 copper centres with in their active site.

Here two copper atoms are each coordinated with three histidine residues (Klabunde et

al., 1998). The crystallographic structural feature (Matoba et al., 2006) of the

Streptomyces tyrosinase is as shown under.

  57

A indicates caddie protein (blue), B and C indicates copper clusters (red) and

histidine residue (orange) and D indicates supra molecular structure of Streptomyces

tyrosinase.

Tyrosinases are bifunctional enzymes, it catalyzes two types of reactions in the

presence of molecular oxygen; the ortho-hydroxylation of monophenols to its

corresponding o-diphenol (monophenolase, cresolase activity) and the oxidation of

diphenols to its correspondent ortho-quinones (diphenolase, catecholase activity).

Quinones are highly susceptible to non-enzymatic reactions, which may lead to the

formation of mixed melanins and heterogeneous polymers (Lerch, 1983). The catalytic

mechanisms behind oxidation of a substrate typically involve formation of a reactive

intermediate by the reaction of a reduced Cu+ centre with molecular oxygen, which

may also be incorporated to the substrate (Mayer, 1987). The catalytic mechanism of

tyrosinase leading to the formation of eumelanins and pheomelanins illustrated by

Lerch (1983) is as follows.

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Bioprocess: A bioprocess is any process that uses complete living cells or their

components to obtain desired products. It is a technique of biological conversion of

complex substrates into simple compounds by various microorganisms such as bacteria,

fungi and actinomycetes (Balakrishnan and Pandey, 1996). The development of

suitable and standard bioprocesses has lead to industrial level production of bioactive

compounds (Demain, 1999). Different types of bioprocesses have been designed and

standardized by various researchers suitably based on the type of substrate, organism

and the desired end product. Solid and submerged states of bioprocesses are

conventionally referred as fermentations, are normally followed for the large scale

production of bioactive compounds. Submerged bioprocess utilizes free flowing liquid

substrates, such as molasses and broths. The bioactive compounds are secreted into the

fermentation broth. The substrates are utilized quite rapidly; hence, need to be

constantly replaced/supplemented with nutrients. This fermentation technique is best

suited for microorganisms such as bacteria that require high moisture content.

An additional advantage of this technique is that purification of products is easier.

Normally Submerged fermentation is primarily used in the extraction of secondary

metabolites that need to be used in liquid form (Subramaniyam and Vimala, 2012).

The process development is the key step in any basic fermentation criteria.

Process development refers to the up-gradation of the factors that influence the

metabolite. It involves optimizing the production parameters and suitably enabling the

organism into a potent producer of the required metabolite in its best possible quality

and concentration. The organisms vary in their abilities to produce the required enzyme

and also their competitive reciprocation to the production environment. To understand

these process variables, knowledge of enzyme kinetics is essential and indeed even

more basic consideration will be the choice of an appropriate environment for an

appropriate enzyme. This choice must take into consideration such factors as

specificity, initial and final pH, activator and inhibitor requirements, availability, cost,

toxicity and ease to control the process (Barrett, 1975). Also, it is important that the

maximum activity is retained during the process development of enzyme production.

Two key elements in an enzyme reaction are the substrate and the enzyme, both

contained in a suitable medium providing the necessary environment obvious for an

effective reaction. Nevertheless, production environment, medium engineering and

biocatalyst engineering are likely to be used as major attributing parameters,

increasingly in a useful fashion for optimizing the production of enzymes.

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Consequently numerous investigations have been carried out to better

understand and define conditions, which would lead to higher enzyme production

efficiency. The extent of enzyme production is dependent upon a variety of factors

including the makeup of the fermentation medium, particularly composition of

the medium and how it was prepared, its physical and environmental conditions

(Gupta, 2002). Enzyme production by microorganisms is modulated by a number of

nutritional factors. The metabolism for the organisms is important, because the carbon

and nitrogen sources supply the components needed of the synthesis of proteins,

nucleic acids, cell wall material and also the reserve food materials. The fermentation

medium must meet the industrial requirement of the fermenting organisms (Frost and

Moss, 1987). It basically contains sources of carbon, nitrogen and metal ions

(Volesky and Luong, 1985). The basic approach to medium optimization involves

change of only one medium component at a time. Optimization strategies have proven

to be the key factors in developing media that fully to achieve high

productivity, consistent and economical fermentation process (Maiorella et al., 1984).

The availability of nitrogen and carbon sources also influences the production of

secondary metabolites (Aharonowitz, 1980). All organisms require energy and the

carbon source for their growth. As a source of cellular material, carbon form major part

of the dry weight of actinomycetes. The range of carbon compounds that can be utilized

is vast. The production of secondary metabolites is increased by the presence of a non-

preferred carbon source or by phosphate starvation (Aharonowitz, 1980). Type source

and nature of carbon are among the most important factors for any fermentation process

and should be given significant consideration. The medium constituent, which has most

influence on enzyme production by actinomycetes next to carbon, is the nitrogen

source. Nitrogen is among the essential, non-metallic component required by

actinomycetes for the structural and functional purpose of the cell. Enzyme production

by actinomycetes depends on limited nitrogen supply and a definite nitrogen source.

The proportions of these have marked influence in enzyme production in

actinomycetes. Nitrogen metabolism by microorganisms has been recently reviewed by

Payne (1980). Yeast extract as a nitrogen source was found to be most suitable for

incliner production. Media used in cultivation of microorganisms must contain all

elements in a form suitable for the synthesis of cell substances and also for the

production of metabolic products. Thus, type source and nature of carbon, nitrogen,

mineral salts are among the most important factors for any fermentation process.

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Daryoush et al. (2013) optimized media components, cultural conditions

including physicochemical and nutritional parameters for the maximum production of

tyrosinase. Shivaveerakumar et al. (2013) carried out process optimization for the

production of tyrosinase. The enhanced productions of tyrosinase by process

optimization of physical and nutritional parameters were carried out in the study.

Effect of pH, temperature, aeration and effect of nutritional factors which includes the

effect of carbon, nitrogen sources and metal ions were optimized in the study.

3.2.2. Enhanced production of tyrosinase

Enhanced production of any bioactive molecules normally achieved by various

physicochemical, nutritional, molecular, immobilization and mutational regulated

operations. Regulation and operation of physicochemical and nutritional variations for

the maximum production is a basic and classical method to increase the production.

Mutations, genetic engineering and immobilization techniques are effective tools to

enhance the production. Recent advances made in software and bioprocess technology

resulted several highly integrated software based techniques to achieve the maximum

production of end product in any bioprocess. Response Surface Methodology (RSM)

with a suitable statistical design is one of the recent and important techniques preferred

to achieve (Aghaei Kohazani et al., 2012) the enhanced production of enzymes.

This technique gives contour plots from linear, interaction and quadratic effects of two

or more parameters and fits the experimental data to calculate the optimal response of

the system. It has been extensively used to investigate the optimization of

physicochemical parameters and factors of several fermentation media with various

microorganisms (Chang et al., 2002).

Several researchers employed Response surface Methodology (RSM) with

different statistical design for the production of various bioactive molecules or

secondary metabolites.

Martorella et al. (2012) optimized the culture medium composition for

manganese peroxidase and tyrosinase production by yeast employing a nine

factor Plackett Burman experimental design with statistical analysis. A total of

26 experiments including two extra replicates as centric points were carried out to

achieve the maximum production of manganese peroxidase and tyrosinase.

The experimental design was studied and interpreted using MINITAB 16 (PA, USA)

statistical software to estimate the response of the dependent variable. Production of

L-dopa by Aspergillus niger was reported (Ali and Haq, 2010) and Plackett Burman

  61

design was employed to understand the parameter significance analysis aiming at

maximum production. The optimal tyrosinase activity was recorded to be 64.55 U/mg.

Statistical optimization of culture conditions employing RSM for the synthesis of

lipase from Enterobacter aerogenes was reported by Annapurna Kumari et al. (2009).

The influence of temperature, oil concentration, inoculum size, pH and incubation time

were examined and optimization of combined physicochemical variables resulted

1.4 fold (27.25 U/ ml) increase in lipase activity.

Enhanced production refers to the improvement in yield or value of yield

greater than earlier achieved by some strategies being followed. Generally fermentation

yield can be enhanced by suitable fermentor design, media optimization, optimization

of process parameters, recombination (Ren et al., 2013), improvement of strain used in

the study by different strategies like mutation, immobilization (Haq and Ali, 2006).

There are very few reports available on the enhanced production of tyrosinase.

High level production of tyrosinase in recombinant Escherichia coli was reported by

Ren et al. (2013). In this study tyrosinase from Verrucomicrobium spinosum was

expressed in Escherichia coli and its production was studied in both batch and

fed-batch cultivations. A comparative study has been reported with wild type tyrosinase

and mutant in respect of temperature sensitivity by Halaban et al. (2002). In this study

co-expression of wild-type protein with temperature sensitive tyrosinase mutants

corrects the mutant conformation defect in an activity dependent manner was

examined.

3.2.3 Purification of tyrosinase

A schematic illustration of the purification of tyrosinase from edible mushroom

reported by Kamahldin et al. (2004) is as presented in the next page.

He has purified tyrosinase from edible mushroom by following a simple

preparative method employing ammonium sulphate, desalting, ion exchange

chromatography, gel filtration and electrophoresis. Important observations such as total

protein, activity and specific activity of the enzyme along with degree of purification at

different purification stages were also reported.

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Jimbow et al. (1981) studied purification of tyrosinase by affinity

chromatography and reported 100% recovery with tyrosine ethyl ester (TEE) and dopa

as affinity media ingredients. Bernan et al. (1985) purified the intra and extracellular

tyrosinase obtained from Streptomyces antibioticus. The molecular weight of tyrosinase

was estimated to be 72000 +/- 500 daltons to 73000 +/- 800 daltons. The molecular

weight of tyrosinase with single polypeptide chain obtained from Neurospora was

reported (Huber et al., 1988) to be 46,000 daltons. Lerch and Ettlingler (1972) revealed

a molecular weight of 29100 daltons with 286 amino acids residues from Streptomyces

glaucescenes. Jolly et al. (1969) reported 287 residues of amino acids from mushroom

tyrosinase. Aline et al. (1992) purified tyrosinase isolated from Streptomyces

antibioticus in two steps by chromatography. A prominent protein band of about

16 kDa in SDS-PAGE gel of intracellular and extracellular protein was revealed.

Kumar and Flurkey (1991) purified commercial mushroom tyrosinase and monitored

by native IEE and denaturing SDS-PAGE. It was reported that commercial tyrosinase

preparation contains several isoenzymes with varied specifications from the cell extract

of Streptomyces nigrifaciens (Nambudiri and Bhat, 1972) and Streptomyces glaucesens

(Lerch, 1995). Chan-Ki Sung and Sung-Hye (1988) studied the purification and

characterization of tyrosinase from Diospyros kaki thunb by ammonium sulfate

precipitation, sephadex G-25 gel filtration and DEAE-Sephacel Chromatography.

  63

It was observed that the molecular weight of tyrosinase was approximately 25,000

daltons with 227 amino acids residues. Masaaki and Kohei (2000) isolated and purified

organic solvent resistant tyrosinase of Streptomyces sp. REN-21 isolated from the soil

of Sakyo-Ku, Kyoto. The tyrosinase was purified by column chromatography and the

molecular weight was reported to be 32000 daltons as a single polypeptide. Dolakshi et

al. (2009) purified the extracellular tyrosinase of Streptomyces albus by aqueous two-

phase system based on polyethylene glycol PEG 8000 (w/w), potassium phosphate

buffer (pH 7.0) followed by ammonium sulphate precipitation and ultrafiltration.

Thereafter, the crude enzyme was loaded on a SEC Sephacryl S-100 column and

ultrafiltration was carried out to obtain 1.17 mg of purified tyrosinase. Claudia and

Gabriela (2011) reviewed recently the production and application of Streptomyces

tyrosinase.

The first bacterial tyrosinases have been purified from cell extracts of

Streptomyces nigrifaciens (Nambudiri and Bhat, 1972) and Streptomyces glaucescens

(Lerch and Ettlinger, 1972). Unlike most eukaryotic tyrosinases, the active form of the

S. glaucescens protein is a monomer, without tendency of concentration dependent

aggregation as shown by analytical ultracentrifugation. The enzyme has a molecular

mass of 29,100 Da in SDS-PAGE. The extracellular tyrosinase of S. glaucescens was

isolated 10 years later from the culture supernatant (Crameri et al., 1984). Bernan et al.

(1985) purified the intra and extracellular tyrosinase of Streptomyces antibioticus.

The extracellular tyrosinase of Streptomyces michiganensis has been isolated from a

fermentation broth (Philip et al., 1991). The purified enzyme exhibited two bands

corresponding to 32,000 and 34,500 Da in SDS-PAGE, however, only one band at

pH 9.0 was confirmed after isoelectric focusing. The enzyme from Streptomyces

castaneoglobisporus has been efficiently over expressed in Escherichia coli and

his-tagged protein purified on a Ni (II)-bound affinity column (Kohashi et al., 2004).

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3.3. EXPERIMENTAL METHODS

3.3.1. Optimization of submerged bioprocess for the production of tyrosinase

A submerged bioprocess was carried out to optimize various physicochemical

and nutritional variables for the maximum production of tyrosinase.

3.3.1.1. Submerged bioprocess for the production of tyrosinase

A submerged bioprocess (Iyer and Singhal, 2010) was carried out for the

production of extracellular tyrosinase by Streptomyces tuirus DBZ39 in 100 ml tyrosine

broth at laboratory scale. Tyrosine broth was sterilized at 121 0C for 15 min. 1 ml

suspension of five days old test isolate with spore count 1x108 spores/ml was inoculated

into sterilized broth and kept for incubation at 35 0C for 120 h in a shaker incubator at

180 rpm. An enzyme assay, as mentioned earlier under the process of screening was

carried out at every 24 h.

3.3.1.2. Optimization of physicochemical variables

Important physicochemical parameters such as pH, temperature and agitation

speed were optimized for the maximum production of tyrosinase in tyrosine broth

under submerged bioprocess as mentioned earlier. A principle of operating one variable

at a time keeping others constant (Liu and Tzeng, 1998) was followed to record the

optimum conditions. A range of, pH from 7.0 to 9.0 with an increment of 0.5,

temperature from 30 to 50 0C with an increment of 5 0C and agitation speed from

150 to 250 rpm with an increment of 25 rpm were optimized.

3.3.1.3. Optimization of nutritional variables

Major nutritional variables including carbon (Starch, glucose, sucrose, fructose,

cellulose and beef extract, from 0.2 to 1.0 %) and nitrogen (ammonium nitrate, casein,

gelatin, arginine, peptone and tyrosine from 0.2 to 1.0%) sources were optimized by

one variable at-a-time approach and keeping others constant, as mentioned earlier.

Further, various minerals such as, CuSO4, MgSO4, FeSO4, MnSO4, KH2PO4 and

K2HPO4, at the concentration from 0.01 to 0.05% were also optimized. The amount of

tyrosinase produced in the medium was determined at every 24 h by following the

procedure as mentioned earlier.

3.3.2. Response surface methodology for the enhanced production of tyrosinase

Enhanced production of tyrosinase, after the manual process of optimization,

was carried out by following automated statistical optimization under submerged

system using Response Surface Methodology (RSM) with Central Composite Design

(CCD).

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3.3.2.1. Response Surface Methodology with Central Composite Design

Submerged system fermentation was carried out to understand the influence of

critical process variables namely tyrosine, beef extract, gelatin and copper sulphate.

Central Composite Design (CCD) was followed (Annapurna et al., 2009) to resolve the

optimum combination of all the critical process variables. The CCD of 30 runs was set

using the Design Expert Software, USA (Version 7.0). All the experiments were carried

out in duplicate and average of tyrosinase produced at 96 h was considered as the

dependent variables or responses (Y). The predicted response was calculated from the

second degree polynomial equation, which included all the terms. Y= β0+∑ βiXi+∑

βiiXi2+∑ βijXiXj; where Y stands for the response variable, β0 is the intercept

coefficient; βi represents the coefficient of the linear effect, βii the coefficient of

quadratic effect and βij the ijth interaction coefficient effect. XiXj are input variables

which influence the response variable Y, βi is the ith linear coefficient. The statistical

and numerical analysis of the model was performed with the analysis of variance

(ANOVA). The statistical significance of the model was analyzed by the Fisher’s

F-test, its associated probability P(F), correlation coefficient R and determination

coefficient R2, which explains the quality of polynomial model. For each variable, the

quadratic models were represented as contour plots (three-dimensional) and response

surface curves were created and the model was validated.

3.3.2.2. Analysis of variance (ANOVA)

The statistical and numerical analysis of the model was performed by means of

the analysis of variance. The statistical significance of the model was analyzed by

Fisher’s F-test, its associated probability p(F), correlation coefficient R and

determination coefficient R2 which explains the quality of polynomial model.

The quadratic models were represented as contour plots (3D) and response surface

curves were generated for each variable.

3.3.3. Criteria for the purification of tyrosinase

Different criteria, mentioned in brief as follows, were employed based on

standard techniques for the purification of tyrosinase from the fermented broth at 4 0C.

3.3.3.1. Ammonium sulphate precipitation

About 500 ml of culture filtrate was centrifuged at 10,000 rpm for 10 min at

4 0C. Finely powdered ammonium sulphate was added to the clear supernatant.

The tyrosinase activity was associated with the fraction precipitated at 60 - 90%

saturation. This was carried out with continuous stirring in an ice bath and it was kept

  66

at 4 0C for overnight. The precipitate was collected by centrifugation at 10,000 rpm for

10 min. The supernatant was decanted; the precipitate was dissolved in minimum

volume of 10 mM Tris-HCl buffer pH 7.0. Protein estimation was done with Folin-

Phenol reagent using BSA (200 µg/ml) as a standard (Lowry et al., 1951).

3.3.3.2. Dialysis

Activation of membrane (cellulose membrane) was performed as follows.

100 ml distilled water was kept for boiling. Dialysis membrane was placed in water for

10 min. Then 2% sodium carbonate was added and further kept for boiling for 10 min.

Finally the membrane was transferred to fresh water. The precipitate obtained after

ammonium sulphate treatment was dialyzed against 50 mM Tris HCl buffer, pH 7.0

with occasional changes of buffer. Protein estimation was done with Folin-Phenol

reagent using BSA (200 µg/ml) as a standard (Lowry et al., 1951).

3.3.3.3. Ion exchange chromatography

The dialyzed ammonium sulphate fraction was applied to a column of DEAE

cellulose. 2 g of DEAE cellulose was suspended in 10 mM Tris-HCl-NaCl, pH-7.0 and

kept at 4 0C overnight. The column was equilibrated with 0.05 M Tris-HCl, pH 7.0

Swollen DEAE cellulose was loaded into a chromatographic column and allowed to

settle. The sample was loaded to top of the column. The unbound protein was eluted

and the bound protein eluted with linear gradient of NaCl. Each fraction was analyzed

for enzyme activity and protein. The tyrosinase activity was assayed by the molecular

extinction coefficient of dopachrome method as described earlier. Protein estimation

was done with Folin-Phenol reagent using BSA (200 µg/ml) as a standard (Lowry et

al., 1951).

3.3.3.4. Gel filtration chromatography

The concentrated enzyme solution was applied to a Sephadex G-200 column

(39x9 cm), the sample was subjected to gel filtration chromatography using

SEPHADEX G-75 (SIGMA ALDRICH). 2 g of SEPHADEX was suspended in 0.05M

Tri-HCl, pH 7.0 and kept at 4 0C overnight. Before loading, the column was well

equilibrated with 0.05 M Tris-HCl, pH 7.0. Swollen sephadex was loaded into a

chromatographic column and allowed to settle. The 3 ml of the sample was loaded to

top of the column. The enzyme was eluted using 0.05 M Tris HCl, pH 7.0. Flow rate

was adjusted to 0.5 ml/min. Different fraction were collected and each fraction was

assayed for protein by taking absorbance at 280 nm. Protein estimation was done with

Folin-Phenol reagent using BSA (200 µg/ml) as a standard (Lowry et al., 1951).

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3.3.3.5. Electrophoresis

SDS-PAGE was performed according to the modified method of Laemmli

(1970), with a separating acrylamide gel of 10% and stacking gel 5% containing 0.1%

SDS. The gel was stained with coomassie brilliant blue R-250 and destained. Catalase

(240 kDa), Bovine Serum Albumin (67 kDa) and Ovalbumin (43 kDa) were used as

standard proteins for determination of the molecular weight of tyrosinase.

Overall scheme presenting the production and purification of extracellular tyrosinase by Streptomyces tuirus DBZ39

Optimization of submerged bioprocess for the production of tyrosinase by Streptomyces tuirus DBZ39

One variable at-a-time experimental protocol (pH, Temperature, Agitation, Carbon and Nitrogen sources

and Mineral salts)

Enhanced production by Response Surface Methodology (RSM) with Central Composite Design (CCD)

(Tyrosine, Beef extract, Gelatin and Copper sulphate as critical process variables)

Purification of tyrosinase (Ammonium salt precipitation, Dialysis, Ion exchange

chromatography and Gel filtration)

Purified tyrosinase

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3.4. RESULTS AND DISCUSSION

3.4.1. Production of tyrosinase

Efforts were made in the present study for the maximum production of

tyrosinase by an efficient isolate Streptomyces tuirus DBZ39 in tyrosine medium, at

laboratory scale under submerged system. Important physicochemical and nutritional

variables were optimized at different levels and concentrations to achieve the maximum

production of tyrosinase, operating one variable at a time and keeping others constant.

Plate 3.1 reveals tyrosinase produced at 96 h of incubation with dark black color in the

test flask and the control flask indicates no production of tyrosinase.

Plate 3.1: Representative flask showing the production of tyrosinase in tyrosine broth by Streptomyces tuirus DBZ39 at laboratory scale

pH in the range of 7.0 to 9.0 with an increment of 0.5; temperature in the

range of 30 0C to 50 0C with an increment of 5 0C and agitation at the speed of 150 to

250 rpm with an increment of 25 rpm were optimized to observe the maximum

production of tyrosinase. Maximum production of tyrosinase was obtained at pH 8.0

(220.5 IU, Figure 3.1), temperature 40 0C (240.12 IU, Figure 3.2) and the agitation

speed 200 rpm (225.34 IU, Figure 3.3). All the variables reached the maximum

production of tyrosinase at 96 h of incubation.

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Major carbon sources, namely beef extract (Figure 3.4), cellulose (Figure 3.5),

fructose (Figure 3.6), glucose (Figure 3.7), starch (Figure 3.8) and sucrose (Figure 3.9)

were examined at different concentration from 0.2 to 1.0% with an increment of 0.2%,

for the maximum production of tyrosinase, which was recorded at every 24 h till 120 h.

All carbon sources showed the maximum production of tyrosinase at 0.2 % and a linear

decrease in the production of tyrosinase was observed along the increase in the

concentrations. Figure 3.10 gives a comparative account on the effect of all carbon

sources at optimum concentration of 0.2% on the production of tyrosinase. Glucose has

showed maximum production of tyrosinase (260 IU) followed by starch (253 IU) and

Figure 3.1: Optimization of pH for the production of tyrosinase by Streptomyces

tuirus DBZ39 

Figure 3.2: Optimization of temperature for the production of tyrosinase by

Streptomyces tuirus DBZ39

Figure 3.3: Optimization of agitation speed for the production of tyrosinase by

Streptomyces tuirus DBZ39

  70

fructose (230 IU). The least production of tyrosinase (198.0 IU) was recorded with

cellulose as carbon sources. However, beef extract and sucrose showed the moderate

production of tyrosinase with 223 and 210 IU respectively. Important nitrogen sources,

namely ammonium nitrate (Figure 3.11), arginine (Figure 3.12), casein (Figure 3.13),

gelatin (Figure 3.14), peptone (Figure 3.15) and tyrosine (Figure 3.16) were assessed at

different concentrations from 0.2 to 1.0% with an increment of 0.2% for the highest

production of tyrosinase. The production of tyrosinase was recorded at every 24 h upto

120 h. The maximum production of tyrosinase was obtained at 0.2% with all the

nitrogen sources. Figure 3.17 depicts the comparative account on the effect of nitrogen

sources at optimum concentration of 0.2% for the production of tyrosinase. Tyrosine

showed maximum production of tyrosinase (275.1 IU) followed by peptone (255.1 IU)

and ammonium nitrate (262.1 IU). The least production of tyrosinase (209.1 IU) was

recorded with casein as nitrogen source. However, arginine and gelatin showed the

moderate production of tyrosinase with 222.78 and 236.11 IU respectively.

Figure 3.4: Optimization of beef extract for the production of tyrosinase 

Figure 3.5: Optimization of cellulose for the production of tyrosinase

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Figure 3.6: Optimization of fructose for the production of tyrosinase 

Figure 3.7: Optimization of glucose for the production of tyrosinase

Figure 3.8: Optimization of starch for the production of tyrosinase 

Figure 3.9: Optimization of sucrose for the production of tyrosinase 

Figure 3.10: Comparative account on the effect of carbon sources at optimum concentrations on the production of tyrosinase

  72

Figure 3.11: Optimization of ammonium nitrate for the production of tyrosinase

Figure 3.12: Optimization of arginine for the production of tyrosinase 

Figure 3.13: Optimization of casein for the production of tyrosinase

Figure 3.14: Optimization of gelatin for the production of tyrosinase

Figure 3.15: Optimization of peptone for the production of tyrosinase

Figure 3.16: Optimization of tyrosine for the production of tyrosinase

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Figure 3.17: Comparative account on the effect of nitrogen sources at optimum concentrations on the production of tyrosinase

Important mineral salts such as, CuSO4 (Figure 3.18), FeSO4 (Figure 3.19),

MgSO4 (Figure 3.20), MnSO4 (Figure 3.21), K2HPO4 (Figure 3.22) and KH2PO4

(Figure 3.23) were assessed for their influence on the production of tyrosinase, at the

concentration 0.01 to 0.05% with an increment of 0.01%. The tyrosinase produced was

determined at every 24 h upto 120 h. All minerals revealed maximum production of

tyrosinase at 0.01% concentration. A linear increase in the production of tyrosinase was

observed from 48 h to 96 h with all minerals and the maximum production was

recorded at 96 h. Figure 3.24 illustrates a comparative account regarding the production

of tyrosinase by different minerals at optimum concentration. The maximum amount

(286.19 IU) of tyrosinase was produced with CuSO4 followed by FeSO4 (270.98 IU),

MgSO4 (253.86 IU), MnSO4 (237.45 IU), K2HPO4 (223.45 IU) and KH2PO4

(207.45 IU).

  74

Figure 3.18: Optimization of CuSO4 for the production of tyrosinase 

Figure 3.19: Optimization of FeSO4 for the production of tyrosinase 

Figure 3.20: Optimization of MgSO4 for the production of tyrosinase 

Figure 3.21: Optimization of MnSO4 for the production of tyrosinase

Figure 3.22: Optimization of K2HPO4 for the production of tyrosinase

Figure 3.23: Optimization of KH2PO4 for the production of tyrosinase

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Figure 3.24: Comparative account on the effect of mineral sources at optimum concentrations on the production of tyrosinase

Fermentation, a conventional nomenclature being currently also used as

bioprocess is an important stage to be developed for a maximum production of any

bioactive molecule in general and enzymes in particular. Submerged and solid state

fermentations are the two types of major bioprocesses generally will be evaluated for

the production of enzymes by any efficient organisms. Physicochemical conditions,

nutritional ingredients including mineral salts are the most important factors considered

to influence the level of production of enzyme. Efforts were made in the present study

to develop a submerged bioprocess, optimizing physicochemical, nutritional and

mineral components for the maximum production of tyrosinase by Streptomyces tuirus

DBZ39, as mentioned earlier. Several investigation have revealed that the production of

tyrosinase by a microorganisms in a suitable growth medium is regulated by the

composition of the medium, the growth duration, pH, temperature, biosynthetic

inhibitors, the density of tyrosinase producing cells and enzyme inducers

(Katz and Benatcourt, 1988). The physical parameters such as pH, temperature,

agitation and incubation period will play a vital role in the production of bioactive

molecules, especially by actinomycetes (Shatta et al., 1990).Initial pH of th medium is

one of the crucial factors for the successful production of tyrosinase. In most of the

industrial fermentation controlling of pH of the medium at optimum levels is essential

for achieving maximum product formation (Shankaranand and Lonsane, 1984).

Inoculum size plays an important role in enzyme production under any bioprocess.

Low desnsity of spores leads to insufficient biomass and end product synthesis, as well

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as permits the growth of undesirable contaminants and too high densities of spores may

cause a quick and too much biomass production thereby leading to fast nutrient

depletion and ultimately reduction in the end product quality. An increase in inoculum

generally improves the growth and growth activities of the organism upto a certain

level and with further increase, there could be a reduction in microbial activity due to

nutrient limitations. Due to lower inoculum size, a longer time is required for the

organism to grow upto optimum number to utilize the substrate and form the desired

product (Kashyap, 2002). Temperature is also an important factor that governs the

process of fermentation as well as the recovery of desired product. Transformation of

substrate into product is under the influence of temperature for biochemical coversion

of nutritional reactants to products. Influence of higher range of temperature on

actinomycetes for the maximum production of enzymes is well established. Agitation is

also an important physical factor required for adequate mixing, mass and heat transfer,

as it prevents formation of gradients of these factors in the bioreactor. As such efforts

have been made in the present investigation to understand the influence of all these

factors on the maximum production of tyrosinase. pH 8.0, temperature 40 0C and

agitation speed 200 rpm were found to be optimum for the maximum production

(225.34 IU) of tyrosinase at 96 h of incubation by Streptomyces tuirus DBZ39.

These observations are in confirmation with the findings of several researchers

(Shivaveerakumar et al., 2013; Katz and Benatcourt, 1988).

In expanding the industrial exploitation of microbes, it is likely that more attention

will be directed towards nutritional parameters. The nutritional parameters are the ones that

significantly affect the enzyme production. In the interests of profitability, it is essential to

supply necessary nutrients to ensure optimal growth of an organism, or to maximize

product yield at minimum cost. This can only be achieved by an understanding of what

microorganisms require as nutrients and in some instances, what they don’t (Aharonowitz,

1980). All organisms require energy and the carbon source for their growth. As a source of

cellular material, carbon form major part of the dry weight of actinomycetes. The range of

carbon compounds that can be utilized is vast. The versatility of the microbial world is

illustrated by the fact that all biological synthesized carbon components are biodegradable.

The source and availability of nitrogen and carbon sources also influence the production of

secondary metabolites (Aharonowitz, 1980). The main product of the fermentation process

will often determine the choice of carbon source, particularly if the product results from the

direct dissimilation of it. Most industrial microbes utilize the commonest form of energy in

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the form of carbon sources such as carbohydrate, lipids and proteins. The rate at which the

carbon source is metabolized often influences the formation of biomass or production of

primary or secondary metabolites (Stanbury et al., 1995). Among all carbon sources,

glucose (0.2%) was found to be most influencing giving maximum production (260 IU) of

tyrosinase at 96 h of incubation. Next to carbon, nitrogen is the source which has most

influence on enzyme production. Nitrogen is among the essential and non metallic

components required by actinomycetes for the structural and functional purpose of the cell.

The requirement of nitrogen source mainly depends on the strain and the substrate

employed during the bioprocess (Pintado et al., 1988). Various nitrogen sources were

examined for the maximum production of tyrosinase in the present investigation. Tyrosine

(0.2%) was proved to be most influencing for the maximum production (275.1 IU) of

tyrosinase. Mineral salts or trace elements have profound effect on the growth and

physiological activities of microorganisms. These much be added as distinct components to

the medium, as they are essential for cell mass formation and also acts as cofactor for

several biosynthetic enzymes. Various mineral salts were examined in the present study to

achieve the maximum production of tyrosinase. Copper sulphate (0.01%) has given the

maximum production (286.19 IU) of tyrosinase at 96 h of incubation.

Several researchers optimized various physicochemical, nutritional and mineral

salts for the production of tyrosinases by bacteria (Liu et al., 2002; Greta et al., 2012)

and fungi (Halaouli et al., 2005; Krishnaveni et al., 2009). However, very few

researchers optimized these conditions for the production of tyrosinase by

Streptomyces (Huber et al., 1985; Claudia and Gabriela, 2011; Shivaveerkumar et al.,

2013; Patil et al., 2012). Recently, Shivaveerkumar et al. (2013) and Patil et al. (2012)

have reported 285 IU and 289 U/mL activities of tyrosinase by different Streptomyces

sp. respectively. In the present investigation, highest production of tyrosinase (286.19

IU) by Streptomyces tuirus DBZ39, at all optimized conditions was reported.

3.4.2. Enhanced production of tyrosinase

An attempt was made to achieve the enhanced production of tyrosinase, after the

optimization of all important physicochemical and nutritional variables. Combined effect of

critical process variables namely tyrosine, beef extract, gelatin and CuSO4 as X1:A, X2:B,

X3:C and X1:D respectively were evaluated (Table 3.1) by employing Response Surface

Methodology (RSM) with Central Composite Design (CCD). In all, 30 runs were operated

and 16th run showed highest production of tyrosinase with 369.41 IU as actual value against

predicted value of 366.22 IU. Critical process variables at the concentration of tyrosine

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0.25%, beef extract 0.25%, gelatin 1% and CuSO4 0.01% found effective in combination for

the enhanced production of tyrosinase. The statistical significance of the model operated for

the enhanced production of tyrosinase was analyzed by Fisher’s F-test, associated probability

and co-efficient of correlation. The important significant values by numerical analysis

(ANOVA) of the model are as presented in Table 3.2. Characteristic response of the model

showed a high co efficient R2 value of 0.970 F-value of 35.72 and lack of fit value 43.38 was

found significant at 0.01%. The probable > F value was less than 0.0500 suggesting the

model terms as significant. Three dimensional contour plots with six response surfaces

including all the possible combinations of critical process variables, as mentioned earlier are

illustrated in Figure 3.25. All the six response surfaces with combination of critical variables

visualize enhanced production of tyrosinase with 369.41 IU. Table 3.1: Combined effect of critical process variables for the production of tyrosinase

Critical process variables Tyrosinase (IU) Run

X1:A:

Tyrosine %

X2:B: Beef

Extract %

X3: C: Gelatin

%

X4: D: CuSO4

% Actual Value

Predicted Value

1 0.25 1.00 0.25 0.01 221.62 253.64 2 0.63 1.38 0.63 0.03 242.42 239.58 3 0.63 0.63 0.63 0.03 369.22 366.22 4 0.25 1.00 1.00 0.01 206.65 196.32 5 0.25 1.00 1.00 0.05 209.93 210.64 6 1.00 0.25 1.00 0.01 336.92 321.41 7 1.00 1.00 1.00 0.01 266.31 264.22 8 0.25 0.25 0.25 0.05 196.92 218.16 9 1.00 0.25 0.25 0.05 269.92 269.44

10 0.63 0.63 0.63 0.03 265.92 366.22 11 1.00 1.00 0.25 0.05 251.21 255.27 12 1.00 1.00 0.25 0.01 336.92 321.97 13 1.38 0.63 0.63 0.03 169.92 185.90 14 0.63 -0.13 0.63 0.03 208.62 279.06 15 0.63 0.63 0.63 0.03 280.62 279.06 16 0.25 0.25 1.00 0.01 369.41 366.22 17 0.63 0.63 0.63 0.03 361.92 366.22 18 0.63 0.63 0.63 0.03 367.71 366.22 19 0.63 0.63 0.63 0.03 363.12 366.22 20 0.63 0.63 -0.13 0.03 307.21 288.96 21 0.25 0.25 1.00 0.05 280.14 281.14 22 -0.13 0.63 0.63 0.03 91.16 70.78 23 1.00 0.25 1.00 0.05 341.60 328.73 24 0.25 1.00 0.25 0.05 235.12 235.87 25 1.00 0.25 0.25 0.01 271.71 290.15 26 0.63 0.63 0.63 -0.01 349.92 348.85 27 0.63 0.63 1.38 0.03 281.13 294.99 28 0.63 0.63 0.63 0.07 341.17 338.39 29 0.25 0.25 0.25 0.01 210.13 194.74 30 1.00 1.00 1.00 0.05 229.71 230.35

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Table 3.2: Analysis of variance of model response data

Source Sum of Squares Df Mean

Square F Value p-value Prob > F

Model 1.451E+005 14 10363.95 35.72 <0.0001

A-Tyrosine 19877.77 1 19877.77 68.51 <0.0001

B-Beef Extract 2338.01 1 2338.01 8.06 0.0124

C-Gelatin 54.48 1 54.48 0.19 0.6709

D-CuSO4 164.01 1 164.01 0.57 0.4638

AB 777.29 1 777.29 2.68 0.1225

AC 0.096 1 0.096 3.312E-004 0.9857

AD 2316.50 1 2316.50 7.98 0.0128

BC 7781.89 1 7781.89 26.82 0.0001

BD 1696.20 1 1696.20 5.85 0.0288

CD 1025.60 1 1025.60 3.53 0.0797

A2 97002.03 1 97002.03 334.34 <0.0001

B2 19588.36 1 19588.36 67.52 <0.0001

C2 9449.69 1 9449.69 32.57 <0.0001

D2 875.59 1 875.59 3.02 0.1028

Residual 4351.98 15 290.13

Lack of Fit 4305.40 10 430.24 43.38 0.0003

Pure Error 49.59 5 9.92

Cor Total 1.494E+005 29

R2=0.9709

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Figure 3.25: Contour plots and response surface curves of the production of tyrosinase

Enhance production of any bioactive molecule, even after optimization of

various process variables is utmost important industrial perspective. They are several

criteria based on physicochemical, biological engineering and software modules for

enhance production of enzymes. Immobilization techniques (Devendra Kumar et al.,

2012), mutation methods (Haq and Ali, 2006), recombinant DNA technology (Ren et

al., 2013) and certain statistical designs (Burkert et al., 2004; Ali and Haq, 2010) of

experiments are the leading criteria based on which the enhance production of enzymes

  81

was achieved by several researchers. To the best of our knowledge, no reports are

available on the enhanced production of tyrosinase by actinomycetes in general and

Streptomyces in particular. Shivaveerkumar et al. (2013) have carried out

immobilization technique as an important means for the enhanced production of

tyrosinase with 310.5 IU by Streptomyces sp. DSV5. Response Surface Methodology is

important software based criteria with a particular experimental design being recently

attracted by various researchers (Annapurna et al., 2009; Chang et al., 2002; Chien et

al., 2006; Madhusudhan et al., 2014) for the production of different bioactive

molecules. In the present investigation response surface methodology with central

composite design was operated effectively to achieve successfully the enhance

production of tyrosinase with 369.41 IU. About 30% increase in the yield of tyrosinase

was achieved when compared to the earlier production at all optimized process

conditions. Tyrosine, gelatin, beef extract and copper sulphate were found to be critical

process variables, which lead in combination to give enhanced yield of tyrosinase.

In addition to the routine all physicochemical and nutritional variables, only few would

act as critical process variables. The critical process variables vary from one organism

to another because of their varied metabolic and physiological nature. Copper sulphate

found to be much more critical process variable for the production of tyrosinase, as

such tyrosinase constitutes copper as an important cluster.

3.4.3. Profile of purified tyrosinase

Table 3.3 illustrates profile of purified tyrosinase. The crude fraction of

tyrosinase gives 369.41 IU/mL activity with a protein content of 15.23 mg/mL and

specific activity of 24.25 IU/mg. The fraction collected after precipitation showed an

enzyme activity of 314.23 IU/mL with a protein content of 9.16 mg/mL and specific

activity of 34.30 IU/mg. Further, after dialysis or desalting the enzyme activity of the

fraction was 242.19 IU/mL a protein content of 1.67 mg/mL and specific activity of

145.02 IU/mg. The fraction collected after ion exchange chromatography and gel

filtration methods, showed enzyme activities of 106.12 IU/mL and 160.60 IU/mL

with a protein content of 0.52 mg/mL and 0.26 mg/mL and specific activity of

352.83 IU/mg and 617.71 IU/mg respectively. Fraction obtained at every stage of

purification exhibit an increase in the degree of purification or fold purification and

naturally a sharp decline in the % yield of enzyme. Ion exchange chromatography and

gel filtration have showed 14.54 and 25.47 fold purification of enzyme respectively.

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The fraction obtained after ion exchange chromatography containing protein

molecules and impurities were subjected for gel filtration chromatography on sephadex

G-100 column. The fraction 24 showed the highest peak indicating maximum activity

of tyrosinase as shown in the Figure 3.26. The purity of enzyme was examined by

SDS-PAGE analysis. The band obtained (Plate 3.2) after separation through

SDS-PAGE reveals the molecular weight of about 140 kDa.

Table 3.3: Purification profile of tyrosinase produced by Streptomyces tuirus DBZ39

Purification stage Volume (ml)

Enzyme activity (IU/ml)

Protein (mg/ml)

Specific activity (IU/mg)

Fold purifi cation

% yield

Crude fraction 100 369.41 15.23 24.25 1 100

Ammonium sulphate precipitation

20 314.23 9.16 34.30 1.41 17.01

Dialysis/desalting 6 242.19 1.67 145.02 5.98 3.93

Ion exchange chromatography

4 186.12 0.52 352.83 14.54 2.01

Gel filtration /permeation 2 160.60 0.26 617.71 25.47 0.86

Figure 3.26: Elution profile of tyrosinase by gel filtration chromatography

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Plate 3.2: SDS-PAGE profile of purified tyrosinase

Purification means the act of cleaning by getting rid of impurities.

Product recovery during any bioprocess is difficult since the diluted and liable products

of interest are always mixed with macromolecules of similar properties. Based on the

production technique, downstream process can be designed. It is usually achieved by

filtration, centrifugation and precipitation and also by chromatographic techniques such

as ion exchange, gel permeation and affinity chromatography. To confirm purity of the

enzyme and to determine the molecular weight, electrophoresis is performed using

denaturing Poly Acrylamide Gel Electrophoresis (Rath et al., 2009). Enzymes are

nature’s catalysts and their ability to catalyze biochemical reactions under mild

conditions in a highly specific and efficient manner has lead to the inherent in their

exploitation as industrial catalysts. Nonetheless, industrial application is often

hampered by a lack of long-term operational stability and difficult recovery and re-use

of the enzyme. These drawbacks can often be overcome by immobilization of the

enzyme (Sheldon, 2007). In the present study, tyrosinase was purified by following

standard protocols.

A greater variation was recorded in the profile tyrosinase at different stages.

Ion exchange chromatography and gel filtration stages revealed a fine purification with

higher fold. However, fraction 24 of gel filtration showed 25.47 fold purification of

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tyrosinase. The purity of tyrosinase was confirmed by SDS-PAGE and it denotes the

molecular weight of about 140 kDa. Purified tyrosinases from different sources were

reported by several researchers (Burnett, 1971; Masaaki and Kohei, 2000; Kamahldin

et al., 2004). It is clear from the available literature that degree of purification, protein

content, enzyme activity and specific are highly varied referred to be based on the

number of amino acid residues with varied number of copper clusters. However,

indepth studies are essential to understand further attributes of purified tyrosinase.

3.5. CONCLUSIONS

Important physicochemical conditions, nutritional variables and minerals salts

were optimized, manually one at a time and keeping others constant, under submerged

bioprocess for the maximum production of tyrosinase. Streptomyces tuirus DBZ39 has

produced 286.19 IU tyrosinase in tyrosine medium at all optimized process variables.

Tyrosine, beef extract, gelatin and copper sulphate were considered as critical process

variables for the enhanced production of tyrosinase employing Response Surface

Methodology with Central Composite Design. Streptomyces tuirus DBZ39 has

produced an enhanced amount of tyrosinase with actual value of 369.41 IU against

predicted value of 366.22 IU, at 16th run. Thus produced tyrosinase showed a molecular

weight of 140 kDa on its purification based SDS-PAGE.

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CHAPTER 4

SYNTHESIS AND CHARACTERIZATION OF GOLD NANOPARTICLES

4.1. PREFACE

There is a tremendous excitement in microbial synthesis of metallic

nanoparticles. Nanoparticles are referred to particle whose dimensions are controlled in

the order of 100 nm or less and it is one billionth of a meter (10-9). These nanoparticles

are attracting significant attention because of their unique structures and exotic

properties, those can be systematically manipulated by adjusting the size, composition

and shape (Jain et al., 2006). The development of techniques for the controlled

synthesis of nanoparticles of well defined size and shape is a big challenge and

numerous chemical methods for the production of metal nanoparticles are not regarded

as being environmental friendly (Gamez et al., 2002). As a result, researchers in the

field of nanoparticles synthesis have turned to biological systems for inspiration.

Most of the biological sources are known to synthesize nanoparticles. Among

microorganisms, actinomycetes, due to their diverse metabolic and physiological

nature, are most attracted recently for the synthesis of extracellular metallic

nanoparticles.

Bioreduction of metal nanoparticles is regarded as an organism’s survival

mechanism against toxic metal ions (Murali Sastry et al., 2003). During the synthesis of

gold nanoparticles, Au+ ions are trapped on the surface of cells by electrostatic

interaction between Au+ and negatively charged carboxylate group present in NADH-

dependent reductase enzyme of the cell wall leading to the synthesis of gold nuclei at

nanoscale (Daniel et al., 2004). Several advanced physicochemical techniques namely

UV-vis absorption, Electron Microscopy, Energy Dispersive X-ray analysis, Fourier

Transmission Infrared analysis and X-ray Diffraction analysis are commonly being

used to confirm and characterize the nanoparticles.

Extracellular synthesis of gold nanoparticles by Streptomyces tuirus DBZ39 in

optimized process conditions and the characterization of gold nanoparticles by various

analytical techniques are presented in this chapter.