production and characterization of melanin pigment...

International Journal of Pharma Research & Review, August 2013; 2(8):9-17

M Kalaiselvam et.al, IJPRR 2013; 2(8) 9

Research Article

Production and Characterization of Melanin Pigment from Halophilic Black Yeast Hortaea werneckii

M. Helan Soundra Rani, T. Ramesh, J. Subramanian, *M. Kalaiselvam

CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai-608502, Tamilnadu, India. _______________________________________________________________________________________________________________________________________________________

ABSTRACT Melanin is nearly a ubiquitous pigment synthesized by living organisms in the course of hydroxylation and polymerization of organic compounds. Melanins have immense application potentials in the field of agriculture, cosmetics and pharmaceutical industries (photoprotection and mosquitocidal activity isolated from Streptomycete). In this study, the halophilic black yeast, Hortaea werneckii produced a diffusible dark pigment on potato dextrose agar. This work was designed to study the ability of the yeast for the production, characterization and optimization of the melanin pigment with different nutrient sources. Characterization of melanin was analyzed by UV spectroscopy, FT IR, SEM and antibacterial potential of melanin was also studied. The favorable condition for the high yield of melanin was found to be glucose as carbon source and peptone as nitrogen source with the optimum parameters like temperature 30ºC, salinity 15‰, pH 7.0, incubation period of 168 hrs and the rice bran as a cheaper substrate, which produced 5.60g/L of melanin. It also showed inhibitory activity against potential pathogens and activity was observed in Salmonella typhi (17mm) and Vibrio parahaemolyticus (15mm). It was concluded that the melanin of Hortaea werneckii isolated from solar salterns possess a high antibacterial activity and could act as a suitable source of new antimicrobial natural products. Keywords: Halophilic fungi, black yeast, Hortaea werneckii, melanin, antibacterial activity. Received 04 June 2013 Received in revised form 27 June 2013 Accepted 29 June 2013 *Address for correspondence: M. Kalaiselvam CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai-608502, Tamilnadu, India. E-mail: [email protected] _______________________________________________________________________________________________________________________________________________________

INTRODUCTION Melanin is a well-known and universal pigment in living organisms. It brings many benefits to human beings and especially plays a key role to protect internal tissues from the harmful effects of ultraviolet rays [1- 4]. The most common form of biological melanin is a complex polymer of either or both of 5, 6-indolequinone and 5, 6- dihydroxyindole carboxylic acid. Melanin pigments are negatively charged hydrophobic [5], and high-molecular-weight compounds. It is used commercially as a component of photo productive creams for anti-melanoma therapy and also reported to possess immuno-pharmacological properties [6]. Recent studies have shown that melanins are highly immunogenic [7] and have anti-inflammatory properties [8, 9]. It has been

shown to protect micro-organisms against UV-radiation [10-12], microbial lysis [13-16], oxidants killing by alveolar macrophages [17, 18] and defense responses of host plants and animals against fungal infection [19-21]. Melanin synthesis in fungi is associated with virulence in a number of human pathogenic fungi, such as Cryptococcus neoformans [22-24] and Sporothrix schenckii [25, 26]. Hortaea werneckii, melanized halophilic black yeast like fungus capable of causing Tinea nigra in humans [27]. This fungus has been reported from several environments around the world and it has shown a preference to hypersaline habitats but not used for the melanin production. They confer certain advantages to fungi such as increasing their survival potential in some



environments and enhancing their virulence [28]. The strain had the highest frequency of isolation and grew at the maximum NaCl concentration (25%) tested (optimum at 10%) and had an optimal growth at 29°C [29]. Cultures of Hortaea werneckii produced more hyphae and became more melanized at 30°C. Ultra structural studies of Hortaea werneckii, cell walls showed the organization of melanin granules is dependent on the concentration of salt in the medium [30, 31]. The membrane fluidity decreased with the rise in NaCl concentration, while in halophilic yeast, such as Hortaea werneckii the fluidity is maintained over increasing salt levels and reduces the porosity. Several types of melanin have been described in bacteria, fungi and animals; eumelanins, phaeomelanins, allomelanins and pyomela-nins. Eumelanins are formed from Quinines and free radicals. Fungal melanins are complex pigments which are produced by two different synthetic pathways, known as the DHN (1,8-dihydroxynaphthalene) and L-DOPA (L-3,4-dihydroxyphenyl-alanine) pathways, depending on the species [32]. The DHN pathway of melanin biosynthesis is very common in the fungi kingdom [33]. When compared to terrestrial fungi the marine fungi are expected to produce novel and potentially active metabolites and also the scarcity of reports regarding melanin from marine habitats especially hypersaline environments leads us to undertake the study. MATERIALS AND METHODS Isolation and Identification: Solar salterns water samples were collected from Marakkanam near Pondicherry, India. Samples were capped and brought to the laboratory. The samples were serially diluted (up to 10-6 dilutions) by adding 1ml of water sample in 9ml of distilled water. About 1.0ml of diluted samples were plated on potato dextrose agar (1M NaCl concentration) media by pour plate technique and added 150mg l-1 chloramphenicol to avoid bacterial contamination by then kept in light chamber at 30ºC about 7-15 days. The isolated fungal strains were identified by standard mycological manual [34].

Extraction and purification of melanin: Due to the black appearance of the colony, the yeast was subjected for melanin extraction. The method of Gadd [35] was used for extracting the melanin pigment. Disc (15mm diam) were cut from 15 days old colonies, boiled for 5min in 5ml distilled water and centrifuged. The pigment was extracted by autoclaving with 3ml of 1M NaOH (20mins, 120ºC). The alkaline pigment extract was acidified to pH 2 with concentrated HCl to precipitate the melanin. The precipitate was washed 3 times in distilled water and dried overnight at 20ºC in a dehumidified atmosphere before further analysis. UV and Fourier- Transform infrared (FT IR) Spectrophotometer: The pigment was subjected in 3 ml of sodium borate buffer (pH 8.0) and its UV-visible spectrum was measured in Perkin Elmer. For the IR investigation, the purified pigment was ground with infra red quality KBr (1:10), pressed into discs under vacuum using SHIMADZO spectrophotometer [36]. The spectral width was 4000-5000cm-1 and the spectral resolution was recorded. Optimization of melanin: To optimize the culture conditions for melanin production, the strain was inoculated at different conditions such as temperatures (25, 30, 35, 40 and 45ºC), salinity (10‰, 15‰ and 20‰), pH range (6, 7, 8 and 9) and incubation periods (24, 48, 96, 120, 144, 168 and 192 hrs) and also tested various carbon sources (dextrose, fructose, sucrose and glucose), nitrogen sources (yeast extract, peptone and corn steep) and cheaper substrates (rice bran, wheat bran and coconut cake). The inoculums were maintained in rotatory shaker. All the experiments were carried out in 1000ml conical flasks containing 100ml of production medium. Antibacterial activity of melanin pigment: Antibacterial activity was tested by well diffusion method. Pathogens like Salmonella typhi, Klebsiella pneumoniae and Vibrio parahaemolyticus were swabbed on a Muller- Hinton agar and 10µl of pigment extract was placed in the well and incubated at 37ºC for 24hrs.



Scanning Electron Microscopy: The melanin sample were fixed in 2% (v/v) glutaraldehyde (pH 7.4) prepared on a phosphate buffer and cell fixation and initial contrasting was performed with 2% water solution of OsO4 for 2hrs. After washing in distilled water, dehydration was carried out in a series of ethanol solutions and twice in acetone. The samples were dried in the grid and the samples were observed under a Scanning Electron Microscope.

RESULTS In this study, hypomycetes black yeast Hortaea werneckii was chosen for melanin study (Fig. 1a, 1b). UV-Vis Absorption spectra: The nature of the pigment was confirmed by spectral properties. Its UV spectrum was typical of the absorption of melanin. Observation of melanin in the UV absorption spectrum exhibited absorption peak of maxima at 235nm-300nm which was shown in the (Figure 2).

Figure 2: Absorption spectrum (UV) of melanin pigment by black yeast H. werneckii

Fig. 1a: Microscopy observation of Hortaea werneckii.

Fig. 1b: Colony morphology of black yeast

Fourier Transform infrared (FT IR) Spectrophotometer: The IR spectrum of the pigment was also characterized as fungal melanin (Fig.3). The chemical properties of the resultant dark pigment were determined including its elemental composition with infrared (IR) spectrum. In this study, IR spectrum of melanin shows peaks near 3,435cm-1 was ascribed to the amino second group (NH). The peak at 2,931cm-1 was assigned to the C-H stretch bond related to methane group. The peak at 1,633cm-1 was the amino group with NH2 stretching. The peak at 1,267cm-1 relates to the anhydride group (C-O) in synthetic melanin and all extracted fungal pigment. This may be a strain related phenomenon, or C/N ratio might have resulted from impurities which were difficult to remove from melanin. Based on this finding, we suspect that the resulted pigment may be Eumelanin not any other types which lacks nitrogen.



Fig 3: Infra-red spectrum (FT-IR) of melanin pigment of Black yeast Hortaea werneckii

Optimization of melanin: The optimized conditions for melanin production were analyzed at different parameters and high quantity of melanin was produced in the presence of glucose (Fig.4), peptone (Fig.5), rice bran (Fig.6) in

15‰ salinity (Fig.7), at 30ºC (Fig.8), pH range 7.0 (Fig.9) and incubation periods (Fig.10). 5.60g/L of crude melanin was extracted under optimum condition.

Fig 4: Effects of different carbon sources Fig 5: Effects of different nitrogen melanin production sources on melanin production

Fig 6: Effects of different cheaper sources Fig 7: Effects of different salinity on

melanin production

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Fig 8: Effects of different temperatures Fig 9: Effects of different pH on on melanin production melanin production

Fig 10: Effects of different incubation periods on melanin production

Antibacterial activity of melanin pigment: The melanin pigment had antibacterial activity against all tested pathogens and the maximum zone of clearance against

Salmonella typhi (17mm), Vibrio parahaemolyticus (15mm) and Klebsiella pneumonia (11mm) were observed which was shown in the Fig. 11.

Fig 11: Antibacterial activity of Melanin pigment against bacterial pathogens Scanning Electron Microscopy: The suspensions of black particulate material at a magnification of 3,000x revealed structures resembling yeast cells

and 1,000x of SEM photograph shown the particulate materials which was shown in the Figure12.

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Fig 12: SEM magnification of Melanin particles in 3000x and 1000x

DISCUSSION In this study, the black yeast Hortaea werneckii produced 5.60g/L of melanin pigment were synthesized at optimized conditions. Glucose was observed as best carbon source for the melanin production when compared to other sources. Similarly, the same results were reported as glucose, the best carbon source for yielding high quantity (7.22g/L) of melanin by Yarrowia lipolytica [37]. Starch was the effective carbon sources for Streptomyces sp. followed by glycerol and fructose [38, 39]. In case of nitrogen source, peptone results in hyper secretion of pigment when compared to yeast extract and corn steep, which is accompanied by the previous reports (40). However, it was feasible to combine yeast extract and peptone into a single medium to increase production and the pigment production [40]. When compared to other substrates rice bran showed more effect on the production and extracted high quantity of pigment. The maximum pigment production was observed at 30ºC, pH 7.0 and salinity of 15‰, where the optimum pH and temperature was found to be pH 7.0 at 32ºC. The maximum amount (20.76g/L) of pigments synthesized by the fungus Aspergillus carbonicus at 15th-25th day’s incubation period [41]. In this study, 5.60g/L of crude melanin was extracted at 6th day. Halophilic black yeast potentially produced active secondary metabolites which possess

high antibacterial activity. Melanin pigment showed significant antibacterial activity against three pathogenic organisms. From the results, it is evident that the melanin extract showed strong antibacterial activity against Salmonella typhi (17mm), Vibrio parahaemolyticus (15mm) and Klebsiella pneumonia (11mm). The purified pigment showed 20mm zone of clearance against Escherichia coli. Similarly, the same results were reported and the zone of inhibition against the Escherichia coli was 20mm [42]. Melanin extracts showed the strong activity against Escherichia coli (18mm), Lactobacillus vulgaris (19mm), followed by Staphylococcus aureus (18mm), Proteus mirabilis (15mm), Vibrio cholera (17mm), Salmonella typhi (12mm), Salmonella paratyphi (11mm) and Klebsiella oxytoca (9mm) [43]. In this study, the absorption spectrum of black yeast showed the absorption maxima at 235-300nm which were highly correlates with the absorption spectrum of Aspergillus carbonicus. It exhibited absorption range at 280-310nm which may be the reason of high intensity light absorption is typical of dark color pigment. It was absorbed strongly in the UV region and progressively less as the wavelength increased. This is due to the presence of much complex conjugated structure in the melanin molecule [44]. The similar reason that all spectra showed a strong UV absorption in the 200-300nm region that can be attributed to the of



the amino, carboxylic and aromatic moieties. The presence of oxygen containing groups may contribute to the dark color of pigments [45]. Likewise, IR spectrum of melanin from black yeast shows peaks near 3,435cm-1 was ascribed to the amino second group (NH). The peak at 2,931cm-1 was assigned to the C-H stretch bond related to methane group. The peak at 1,633cm-1 was the amino group with NH2 stretching. The peak at 1,267cm-1 relates to the anhydride group (C-O) in synthetic melanin and all extracted microbial pigment, and the similar results were found with the peaks near 3,381cm-1 was ascribed to the OH bond [46]. The peak at 2,925cm-1 was assigned to the C-H stretch bond related to methane group. The peak at 1,633cm-1 was the amino group with NH2 stretching and the melanin bright spectral absorption lines of 2,925cm-1 to 2,938cm-1 peaks related to hydroxyl group (OH) peak at 3,344 to 3,436cm-1 relates to the amino second group (NH) and peaks at 1,243cm-1 to 1,305cm-1 relates to the anhydride group (C-O) in synthetic melanin and all extracted microbial pigment. Also, there were 2,925cm-1 to 2,938cm-1 peaks relating to the methane group (CH) 1,628cm-1 to 1,651cm-1 peaks relating to the amino group (NH) in the pigment extracted from Kluyveromyces marxianus and Streptomyces chibaensis [47]. Oxidation of OH group to carboxyl group enhances the color intensity because of the appearance of C-O double bonds. This provides an explanation for the correlation between extinction coefficients and contents of COOH groups in melanin. Thus the melanin with a high content of COOH groups is characterized by the greatest value of the extinction coefficient and this is due to the presence of many complex conjugated structures in the melanin molecule. CONCLUSION It is inferred from this study, that the Hortaea werneckii strain have the potential for melanin production. Synthesized melanin showed the promising antibacterial activity against life threatening bacterial pathogens of Vibrio parahaemolyticus, Klebsiella pneumoniae and Salmonella typhi. Rice bran acts as the cheapest source for increased production of

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M. Kalaiselvam et al., J. Sci. Res. Phar. 2013, 2(1), 8-11

Journal of Scientific Research in Pharmacy 2013, 2(1) 8-11

Journal of Scientific Research in Pharmacy Research Article Available online through ISSN: 2277-9469

www.jsrponline.com

Studies on Pectinase production from halotolerant Aspergillus terreus by Submerged Fermentation

M. Helan Soundra Rani, A. Elavarasi and M. Kalaiselvam* CAS in Marine Biology, Facul ty of Marine Sciences, Annamalai University, Parangipettai -608 502, Tamilnadu, India.

Received on: 07-02-2013; Revised and Accepted on: 20-02-2013

ABSTRACT

Pectinase enzymes which catalyzing the degradation of pectic subs tances are one of the most important enzyme for industry. They contribu te

approximately 25% in the global sales of food enzymes. The aim of the work was to evaluate the potentials of fungi to produce pectinase Banana peels as

substrate. In this study, among the four fungi Aspergillus terreus showed the pecti nase enzyme production. Production of the enzymes and characterized the

enzyme produced by halophilic fungi Aspergillus terreus isolated from solar saltern of Marakkanam, Pondicherry, India. Optimization of temperature, pH,

salinity, incubation period and different cheaper subs trates were also determined. High pectinase production was observed at 72hrs incubation at 30°C and

the pH of 6. In case of cheaper substrates, maximum enzyme production was observed in banana peel waste (206 U/ml). Purified pectinase showed a

molecular weight of 37 kDa observed by means of SDS-PAGE.

Keywords: Pectinase, Solar saltern, Aspergillus terreus, Banana peel waste.

INTRODUCTION

Hyper saline environments provide excellent sources for the various microbial primary and secondary metabolites especially enzymes, that highly stable in many stress like unbalanced pH, temperature and salinity range. However, only a limited number of enzymes have been well characterized and only a few of them are exploited commercially, mainly because research has largely focused on microbial diversity in hypersaline environments. Pectinases or petinolytic enzymes hydrolyze pectic substances which are widely distributed enzymes in bacteria, fungi and plants. Protopectinases, polygalacturonases, lyases and pectin esterases are extensively studied pectinolytic enzymes [34]. Pectinases are group of enzymes that attack pectin and depolymerize it by hydrolysis and trans-elimination as well as by de-esterification reactions, which hydrolyses the ester bond between carboxyl and methyl groups of pectin [10]. Pectinolytic enzymes catalyzing the degradation of pectic substances are of great industrial importance [3]. Pectinases have extensive applications in fruit juice industries in order to improve fruit juice yield and clarity [2]. Pectinase treatment accelerates tea fermentation and also destroys the foam forming property of instant tea powders by destroying the pectins [8]. Pectinases are used broadly in the paper and pulp industries in many countries [5, 21]. During paper making, pectinase can depolymerize the polymers of galacturonic acids, and subsequently lower the cationic demand of pectin solutions and the filtrate from peroxide bleaching [35,

42]. Pectinase producing bacteria and fungi for its pectinolytic a ctivity using fruit wastes were also determined [13].

Many microbes have been reported in ability of pectinase production, Bacillus sp. [29], Aspergillus fumigatus [32], Aspergillus sojae [39], Aspergillus clavatus, Fusarium sp, Penicillum chrysogenum, and Trichoderma sp [28, 7]. Aspergillus sp is one of fungi group that often identified as source microbe in pectinase activity . Amongst these, the filamentous fungi are most commonly employed. Production of pectin enzymes by fungi such as Alternaria sp, Clados porium, Colletotrichum, Mucor, Penicillium, and Trichoderma was confirmed [16, 19]. Many filamentous fungi like Aspergillus niger, Aspergillus awamori, Penicillium restrictum, Trichoderma viride, Mucor piriformis and Yarrowia lipolytica are used in both submerged as well as solid state fermentation for production of various industrially important products such as citric a cid,

*Corresponding author: M. Kalaiselvam CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai-608 502, Tamilnadu, India. Mobile: +91 9894445103 Fax: 04144-243999. *E-Mail: [email protected]

ethanol and recorded maximum pectin esterase and pectic lyase activity etc. Aspergillus terreus has been employed and gave the best results in enzyme production. The compounds isolated from A. terreus mostly possess pharmacologi cal and commercial values. Lovasta tin (an ti-hyperlipidaemic drug), terreulactones A, territrem A and their derivatives likely Terreusinone (UV-A absorbing activity) and also mycotoxins such as citreovividin and terretonin etc, were synthesized [11,

20]. Submerged fermentation (SmF) and solid state fermentation (SSF) have been successfully used in pectinase production by fungi [12, 9, 38, 30]. Various substrates that are being used are sugarcane bagasse, wheat bran, rice bran, wheat straw, rice straw, saw dust, corn cobs, coconut coir pith, banana waste, tea waste, sugar beet pulp, apple pomade, orange peel etc [33]. Maximum production of pectin esterase in Penicillium chrysogenum at 0.1 percent citrus pectin and 0.2 percent of yeast extract [15]. Hyper saline environments might induce biosynthetic pathways of halotolerant microbes to produce structurally unique compounds. It is generally believed that this sta bility is gove rned by the high acidic amino acid content of halophilic enzymes [41]. Acidic amino acid residues have a high water binding capacity when they are deprotonated and can thus form a solvation shell on the surface of the proteins and keep them hydrated under high salt conditions [23]. The common characteristics of halophilic enzymes include: (i) optimal activity at high NaCl concentrations, (ii) higher reversibility to denaturing stresses, (iii) higher stability in the presence of NaCl and (iv) slow mobility on SDS-PAGE due to lower SDS binding [40]. Fungi like Aspergillus niger, Aspergillus oryzae , Penicillium expansum, which are generally regarded as safe (GRAS) by United State s Food and Drugs Administration (USFDA) which are employed in food industry.

MATERIALS AND METHODS

Sample collection: Solar saltern water samples were collected from

Marakkanam, Pondicherry, India. Sample were capped and brought to the laboratory set up. The samples were serially diluted (upto10 -6

dilution) by adding 1ml of water sample in 99ml of distilled water. About 1.0ml of diluted samples were plated on potato dextrose agar (1M NaCl concentration) medium (containing 150mg l-1 Chloramphenicol to avoid bacterial contamination) by pour plate technique and kept in light

chamber at 30ºC about 7-15 days.

Isolation of pectinolytic fungi: Preliminary screening: [31]

Pectin agar was prepared and autoclaved. The test fungi are Aspergillus ochraceus, Aspergillus flavus, Aspergillus terreus and Penicillium crustosum were inoculated on to the agar plates and incubated for 7 to 10 days. The colony diameters were recorded and Aspergillus terreus showed the zone which indicates the pectinase production. The agar plates were flooded with 0.1 M aqueous malic acid

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and left to stand at room temperature for 1 hour. The malic acid was drained and the plates were flooded with 0.1% aqueous ruthenium red and left for 2 days at 4ºC. The ruthenium red was drained off and the plates were washed for 1 hour in distilled water. They were washed with 0.1% aqueous ammonium per sulphate to increase contrast. The agar plates are pink after staining and dark pink zones around colonies indicate pectinase activity.

Shake flask studies: [14]

The medium was composed of 2%NaNO3, 1%K2HPO4, 5%MgSO4, 5%KCl, 0.001%FeSO4 and 15%pectin. In 250ml flasks 200 ml of the medium was added and inoculated with spore suspensions. Two flasks for strains and one flask for control. The cultures were incubated in a rotary shaker (150 rpm) at 30ºC for a week. After every 24 hrs (till 168 hrs) 10 ml broth was separated by filtrations and culture filtrate used as source of crude enzyme. On 8th day of fermentation, the mycelial mat was filtered through whatman No. 1 filter paper and the filtrate was

used for future.

Extraction: 1) Extraction of crude enzyme: Culture filtrate centrifuged at

10,000 rpm for 10 min at 27ºC supernatant was taken in test tube. Supernatant is used for further investigation.

2) Purification: Culture filtrate was cooled to 4ºC for 30 min. Treated with three volumes of chilled ethanol and allowed to stand for 15 min. The precipitation obtained by centrifugation (5000 rpm for 10 min) and was dissolved in

distilled water for further study.

Enzyme Assay: [37]

The Standard protocol of Sigma Quality Control was used for the enzyme assay 0.5% pectin solution, 50mM iodine with 200mM potassium iodide, 1M sodium carbonate, 2M sulfuric acid, 100mM sodium thiosulfate, 1% starch indicator, pectinase solution. Mix by swirling the mixture and titrated the test and blank with reagent Na2S2O3 until it is light yellow. Then added 1 drop of starch indicator and continuously titrate it with reagent Na2S2O3 until solution becomes

colorless.

Ammonium Sulfate Fractionation: Various percentage of Ammonium Sulfate was being used for

the precipitation of the enzyme sample.10 ml crude enzyme solution (cell free filtrate) was taken in centrifuged tube add (40%, 50 %, 60 %, and 70%) ammonium sulphate to enzyme solution. Keep at 40ºC for overnight. Then centrifuged at 12,000 for 15 mins, pellet was dissolved

using sodium acetate buffer for further.

Optimization for culture conditions: The culture conditions (incubation period, pH, temperature and different cheaper source were optimized for maximum enzyme production. Pectinase production was studied at different pH (5, 6, 7, 8,

and 9), temperature (20ºC, 30ºC, 40ºC and 50ºC), Incubation period (24, 48, 72, 96, 120, 144 and 168hrs) and different cheaper sources like rice bran, groundnut cake, fruit waste (Banana peel), and wheat bran was

used in this study.

Production of pectinolytic enzymes by submerged fermentation: To find out the efficiency of fungal cultures in producing pectinolytic enzymes from fruit waste by submerged fermentation, 50g of fruit peel waste was taken and powdered. To this, 100ml of water was added and pH was adjusted to 5.0. It was sterilized at 120ºC for 15 minutes and then inoculated with fungal cultures and incubated at room temperature. On 8th day of fermentation, it was filtered through Whatman No. 1 filter paper and then centrifuged at 10,000 rpm for 15 minutes. This crude enzyme extract was used for measuring pectinase activity. All the dried material was sieved through 2mm mesh to get uniformity. Those materials were as follows;

a) Banana peel –Shade dried and ground b) Wheat bran- Dried and used untreated c) Rice bran - Dried and used untreated

d) Groundnut cake – Shade dried and untreated

SDS-PAGE: The purity of enzyme was checked by using SDS PAGE. The

standard protein marker and purified enzyme were allowed to run simultaneously for determination of molecular weight. The protein bands on gel were visualized by staining it with coomassie brilliant blue.

RESULTS

Screening of Strains: Aspergillus terreus demonstrated a large zone of hydrolysis

around the large colony on pectin agar medium. The zone of clearance shows the degradation and was the evidenced by a clear zone around fungal growth. Diameter of zone of clearance was 3.0 mm.

Media Optimization: Among the different incubation period studied, the optimum

incubation period for the growth and the maximum pectinase production was 72hrs (Fig: 2). Fruit waste (Banana peel) (Fig: 3) shows the maximum enzyme production among other cheaper substrates. The optimum pH for the pectinase enzyme production and was found to be pH 6 (Fig: 4).The optimum temperature for the pectinase enzyme production 30ºC (Fig: 5) were observed. 206 U/ml was screened using

wastes as substrate compared to other substra tes.

SDS PAGE: According to this study, the molecular weight of the pectinase

was known by the formation of single band in gel adjacent to marker and was found to be 37 kDa which was shown in the Fig: 6.

Fig 1: Colony morphology Fig. 1a: Microscopy observation of Aspergillus terreus



Fig: 2 Effe cts of different incubation periods on pectinase production Fig: 3 Effects of different cheaper sources on pectinase production.

Fig: 4 Effe cts of different pH on pectinase on pectinase production Fig: 5 Effects of different temperature production

Fig: 6 Purified protein showed only one band which had MW 37Kda.

DISCUSSION

Pectinase production occupies about 10% of the overall manufacturing of enzyme preparations and used in the food industry for juice and wine production [36]. Pectinase is also used for improvement of tea leaves fermentation [4]. Microbial pectinase can be stated as the most important enzyme for the juice industry. Of these, strains of Aspergillus species dominate the industrial sector. In this study, the optimized conditions for pectinase production were analyzed and the high enzyme productions were observed in 72hrs and similar results were observed by [27, 6] and 96 hrs incubation provide the maximum yield in Bacillus sp. 206 U/ml was screened using Banana peel as substrate compared to other carbon sources. Same results evidenced the high quantity of enzymes were produced using banana peel as substrate [25]. Banana peel and sugar cane bagasse in the ratio 1:1 showed the maximum production

[27]. Maximum enzyme production was obtained in the medium containing wheat bran as substrate compare to rice bran [17]. In case of solid state fermentation, Trichoderma harzianum showed maximum production with citrus peel as substrate. Orange bagasse was the best

substrate for the production [27]. Sugarcane bagasse gives maximum pectinase yield during the fermentation period in the pH 5 at 28ºC. [6]. Cassava waste provided the optimum condition for enzyme production [25]. Orange peels provided the best environment for the production of pectinase [1]. In this study, the production increases in the pH 6 at 30ºC. Optimum condition for the production was observed in the range of pH 6.0 at 35ºC [27]. Again, the production was high in the pH 5.5 at 28ºC [26].The bacterium Bacillus sp. MFW7 produced significant amount of pectinase pH 6.5 at 35ºC [25]. At pH 7 at 40ºC the pectinase productions were observed [26]. In our results, the molecular weight of purified enzyme was determined to be 37 kDa by SDS-PAGE and the similar results were reported [25]. The purified pectinase showed a molecular weight of 35 kDa [27]. The microbial pectinase possesses the molecular weight ranges between 35 and 79 kDa [18].

CONCLUSION

Halophilic enzymes remain highly stable under certain stressful conditions. While, the produced enzymes may be withstand the high temperature and pH as well as salinity. In this study, maximum production of pectinase was obtained from Aspergillus terreus (206 U/ml) and this strain may be used for commercial production of pectinases. This study indicates the utilizing agricultural waste provides cheaper and eco-friendly method for pectinase production on large scale. Future studies on pectic enzymes should be devoted to the understanding of the regulatory mechanism of the enzyme secretion at the molecular level and the mechanism of action of different pectinolytic enzymes on pectic substance s.

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Conflict of interest: The authors have declared that no conflict of interest exists.

Source of support: Nil

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