INTRODUCTION:

Enzymes are being considered as nature’s potential catalysts^[1]. Most of the enzymes produced today are by the Bio-based materials fermentation. The costs of waste treatments have been reduced due to the usage of enzyme based processes. Microbial enzymes show more stability compared to animal and plant enzymes. The productions of these enzymes are safer and convenient^{[2, 3]}. Most of enzyme sources tested constitute only of about 2% of world’s microbial population. Approximately, 500 industrial products are being produced by the microbial enzymes^{[4, 5]}. The most generally used microorganisms are bacteria and compared to yeasts they have higher activity^[6].

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are carboxylesteraces which can catalyse hydrolysis of acylglycerides with acyl chains which have more than 10 carbon atoms. They have the ability to catalyse the reverse reactions also^[7]. Lipases have catalytic role in different reactions like inter-esterification, alcoholysis, acidolysis, production of biodiesel and aminolysis. Lipases are considered to be the third largest group based on total sales volume following the proteases and the carbohydrates^[8]. It has wide range of applications in food, pharmaceutical, textile, leather, dairy and detergent industries. They play an important role in commercial ventures. There are many microbes which has the ability to produce different lipases. The main aspect of the lipolytic enzyme is unique physicochemical parameter of catalysing lipid-water interfaces which involves interfacial adsorptions^[9].

They are also used in making biosurfactants, biocide paper making, biosensor modulation^[10], production of lipid derived flavours and fragrances and olive mill waste water treatment^[11]. Microbial lipases have received much attention because of its easy cultivation, no seasonal fluctuations, and ease of genetic manipulations and also because of their high yields. Researchers have done many attempts to distinguish lipolytic and esterolytic enzymes ^[12]. Lipases are very thermostable and they require less amount of water. They are also used in organic chemical synthesis^[7]. They play an important role in digestion, dietary lipid processing and transport. Lipases also have a major role in bioremediation which is actually degradation of contaminants with the help of microorganisms in to non-hazardous substances ^[13]. Due to the high biotechnological potential, lipolytic enzymes are now receiving massive attention. Most of the biotechnological applications are based on the catalytic properties of lipolytic enzymes ^[14].

Yeasts have unique characteristic of unicellular growth and belongs to polyphyletic group of basidiomycetous and ascomycetous fungi. There are 100 genera and 800 species being discovered. Some of which are pathogenic to animals and plants. They have immune stimulatory properties due to their complex composition ^[15] Yeasts are omnipresent in their population and distribution depending on different concentrations and types of organic materials present. The term ‘yeast’ has been derived from the German word ‘gischt’ and the Dutch word ‘gist’ which means fermentation ^[16]. 71% of the earth`s surface is covered by oceans, they are abundant in biotic resources. They even have yeast gene and enzymes. Recent studies have discovered that yeast strains obtained from marine environment has the ability to produce many kinds of extracellular enzymes for the hydrolysis of various polymers ^[17]. Marine yeasts can survive in sea water for a longer time than in fresh water. They have unique features in producing enzymes compared to terrestrial yeasts ^[18]. Some examples of marine yeast genera are Candida, Clavispora, Mrakia, Cryptococcus and Saccharomyces sp. ^[8].The genus Candida is most used commercial producer of lipases ^[19].

MATERIALS AND METHODS:

Sample collection:

Marine water samples were collected from the coast of Port Blaire (11.6234° N, 92.7265° E), Andaman and Nicobar Islands, India.

Isolation of lipase producing marine yeast:

The marine water samples were serially diluted and plated on yeast malt agar (YMA) medium and were incubated for 3-5 days at room temperature for the growth of microorganisms^[20]. The medium was supplemented with chloramphenicol antibiotic to avoid bacterial growth. Yeast colonies grown on the medium were selected based on the colony morphology and sub cultured. They were then subjected for screening to check the lipolytic activity of yeast isolates.

Primary screening (TBA plate assay):

Primary screening of the selected yeast isolates was done through tributyrin agar plate method ^[21]. Potent marine yeast isolates were selected based on the colony morphology. Then, they were streaked onto tributyrin agar plates. The plates were incubated for 48 hr at room temperature and were observed for the hydrolysis zone around the colonies.

Secondary screening:

The selected isolates of yeast from primary screening were then grown in production media which contains (g/l) 10 glucose, 3 yeast extract, 3 malt extract and 5 peptone. The isolates were inoculated in the media and were kept at room temperature in shaking condition for 3-5 days. Then the cultures were centrifuged for 15 min at 8000 rpm and the supernatant was collected.

Lipase assay:

Lipase assay is done using Tris-HCl buffer. For the assay, the substrate solution was prepared with a buffer consisting of triton X-100 and phenyl acetate was dissolved in that to make the concentration 200µM. Finally, lipase enzyme was added and the activity was measured colorimetrically ^{[22, 23]}.

Identification and characterization of the yeast isolate:

a. Colony morphology:

The colony morphology of the cultures was observed with respect to their shape, opacity, colour, consistency and elevation.

b. Microscopic observation:

Gram’s staining was performed for the identification of yeast colonies.

c. Biochemical tests:

The biochemical tests like methyl red, indole, voges-proskauer, carbohydrate fermentation test and citrate utilization test was done for the characterization.

Lipase enzyme extraction:

The crude lipase enzyme was extracted by using different solvents like chloroform, petroleum ether, ethyl acetate and methanol (non-polar to polar). The solvents were mixed to the culture supernatant in the ratio of 1:1 (v/v). Then the mixture was vigorously shaken for sometimes. The phase which contained the compound that was extracted was separated and was collected ^[12].

Optimization:

Optimization was done for the selected yeast isolate using different pH, temperature and carbohydrate source. To study the effect of carbon source on protease production, 1% of different carbon sources (glucose, lactose, galactose, fructose, sucrose, mannitol and arabinose) were added to the production medium at room temperature (28°C). The yeast isolate was exposed to selected temperatures (0°C, 4°C, 30°C and 37°C) and pH (2.0, 4.0, 6.0, 8.0 and 10.0) of the medium were adjusted. The culture was incubated in various conditions and was centrifuged. The lipase assay was performed to check the optimized environmental conditions ^[24].

Antioxidant assay:

The antioxidant assay was done to determine the enzyme activity with the crude enzyme. This was done to determine the activity of the enzyme.

1. DPPH activity:

The scavenging of DPPH radical was used to determine the antioxidant ability of the samples. DPPH is a free radical and it is violet in colour. The change of colour from violet to yellow indicates the positive result ^[25] (Sabu et al., 2000). The supernatant collected from the secondary screening was used for DPPH assay. 1ml of the culture supernatant was taken and 2ml of DPPH reagent (2, 2-diphenyl-1-picrylhydrazyl) was added to it. Then the test tubes were incubated in dark for 20 minutes and the spectrophotometric reading was taken at an absorbance of 517nm ^[26].

% scavenging rate= (control-Absorbance of test/control) x100

ii. Reducing power assay:

The supernatant collected from secondary screening was used for testing the reducing power of the sample.1ml of sample was taken in which 1 ml of phosphate buffer and 1 ml of potassium ferricyanide was added. The mixture was incubated at 50°C for 20 minutes then the tubes were taken in which 1 ml of Trichloro acetic acid (TCA), 1ml of distilled water and 0.1ml of 0.1% ferric chloride was added. Incubate the mixture at50°C for 10 minutes. The spectrophotometric reading was taken at an absorbance of 700nm ^{[27, 28]}.

RESULTS AND DISCUSSION:

Marine yeast isolation:

A total of 26 yeast isolates were isolated from the marine water sample of Port Blair, Andaman and Nicobar islands. From them 15 isolates were selected based on the colony morphology for primary screening. They were subcultured and maintained in Yeast Malt Agar (YMA) media.

Fig 1: Isolated marine yeast colonies

Primary screening:

The 15 isolates selected were screened to check their lipase activity. Zones of hydrolysis were observed in 2 of them. It was about 15mm and 13mm for SJP B and SJP A respectively (Fig 2).

Fig 2: Isolated marine yeast colonies.

Secondary screening:

Lipase assay:

The secondary screening of the lipase producing organisms was done using the lipase assay method and the marine yeast isolate SJP B showed highest lipase enzyme activity of 65% while SJP C showed 59% activity and SJP A showed 48% activity (Fig 3). The lipase activity of the yeast isolates SAGB1 showed 60% and SAGB2 showed 65% of lipase activity in phosphate buffer and in Tris-HCL buffer it showed 58% and 62% of lipase activity respectively ^[22].

Fig 3: Lipase enzyme activity of the 3 marine yeast isolates

Characterization:

a) Colony morphology:

The morphology of the 3 isolates was observed to be slimy, white and opaque. The isolates were round and circular in shape. The colony morphology of the 3 isolates has been shown in the Table 1.

b) Microscopic observation:

By performing Grams staining, it was confirmed as yeast isolates due to the appearance of budding cells.

c) Biochemical tests:

The biochemical tests, including indole, methyl red, voges-proskauer, citrate utilization, carbohydrate utilization were done. All the three isolates, A, B, C were negative to indole, VP, citrate utilization tests. They gave positive results to MR and Carbohydrate fermentation tests (Table 2).

Table 2: Biochemical tests

Test	SJP A	SJP B	SJP C
Indole	_	_	_
MR	+	+	+
VP	_	_	_
Citrate	_	_	_
Glucose	+	+	+
Lactose	+	+	+
Sucrose	+	+	+

Antioxidant assay:

i. DPPH assay:

From the result obtained SJP A showed 78%, SJP B showed 77% and SJP C showed 66% of DPPH activity (Fig 4). So, SJP A showed highest activity among the three. All of these three isolates showed higher percentage of DPPH activity compared to ^[22].

Fig 4: DPPH activity of lipase enzyme producing yeast isolates

ii. Reducing power assay:

The reducing power assay performed and the OD values were taken at 700nm. Here, unlike the DPPH assay, SJP B showed the highest activity of 65%.

Fig 5: Reducing power assay of lipase enzyme producing yeast isolates

Optimization:

The present results showed maximum protease activity at optimum pH 8.0 [Fig. 6 (a)]. Similarly, 37°C temperature was found best-suited for growth of strain SJP A. However, we can see a good amount of enzyme production at 0°C [(Fig. 6 (c)]. Present results showed increased yield of enzyme production when lactose was used as substrate [Fig. 6 (b)]. From previous reports it is evident that marine bacteria Bacillus sp. when optimized with different carbon source it can synthesize enzymes. The results were similar to that of the earlier studies conducted with Bacillus sp. ^[7,
12]. In another study, the optimal pH and temperature produced some marine yeast were shown to be in the range 6 to 8.5 and 35°C to 40°C ^[29] which is also quite similar to the results we got.

Solvent extraction:

The solvent has extracted using Petroleum ether, ethyl acetate, chloroform and methanol.. The extracted solvent has been further send for HPLC analysis (High Performance Liquid Chromatography).

HPLC:

HPLC was done for the identification of the extracted compound (Fig 7).

FT-IR:

The extracted compound is characterized by the presence of following functional groups (Fig 8)-

3273.20 cm^-1 – O-H (hydroxyl group) Strong, very broad.

2920.23 cm^-1– =CH₃group

1539.20 cm^-1 – Aromatic ring

1037.70 cm^-1 – C-F alkyl halide Strong intensity

CONCLUSION:

In the above study, lipolytic marine yeasts were isolated from marine sediment sample collected from coast of Port Blair, Andaman-Nicobar islands. Among 26 isolates only 3 isolates showed lipase enzyme activity. These 3 isolates are potent in lipase enzyme production. The strains were used for performing Antioxidant assays viz. DPPH assay and reducing power assay. SJP A had more scavenging activity compared to that of SJP B and SJP C so it was taken for further studies. The marine yeast isolate SJP A was then optimized and partially purified. The strain gives positive carbohydrate utilization test so we may conclude, strains belong to Saccharomyces sp.

REFERENCES:

1. Anbu P, Gopinath S. C. B, Cihan A. C, Chaulagin B. P. Microbial enzymes and their applications in industries and medicine. Biomed Research International. 2013.

2. Adrio J. L, Demain A. L. Microbial enzymes: tools for biotechnological processes. Biomolecules. 2014; 4: 117-139.

3. Demain A. L, Adrio J. L. Contributions of microorganisms to industrial biology. Molecular Biotechnology. 2008; 38: 41-45.

4. Johannes T. W, Zhao H. Directed evolution of enzymes and biosynthetic pathways. Current Opinion Microbiology. 2006; 9: 261-267.

5. Kumar A, Singh S. Directed evolution: tailoring biocatalysis for industrial application. Critical Reviews in Biotechnology. 2013; 33: 365-378.

6. Hasan F, Shah A.A, Hameed A. Industrial applications of microbial lipases. Enzyme and Microbial Technology. 2005; 39: 235–251.

7. Bussamara R, Fuentefria A.M, Eder S.d.O, Broetto L, Simcikova M, Valente P, Schrank A, Vainstein M. H. Isolation of a lipase-secreting yeast for enzyme production in a pilot-plant scale batch fermentation, Bioresource Technology, 2010; 101: 268–275.

8. Zhenming C, Sheng J, Yan K, Wang X, Gong F, Li J. Use of response surface methodology for optimizing process parameters for high inulinase production by the marine yeast Cryptococcus aureus G7a in solid-state fermentation and hydrolysis of inulin. Bioprocess and Biosystem Engineering. 2009; 32: 333-339.

9. Hasan F, Shah A. A, Hameed A. Methods for detection and characterization of lipases: A comprehensive review. Biotechnology Advances. 2009; 27: 782–798.

10. Vakhlu J, Kaur A. yeast lipases: enzyme purification, biochemical properties and gene cloning. Electronic journal of Biotechnology. 2006; 9: 69-85.

11. Hasan F, Shah A. A, Hameed A. Industrial applications of microbial lipases. Enzyme and Microbial Technology. 2006; 39: 235-251.

12. Kumar D, Kumar L, Nagar S, Raina C, Parsha R, GuptaV. K. Screening, isolation and production of lipase/esterase producing Bacillus sp. strain DVL2 and its potential evaluation in esterification and resolution reactions. Archives of Applied Science Research. 2012; 4 (4): 1763-1770.

13. Vidali M. Bioremediation: An overview. Pure Applied Chemistry. 2001; 73(7): 1163-1172.

14. Sivasubramani K., Singh R. J., Jayalakshmi S, kumar S.S, Selvi C. Production and Optimization of Lipase from marine derived bacteria. International Journal of Current Microbiological Applications and Sciences. 2013; 2(4): 126-135.

15. Kutty S. N, Philip R. Marine yeasts-a review. Yeast. 2008; 25: 465-483.

16. Anusha S. U, Sundar S. K, Williams G P. Studies on the isolation and characterisation of marine yeast, glucan production and immunostimulatory activity on Carassius auratus, International Journal of Current Microbiological applications and Sciences 2014; 3(9): 230-240.

17. Sheng J, Chi Z, Yan K, Wang X, Gong F, Li J. Marine Yeasts and Their Applications in Mariculture. Bioprocess Biosystem Engineering. 2009; 32: 333–339.

18. Kutty N. S, Philip R, Damodaran R. Marine yeasts in the slope sediments of Arabian sea and bay of Bengal. European Journal of Experimental Biology. 2013; 3(3): 311-327.

19. Larios A, Garcia H. S, Oliart R. M, Valerio-Alfaro G. Synthesis of flavor and fragrance esters using Candida antarctica lipase. Applied Microbiology and Biotechnology. 2004; 65(4): 373-376.

20. Zaky A. S, Tucker G. A, Zakaria Y D, Du C. Marine yeast isolation and industrial application. FEMS Yeast Research. 2014; 14: 813-825.

21. Fryer T. F, Reiter B, Lawrence R. C. Lipolytic activity of lactic acid bacteria. Journal of Dairy Science. 1967; 50(3): 388-389.

22. Raj A, Baby G, Dutta S, Sarkar A, Bhaskara R.K.V. Isolation, characterization and antioxidant activity of lipase enzymeproducing yeasts isolated from spoiled sweet sample. Der Pharmacia Lettre. 2016; 8 (10): 129-135.

23. Mohamed A. Abd-E, Ahmed M. E, Taher A. A. New colorimetric method for lipases activity assay in microbial media. Americal Journal of Analytical Chemistry. 2013; 4: 442-444.

24. Richa K, Bose H, Singh K, Karthik L, Kumar G, Rao K. V. B. Response surface optimization for the production of marine eubacterial protease and its application. Research Journal of Biotechnology. 2013; 8: 78-85.

25. Sabu A, Chandrasekaran M, Pandey A. Biopotential of microbial glutaminases. Chemistry Today. 2000; 18(21).

26. Sarkar A, Abhyankar I, Saha P, Kumar S, Rao K. V. B. Antioxidant, haemolytic activity of L-glutaminase producing marine actinobacteria isolated from salt pan soil of coastal Andhra Pradesh. Research Journal of Pharmacy and Technology. 2014; 7(5): 544-549.

27. Daljit S. A, Priyanka C. Antioxidant activity of Aspergillus fumigatus. Microbial Technology. 2011.

28. Chang L. W, Yen W. J, Huang S. C, Duh P. D. Antioxidant activity of sesame coat. Food chemistry. 2006; 78: 347-354.

29. Wang L, Chi Z, Wang X, Liu Z, Li J. Diversity of lipase-producing yeasts from marine environments and oil hydrolysis by their crude enzymes. Annals of Microbiology. 2007; 57 (4): 495-501.

Received on 22.08.2017 Modified on 29.09.2017

Research J. Pharm. and Tech 2018; 11(2):593-598.

DOI: 10.5958/0974-360X.2018.00109.9

Isolates	SHAPE	COLOUR	CONSISTENCY	ELEVATION	OPACITY
SJP A	Round	White	Slimy	Convex	Opaque
SJP B	Round	White	Slimy	Raised	Opaque
SJP C	Round	Creamy	Slimy	Convex	Opaque