Optimization for Lipase

Pratyoosh Shukla and Kshitiz Gupta, 2007. Ecological Screening for Lipolytic Molds and Process Optimization for Lipase Production from Rhizopus oryzae KG-5.

Journal of Applied Sciences in Environmental Sanitation, 2 (2): 35-42.

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Research Paper

ECOLOGICAL SCREENING FOR LIPOLYTIC MOLDS AND PROCESS

OPTIMIZATION FOR LIPASE PRODUCTION FROM Rhizopus oryzae KG-5

PRATYOOSH SHUKLA* and KSHITIZ GUPTA Department of Biotechnology, Birla Institute of Technology, (Deemed University), Mesra, Ranchi-835215,

Jharkhand, India. *Corresponding Author: Phone: +91-651-2276223; Fax: +91-651-2276590; E-mail: [email protected]

Received: 5th May 2007; Revised: 14th May 2007; Accepted: 15th May 2007

Abstract: The lipases of the Rhizopus species family are important and versatile enzymes that are mainly used in fat and oil modification due to their strong 1,3-regiospecificity. In the present study 20 samples were collected randomly from different ecological niche of Ranchi, Jharkhand, India during July, 2005 to March 2006 and were primarily screened by sprinkling method and serial dilutions techniques for isolation of lipase producing indigenous species of fungi from Jharkhand. Out of 20 samples collected 18 isolates were primarily identified as excellent producers of lipase in modified lipase assay media. Further it was found out that among these isolates 13 species of fungi were producing lipase extracellualarily and their activity was tested using titrimetric assays. Out of these fungi Aspergillus niger KG-2, Rhizopus oryzae KG-5, Rhizopus oryzae KG-10 were found to be the excellent producers of lipase. Due to amazingly high yields of lipase from Rhizopus oryzae KG-5 it was further selected for further physicochemical studies. It was found that Rhizopus oryzae KG-5 has maximum activity of 48.66 I.U. and was further found as the prominent producer of lipase in liquid media (modified lipase production media). Various physicochemical parameters such as pH, temperature, effect of media components and time were also studied. Keywords: Environmental samples, enzyme assays, physicochemical parameters

INTRODUCTION

Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) occur extensively in nature in animals,

plants and microorganisms and catalyse the hydrolysis and synthesis of esters formed from

glycerol and long chain fatty acids [1-3]. Lipases from fungi are find use in diverse range of

industries like detergents, pharmaceuticals, beverages, dairy etc. [4-7] which makes them

commercially important enzyme and further these are involved in the breakdown and mobilization

of lipids within the cells of an individual as well as transfer of lipids from one organism to another.

Apart from their prevalent use in detergents, dairy foods bakery and beverages, lipases also find

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use in health foods, chemicals and pharmaceuticals for transesterification and enantioselectivity

[8-10]. Research reports on lipase producing fungi have been reported with main focus on Kinetic

parameters, sequencing and cloning of lipase genes, enzyme action, and structural

characterization [11,12] but the screening for isolating lipolytic molds from different environmental

samples of different ecological habitats have been left relatively unexplored. The present study

will reveal some of such interesting screening results for the first time in this area of India which is

a vegetation rich area with moderate climate for the growth of microflora. In the present study we

have also discussed the method for isolation of lipase producing fungi, lipase essay, activity

measurement and effect of various parameters on production and activity of lipase with one of the

best lipase producing strain Rhizopus oryzae KG-5. Now a days fungal lipase are preferred over

bacterial lipases due to their better protein processing and wider range of activity so such

research findings will definitely enhance the future prospects of isolating novel fungal species

from the environmental samples.

MATERIALS AND METHODS

Sample collection

Samples were collected from the sites contaminated with lipids, oils, and decaying organic

matter from 10 different sites in Ranchi, Jharkhand, India during July, 2005- December 2006. The

soil samples were collected from one inch below the soil surface in sterile polybags whereas

samples of decaying fruits and vegetables were collected from local shops.

Isolation of lipolytic molds

Dilution plate method was used in this study and the lifting of conidia from sporulating

conidiophores for isolating various fungal forms was also considered for effective isolation of

different molds [13,14]. This methodology helps in getting cultures with less contamination and

abundance of bacteria can be minimized by using antibiotics in culture medium.

The samples were plated simultaneously on Rose Bengal chloramphenicol agar and Potato

Dextrose agar (PDA) media supplemented with streptomycin (40 L mL-1) and tetracycline (20 L

mL-1) with a concentration of 20 g mL-1 of media and incubated at 28oC for 3-5 days and stored

at 4 oC. Single colony forming units were picked up aseptically and were transferred on fresh

PDA.

Identification of fungi

After ascertaining the purity of cultures the fungi were identified on the basis of cultural and

morphological features with the help of suitable literature [15].

Enzyme assay and activity measurements

Modified lipase assay media (15g Peptone, 5g NaCl, 1g CaCl2, 10 mL Tween 20, 15g Agar, and pH 7.0) was used for enzyme assay. Out of 20 test fungi 18 fungal species gave promising results for lipase production while 13 other species showed a distinct zone of lipolysis indicating extracellular production of lipase. These 13 species of fungi were selected for further study and were maintained on PDA at 4C .The lipase activity was detected due to occurrence of a zone of



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clearance around the colony and subsequent formation of white precipitate of calcium monolaurate around the colony [16,17].

Enzyme activity

Lipases require oil-water interface for their action. The enzyme activity was measured by universal titrimetric method [16,17]. The oil-water emulsion and enzyme extract (0.1 mL: 9.9 mL: 1 mL) was titrated at constant temperature against 0.1 N NaOH using phenophthalein indicator. A blank (9.9 mL water; 0.1mL Tween 20; and 1 mL sterilized broth media) was previously run to find the standard deduction in titer value. Due to this phenomenon lipase releases free fatty acids from the tween 20 and causes change in pH to acidic, which was further neutralized by titrating with NaOH through constantly stirring the vessel. Each reading was taken in triplicate and the activity was measured as amount of enzyme required liberating one micromole equivalent fatty acid per mL/min. Effect of physico-chemical parameters

Various parameters like pH, time, temperature and media were altered [18,19] to obtain the optimum parameters. For this the production/activity was checked for every sample in which a parameter was varied these values were also obtained in triplicate. Effect of time

The production of lipase was carried out by using liquid sate fermentation at time duration of 24 hrs, 48 hrs, 72 hrs at 37 oC, 150 rpm and pH 7.

Effect of pH & temperature

The effect of pH on lipase activity was determined by incubating 0.1 mL of protein precipitate in 0.4 mL of appropriate buffers (0.1 M citrate-phosphate buffer: pH 4 and 5; 0.1 M phosphate buffer: pH 6.7 and 8; 0.1 M Tris-HCl buffer: pH 6.7 and 8; 0.1 M Tris-HCl buffer: pH 9; 0.1 M glycine-NaOH buffer: pH 10; 0.1 M HCl-NaOH buffer: pH 11 and 12). To this, 0.5 mL of tween 20 (1% w/v) was dissolved and the reaction mixture was incubated at 50 C for 5 min. The effect of temperature was determined by incubating 0.5 mL of proper diluted enzyme and 0.5 mL of tween 20 (1% w/v in 200 mM sodium acetate buffer pH 5) for 5 min at different temperatures. Effect of different media components

The media can be classified into 3 categories that are carbon, nitrogen, and effectors. The nitrogen source like peptone is necessary for extracellular production of lipase. While effectors like sodium chloride and calcium ions enhance the production of lipase in the broth media. Hence these sources were left unaltered and carbon sources were varied to check the effect on production. Various sources like olive oil, Tween 20, and coconut oil were used to analyze the activity.

RESULTS AND DISCUSSION

The distribution of different lipolytic fungi was able to be identified in various environmental

samples (Table 1). The production of lipase was affected by time. It was noted (Fig. 1) that 90%

of the fungal species gave highest production after 24 hr of incubation. It was also noticed that

there was abrupt loss of activity at 72 hrs in nearly all the strains except a few. The effect of pH

and temperatures on lipase activity was shown (Fig. 2). For various carbon sources, it was found

the highest activity was detected in Tween 20 followed by olive oil and coconut oil (Fig. 3).



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Fig. 1: Enzyme activity profile of different isolates

Fig. 2: Effect of Temperature and pH on activity of Rhizopus oryzae KG 5



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Fig. 3: Effect of various carbon substrates on activity of Rhizopus oryzae KG 5 Out of 18 fungal species isolated from different environmental samples 13 showed most

optimum production profile for extracellualar lipase. The best producing fungal isolates namely

Rhizopus oryzae KG-5 sp I, Rhizopus oryzae KG-10, Candida spps, KG-11 and Fusarium spps

KG-15 were further selected for our enzymatic studies. Among them the present fungal isolate

Rhizopus oryzae KG-5 showed very good production profile for lipase under variable pH and

temperature conditions which is to the best of our knowledge nearly twice the value of their

known species found elsewhere. Further it was also revealed by our studies that these molds

predominate the fungal flora of Ranchi, (Jharkhand, India) during the months of December &

February which provides them the favourable temperature and relative humidity for their growth.

Moreover it was found that Rhizopus oryzae KG-5 sp I was showing optimum production profile

which was obtained at pH 7, 37 degrees centigrade and using tween 20 as the carbon source. It

is however clarified that tween 20 is not a specific substrate for lipase production.

Of these 18 strains which thrived very well on the modified lipase assay media only 13

showed the extracellular of lipase, as only extra cellular sources were desired, research was

narrowed down these 13 strains only. It was found that species of the order Mucorales gave the

higher yield as compared to other strains. It was also deduced that species namely Aspergillus

niger KG-2, Rhizopus spps KG-4, Rhizopus oryzae KG-5, Rhizopus oryzae KG-10, Mucour

muceo KG-8, Rhizopus stolonifer KG-9, Candida spps KG-11, Chaonephora spps KG-7,

Fusarium oxysporum KG-13, Fusarium spps KG-15, Rhizomucour spps, KG-16, Aspergillus niger

KG-17, Aspergillus flavus KG-18 were also showing very nice production profile with an average

13 I.U which are predominating in the microbial flora of Ranchi. Moreover the Rhizopus Lipase

has broad range of specificity and hence can act on a number of substrates which can be applied

in various industries. While studying the effect of time on the production it was found that there

was sudden loss of activity after 72 hours of incubation in liquid media. Since the Rhizopus

Lipase showed good activity when carbon source substrates were altered i.e. Tween 20, coconut

oil, olive oil it can be safely assumed that the enzyme had a broad range of specificity.



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CONCLUSIONS

Screening various environmental samples for lipolytic molds is a novel and most interesting

research area with lots of potential towards studying biodiversity of soil mycoflora. There are

certain environmental factors like pH, Temperature, Aw (availability of water) and R.H. (Relative

humidity) which play a major role during enzyme production and metabolic activities. Such

studies are aimed at assessing the potential fungal isolates which can be helpful in degrading

lipid wastes from oil mills, vegetable and fruit shops and other lipid containing garbage. In near

future this will definitely help to organize a cleaner environment with waste utilization by these

fungal species. On the other hand Lipases from Rhizopus oryzae are one of the most versatile

enzymes which are commercially used in vast spectrum of industries and assume great value in

high end product oleo chemical industries. As these lipases are enantioselective and

regioselective it makes them highly suited for biopharmaceutical industries. Efforts have been

carried out for better lipase producing strains by natural selection using UV, NTG treatment [20]

but to the best of our knowledge strategies on screening lioplytic molds direct from environmental

samples has not been fully utilized till date. In this study we have some crucial results which

indicates the ability of indigenous isolate Rhizopus oryzae KG-5 to grow and utilize various

substrates at various varying environmental conditions to produce extracellular lipase.

Bioprospecting for industrial enzymes from microorganisms is a promising area of research and it

has important role to play in near future Regarding the isolation of lipolytic mold from different

environmental samples is concerned it is equally important because lipase based processing has

highly promising future and now a days various genetic engineering tools can be used to enhance

the production of lipases in eukaryotic expression vectors like Pichia pastoris but there are

various limitations like high cost of products, lack of enzymes with optimum temperature range

and wide range of catalytic properties.

A perusal of the present study it was found out that these lipolytic molds isolated from

different environmental samples of Jharkhand, India had a very good lipase production profile.

Isolate Rhizopus oryzae KG 5 and Rhizopus oryzae KG 10 were found to be a promising isolate

and further work on characterization of the enzyme is in progress.

Acknowledgement: We gratefully recognize the support received in the form of R & D Grants from

Department of Agriculture, Government of Jharkhand & Sub-Distributed Information Center (BTISnet

SubDIC) of Department of Biotechnology, Government of India.

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Optimization for Lipase

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