INTRODUCTION:

Lipases [triacylglycerol acylhydrolases; EC: 3.1.1.3] belong to the class of hydrolytic enzyme which catalyze the hydrolysis of long-chain triglycerides¹. In certain conditions, lipases catalyze reaction such as trans-esterification, esterification, lactonization and aminolysis². Lipases are the class of serine hydrolyses and it does not require any co-factor³. Productions of lipases are by lipidic carbon such as fatty acids, oils, glycerol or tweens in the organic nitrogen source⁴. Initially plant seed and animal pancreatic extract were used as a source for lipase production. Demand for lipases increases and microorganisms are used as a source for industrial production⁵.

Special attention have been gained by microbial lipases because of their unlimited supply, simple extraction process, capability towards extreme pH, temperature, metal ions, organic solvents⁶. Microorganisms like bacteria, fungi, yeast and actinomycetes are used for the production of lipase. Among the microbial sources fungal produced efficient lipases^5,7. Microorganisms used for lipase production have been found in wide ranging habitats such as vegetable oil processing factories, soil contaminated with oil, industrial wastes⁸. In 1950, the fungal lipase production was started but the availability of fungal lipase was increased in 1980’s^7,9. Aspergillus niger, Aspergillus flavus, Penicillium restrictum, Rhizomucar rhizopodiformis, Rhizopus oligosporous, Candida rugosa, Rhizopus oryzae, Fusarium oxysporum are few fungal species involved in the production of lipase. According to the composition of the growth medium, temperature, pH strain, cultivation condition and the kind of carbondioxide and nitrogen sources, the production of lipase by fungal species will vary¹⁰. Lipases are widely used in the hydrolysis of fats and oils, detergents and degreasing formulations, biodiesel production, pharmaceutical industry, cosmetic industry, region selective vacillations, pulp and paper industry, flavor enhancement, oleo chemical industry, anti-asthma drug and dairy industry¹¹. Each year approximately 1000 tons of lipases are used for the production of detergent¹². In food processing industry lipases are used for the breakdown and modification of bio-materials. It is also used as a diagnostic tool¹³. For biotechnological applications lipases compose the most important group of bio-catalyst¹⁴.

SOURCES OF LIPASE:

Plant source:

Various plant tissues are used as a source for the lipase production. Mainly seeds are used as a source due to its higher concentration of triacylglycerol¹⁴. Production of lipase from seeds is more advantageous than animal source. Oil seed, Bean, Sunflower seed, Barbados nut, Lupin, Linseed, Coconut, Almond are some seeds used in lipase production¹⁵.

Animal source:

In 1856, Claude Bernard discovered the pancreatic lipase and it was one of the earlier enzyme produced from animal source. Hog pancreas, dog pancreas, human pancreas and mucous cells of gastric mucosa are some of the sources used for the lipase production¹⁶. Ion exchange chromatography and HPLC molecules seive chromatography are used to purify the pancreatic lipase from human pancreatic juice¹⁷.

Microbial source:

The attention for lipase production from microbial sources has increased in last decade, due to its manufacturing applications as food additives, waste water treatment, pharma, medicine, fine chemicals, leather and cosmetics. It have great importance in qualitative determination of triacylglycerol in food industry and clinical diagnosis. Lipase are used in cosmetics for personal care such as cleaning, softening, aroma and coloring. In textile industry lipases are used to remove the size lubricants in order to provide the fabric. The advantage of using microbial sources including produce high yield, enhanced stability, broad substrate specificity, reduced production costs and ability to catalyze diverse reactions¹⁸.

Bacterial source:

Bacterial lipases are extracellular, intracellular or membrane bound¹⁹. Most of the bacterial lipases are glycoproteins and few extracellular bacterial lipases are lipoproteins²⁰. Bacillus subtillis, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus alcalophilus, Bacillus pumiilus and Bacillus coagulans are commonly used as a source for the production of lipase²¹. Among the bacterial source Pseudomonas species are the highest producer of lipase²².

Algal source:

Lipases are isolated from marine algae. It is easily obtainable in shallow culture. The novel routes for industrial production has been developed through the isolation of lipase from seaweeds. People are not uses the seaweed as food in India so it give a chance to utilize it for other purposes. Isolation of lipases from marine algae in industry purpose is yet unknown²³.

Fungal source:

Aspergillus niger, Aspergillus carneas, Aspergillus terreus, Candida cylindracea, Rhizopus arrhizus, Mucor miehei, Rhizopus japonicus, Rhizopus delemar, Rhizopus niveus and Rhizopus oryzae are commonly used source for the production of fungal lipase⁷. Fungi are the best source for the production of lipase and it used ideally for the industrial applications¹¹.

FUNGAL LIPASE:

The first commercial recombinant lipase ‘Lipolase’ which arised from the fungus thermomyces introduced by Novo Nordisk in 1994 and it was exhibit Aspergillus oryzae¹⁸. Fungi are a fascinating source of lipase because they produce extracellular enzyme²⁴. The present day technology favours the use of batch fermentation and low cost extraction methods due to this fungal lipases have benefits over bacterial one. According to the composition of growth medium, cultivation conditions, strain, temperature, pH, kind of carbon and nitrogen sources the production of lipase from fungal will varies⁹. Candida antarctica, Candida rugosa, Mucor, Geotrichu, T. lahuginosus, Aspergillus niger, Aspergillus flavus, Rhizomucar rhizopodiformis, Rhizopus oligosporous, Rhizopus oryzae, Aspergillus oryzae, Fusarium oxysporum and Penicillum restrictum are few fungal species used for the production of lipase^10,11,25. Among the fungal species, Aspergillus species produced high concentration of lipase²⁶. Aspergillus species which are isolated from terrestrial sources have notable properties fit for biotechnological applications^6,26.Solid state fermentation and Submerged fermentation are widely used for the production of lipase while fungi are better to grow on solid state fermentation than yeast and bacteria. Solid state fermentation is more preferable than Submerged fermentation due to lower operating costs, superior production yields and productivities, higher oxygen distribution, simpler equipments and control systems, lower energy consumption, cheaper fermentation media, less demands for asepsis control, fewer operational troubles²⁴.

Extraction of Lipase from Aspergillus species:

Lipase production by Solid state fermentation:

Solid state fermentation slightly varies in environment than submerged fermentation for metabolism and cell growth²⁷. Solid state fermentation provides enormous potential for the production of enzymes²⁸. The general aspects of solid state fermentation are selection of suitable microorganisms and substrate, optimization of process parameters and isolation and purification of the product. Fungi and yeast were referred as a suitable microorganisms for solid state fermentation by theoretical classification based on water activity²⁹. Solid state fermentation is well adapted for fungi grow in a nature on solid substrate such as seeds, roots, pieces of wood and dried parts of animals such s bones, fecal material and skin (i.e) low in moisture. The focuses of solid state fermentation is to bring cultivated fungi in solid contact with insoluble substrate and to attain the higher concentration of nutrient from the substrate for fermentation³⁰. It have enormous advantages than submerged fermentation including relatively higher concentration of the products, including high volumetric productivity, requirements for simple fermentation equipments and less effluent generation²⁸. The production of lipase by solid state fermentation was stable for a period of 15 days. It is 64% more thermally stable and 24% more active than lipase produced by submerged fermentation²⁹. Oil cakes of coconut, fungal infected coconut, soil from sesame oil mill, castor oil soaked cloth, groundnut and sesame, castor oil soaked cloth are the substances which are used for the isolation of lipase producing fungi. For enrichment substrate are inoculated into yeast extract malt extract [YEME] broth. Each substrate is added separately to a test tube which contains 5ml of YEME broth. It is incubated for one week. Serial dilution will be done for broth and 1ml of diluted is taken and it is added to the medium which contains 50ml of molten potato dextrose agar [PDA] containing 50µg/ml of rifampicin and poured to petriplates. The plates are incubated for 7 days at 30℃. The colonies are transferred onto PDA slants and incubated for 3 days at 30℃. Lipolytic activity of isolates was tested.

Lipase production by submerged fermentation:

In submerged process microorganisms are cultivated in liquid nutrient broth³¹. Free flowing liquid substrates are utilized by submerged fermentation such as molasses and broth. Bacteria require high moisture content, so it is best suitable technique for the fermentation process. Submerged fermentation is generally used in the extraction of secondary metabolites and it must be in liquid form. In submerged fermentation process purification of product is more easier and it is one of the advantage of this technique³². Submerged fermentation is used to produce industrial enzyme. Submerged fermentation process takes place in closed vessel which containing a rich broth of nutrients and high concentration of oxygen³¹. Lipase which exhibit lipolytic activity are subjected to submerged fermentation. Preparation of spore suspension is done by adding 5ml sterile water to 72 hrs culture and it is added to 45ml inoculum medium in Erlenmeyer flask and it is incubated for 48 hrs for 30℃ in rotary shaker. To 45ml production medium 10% of inoculum is added and incubated for 96 hrs at 30℃. After 96 hrs lipolytic activity is tested. 10g of solid substrate and 1ml of salt solution is take in Erlenmeyer flask. The component present in the salt concentration are magnesium sulphate, potassium dihydrogen phosphate, calcium chloride, disodium hydrogen phosphate. After sterilization the flasks are cooled and it is inoculated with 10% inoculum of 48 hrs culture. It is incubated for 96 hrs at 30℃³³. Lipase production from different species of Aspergillus under different fermentation conditions is shown in Table 1.

APPLICATIONS:

Food industry:

In food processing industry majority of enzymes are used for the modification and breakdown of bio-materials⁴¹. Lipases have benefits in the food industries where they have quantitative and/or qualitative impacts. To breakdown the milk fats lipases are used and it give characteristic flavour to the cheese⁴². Nowadays lipases are used in the acceleration of cheese ripening and the lipolysis of fat, cream and butter⁴³. To create new functional ingredients and functional foods lipases are being developed. It have great significance in the egg processing sector to enhance the emulsification properties of egg yolk lipids⁴². By esterification, lipases liberate the short chain of fatty acids this action of lipase enhance the flavour content of bakery products and it also prolong the shelf life of the bakery products⁴³. For the production of mayonnaise and other emulsifiers, lipases are used⁴¹.

Pharmaceutical industry:

In pharmaceutical industry, lipases play a prime role in modification of monoglycerides for use as a emulsifiers⁴⁴. PUFAs are used in pharmaceutical due to their metabolic benefits⁴⁵. Lipases are used in the synthesis of lovastatin which is a drug that lower the serum cholesterol level⁴⁶. Lipases are also used for the production of digestive aids and separating lipids⁴⁴. It is used as a therapeutics in the treatment of dyspepsia, cutaneous manifestations of digestive allergies, gastrointestinal disturbances etc⁴⁷. It acts as a key ingredient in formulating active pharmaceutical compounds due to their high level of specificity⁴².

Detergents:

When compared to synthetic detergents enzyme based detergents have better cleaning properties⁴⁸. The common commercial applications for detergents are in dish washing, decomposition of lipid contaminants in dry cleaning, degradation of organic wastes on the surface of exhaust pipes, removal of dirt manure from domestic animals, a bleaching composition, contact lens cleaning, cleaning of drains clogged by lipids in food processing⁴⁷. The enzymatic detergent aimed towards lower wash temperature which save the energy and it also maintains the texture and quality of fabrics⁴⁶.

Bio-diesel production:

Lipases play a significant role in the bio-diesel production and it produce bio-diesel from various feed stocks such as animal fats or palm oil^42,49. Due to the limitation of resources of fossil fuels bio-diesels are preferred and it is eco-friendly⁴⁷. To optimize the application of enzymes in bio-diesel production thermostable lipases are being developed⁴².

Pulp and paper industry:

In paper industry lipases are used to remove the pitch from the pulp⁵⁰. Pulp manufacturing technology is highly diverse and tremendous opportunity are exist for the application of microbial enzymes⁴⁷. Lipases are used to remove the undesirable triglycerides⁴². Application of lipase in wastepaper drinking can increase the whiteness and intensity, decrease chemical usage, increase the pulping rate of pulp, prolong equipment life, reduce pollution level of waste water, save energy and time and reduce composite cost⁴⁷. Nippon paper industries japan introduced a pitch control method which uses the fungal lipases to hydrolyze up to 90% of wood triglycerides⁴⁶.

CONCLUSION:

Fungi is the best source to face the increasing demand of lipase. The great advantage of fungal lipase is that they are easily amenable to extraction due to their extracellular nature, which will significantly reduce the cost and make the lipase more attractive than other sources. Lipases from fungal origin are much embraced owing to their high stability. Versatility of fungal lipases has led to the development of various new applications industrially. Aspergillus species produce high amount of lipase when compared to other fungal species and it play a vital role in industrial field. Lipases obtained from the genus Aspergillus present a remarkable importance in biotechnological applications and numerous studies have reported the importance of the fermentation parameters such as nutrient, temperature and fermentation time. Specifically lipase produced from Aspergillus species have notable properties to fit for biotechnological applications.

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Received on 02.01.2020 Modified on 26.03.2020

Research J. Pharm. and Tech. 2021; 14(8):4471-4475.

DOI: 10.52711/0974-360X.2021.00777

Microorganisms	Type of Fermentation	Lipase Production	pH	Temperature	References
Aspergillus fumigatus	SSF	8.13 U/ml	9	40℃	34, 35
Aspergillus niger	SSF	9.14 IU/g ds	4-7	30℃	36
Aspergillus flavus	SSF	7.00±0.252 U/g	7	37℃	37
Aspergillus oryzae	SSF	150 U	7	30℃	38
Aspergillus nidulans	SMF	27.02±2.77 U/ ml	7	40℃	39
Aspergillus terreus	SMF	15.463±0.39U/mg	7-9	30 - 45℃	40

Department of Bio-Engineering, School of Engineering, Vels Institute of Science,

Technology and Advanced Studies (VISTAS), Chennai, India.