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- 1. Presentation on Production of Enzyme - Lipase
- 2. Contents Introduction Historical Background Properties Reactions Catalyzed Classification Sources Production Technology Submerged Fermentation Solid-State Fermentation Downstream Processing Factors affecting production Applications
- 3. INTRODUCTION • Lipases are hydrolases capable of catalyzing the hydrolysis of Triglycerols (TAGs) into Glycerol and Fatty acids (FAs). • These enzymes operate at the interfaces of Biphasic systems, which is a phenomenon known as interfacial activation. • These do not require co-factors and are easily immobilized on different matrices. • The active sites of lipases are generally characterized by amino acid triad composed of serine, histidine and aspartate. • Lipases exihibit region-selective properties and enantioselective catalytic behaviour and are considered to be the most versatile catalyst in lipid biotechnology. • These enzymes can be employed in a large number industrial processes ( production of agrochemicals, cosmetics , biodiesel etc.)
- 4. HISTORICAL BACKGROUND • In 1856, Claude Bernard first discovered a lipase in pancreatic juice as an enzyme that hydrolyzed insoluble oil droplets and converted them to soluble products. • In 1901, the presence of lipases has been observed for Bacillus prodigiosus , B.pycocyancus and B.fluorescens which represents today’s best studied lipase producing bacteria now named Serratia marcescens , Pseudomonas aeruginosa and P.fluorescens. • Lipase have traditionally been obtained from animal pancreas and are used as a digestive aid for human consumption either in crude mixture with other hydrolases (pancreatin) or as a purified grade. • Lipolase was the first commercial recombinant lipase industialized from the fungus Thermomycesl anugiwnosus and expressed in Aspergillus oryzae in 1994.
- 5. PROPERTIES • pH optima • Temperature optima and thermal inactivation • Activation and inactivation of the enzyme • Substrate specificity
- 8. SOURCES Plant lipases: These have been isolated from the leaves, oils, latex and seeds of oleaginous plants and cereals. Yeast Lipases: These include species Candida antartica, Candida rugosa, Candida utilis and Saccharomyces species. The production of Biodiesel includes lipases from Thermomycesl anuginosus. Image Source: Research Gate Image Source: Wiley Online Library
- 9. Fungal Lipases: Filamentous fungi are considered to be the best source for production of lipases. The genera includes Aspergillus, Rhizopus , Penicillium , Mucor, Geotrichum and Yarrowia etc. Bacterial Lipases: The genera Pseudomonas and Burkholderia are the most widely used for the production of bacterial lipases. P.aeruginosa produces a cystiene hydrolase solvent tolerant lipase. Animal Lipases: These include pancreatic and pregastric lipases. Porcine and Human pancreas were the first sources of lipases used in food processing. Image Source: Health Jade Source: Microbewiki -Kenyon College Source: Research Gate
- 10. PRODUCTION TECHNOLOGY • Screening • Strain selection • Inoculum preparation • Immobilization • Solid-State Fermentation • Submerged Fermentation • Filtration • Centrifugation • Chromatography • Aqueous two phase Upstream Processing Production Downstream Processing
- 11. Raw Materials and Nutrients • Olive oil, Palm oil, Coconut oil • wheat Bran, rice bran • yeast extract, peptone • Urea, NaNO2 • Sucrose , glucose , fructose • KH2PO4 • MgSO4 .7 H2O Microbial Sources Aspergillus sp. Bacillus sp Candida antarctica Pseudomonas aeruginosa Staphylococcus aureus Aspergillus awamori Bacillus subtilis Aureobasidium pullulans Yarrowia lipolytica
- 12. PRODUCTION • Production can be carried out through two modes: • Submerged Fermentation • Solid-State Fermentation • Submerged fermentation could be conducted by batch, but productivity is increased by the fed batch or continuous processes. • Greater enzyme production is obtained using defatted soyabean flour as a substrate. Submerged Fermentation
- 13. Solid-State Fermenation • Lipases produced had greater thermal stability. • Use of agro-industrial residues as substrates greatly reduces the cost of production.
- 14. Conditions For SmF • Species: Aspergillus sp. • Carbon Sources: Fructose, Glucose , galactose • Inducers: Olive oil , palm oil , corn oil , coconut oil • Nitrogen Sources: Yeast extract , peptone , urea , tryptone , NaNO2 • Surfactants: Tween-80 , SDS , TritonX-100 , C-TAB • Substrates: Wheat bran , rice bran , wheat straw, pectin , xylan • pH: 6.0-7.2 • optimum temperature: 37o C • Incubation Time: 24-48 hours • Corn steep liquor,K2HPO4,KH2PO4 • MgSO4.7H2O. Image Source : Research Gate
- 15. Conditions For SSF • Species: Aspergillus sp. • Carbon sources: glucose, fructose, galactose • Inducers: olive oil , soyabean oil , cococnut oil • Nitrogen sources: Urea , yeast extract , ammonium sulphate , sodium nitrate • Support: sugarcane bagasse • Substrates: lignocellulose, wheat straw, rice straw, soyabean bran, lemon peel • pH: 6 • Optimum temperature: 35- 40 oC • Incubation time : 72 hours • Rice husk, KH2PO4 , MgSO4 Image Source : Research Gate
- 16. Maximum Growth Period • The lipase production occurs during the late logarithmic phase or stationary phase. • Thus, the cultivation period may vary according to the microorganism and its growth rate. • Inducers also induce and increased production of lipases, such as free fatty acids, hydrolyzable esters, bile salts and glycerol.
- 17. Fermentation by Cell Immobilization • Enzyme stability and activity of lipases can be improved through the immobilization process. • The immobilization process is advantageous, as it can prevent biomass washout at high dilution rates. • The separation of biomass from the medium is favored due to the high cell concentration in the reactor. • For example: Cell growth and immobilization of Rhizopus oryzae fungus cells for the production of biodiesel through methanolysis of soybean oil. • Fibrous nonwoven fabric used as the immobilization matrix in a circulating packed-bed bioreactor.
- 18. Downstream Processing • Concentration • Chromatography • Aqueous Two-Phase Systems • NOVEL PURIFICATION STRATEGIES • Reverse Micellar System • Membrane Processes • Extractive Fermentation
- 19. • Reverse micellar extraction is a liquid–liquid extraction method that uses an organic solvent containing water droplets stabilized by a layer of surfactant molecules (CTAB). Membrane Processes • The polyvinylidene fluoride,regenerated cellulose and glass fiber membranes are usually applied in microfiltration and ultrafiltration. • The principle of membrane separation is based on the different permeability of substances through the membrane material and the driving force for the separation is either pressure, or a difference in concentration, and/or electric potential. Reverse Micellar Systems Source : Research Gate Source : Science Direct.com
- 20. Aqueous Two-Phase Systems • The two immiscible aqueous phases of an aqueous two phase system (ATPS) are considered an ideal liquid–liquid purification technique for the separation, concentration and extraction of biomolecules due to the high productivity, simplicity, short processing time, scalability, cost effectiveness and versatility of the system. • An ATPS consists of a mixture of polymers and salts. • The polymers can be dextran, polyethylene glycol and polypropylene glycol. The salts can be phosphates, sulfates, surfactants (n-decyl tetraethylene oxide and octylphenol ethoxylate) and ionic liquids (1-ethyl-3-methylimidazolium acetate and 1-butyl-3-methylimidazolium hexafluorophosphate). Low-molecular-weight alcohols (i.e., ethanol and propanol) may also be present. Image Source : Science direct.com
- 21. Improved Lipases-Genetic Recombination • Modern genetic recombination systems are used for insertion of gene of interest into the microorganisms to enhance the production. • For Example: • A bacterial lipase gene from Bacillus subtilis was expressed in Saccharomyces cerevisiae and a significant increase in lipase production was observed. • A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). • Expression of cellobiohydrolase was suppressed via RNAi method. • The reconstructed strains with decreased CBHI production exhibited increase in lipase production than that of parental strain.
- 22. Methods for detection of microbial lipase production • Different techniques have been developed for the screening of microorganisms for lipase production. • Qualitative screening of microorganisms on selective growth media: • In this technique, lipolysis is detected by changes in the appearance of the substrates (such as tributyrin and triolein) that are emulsified in the growth media. • The formation of clear halos around the colonies cultivated on the agar plate is an indication of lipase production. • For Example : Lipolytic Bacillus sp. LBN 4 was isolated on tributyrin agar medium using glycerol tributyrate as substrate. • Solid media supplemented with dyes such as phenol red, Victoria Blue B, Spirit blue, or Nile blue sulfate as pH indicators are also used for determination of lipolytic activity. For example : used for screening of Bacillus strain.
- 23. Quantitative titrimetric assay • Lipase activity is measured quantitatively on a continuously stirred triacylglyceride emulsion by neutralization of free fatty acids released following addition of titrated NaOH (in order to maintain the pH at a constant end point value). • olive oil is used as a substrate for the titrimetric analysis. • For Example : • Lipolytic activity of Pseudomonas monteilli 2403- KY120354 was measured in a reaction mixture containing olive oil emulsion incubated at 37 °C for 1 h. Enzyme activity was terminated after addition of 20 mL acetone: ethanol mixture (1:1). The liberated free fatty acids were titrated against 0.1 M NaOH using phenolphthalein.
- 25. Applications • Food Industry: Used in the production of dairy products, baked foods and fruit juices as well as the interesterification of fats and oils to produce modified acylglycerols. Application Process details Species Food Industry Synthesis of methyl acetate Candida rugosa Food Industry Glycerolysis of corn oil Candida antarctica A human milk fat substitute, was the first commercial product made by 1,3-specific lipases treatment of tripalmitin with unsaturated FAs.
- 26. Biodiesel Lipases are able to synthesize biodiesel in the presence of high water content, which is a useful strategy when waste oils are used, since they usually contain a high amount of water molecules. Application Process Details Species Biodiesel Methanolysis of soybean oil for biodiesel production Burkholderia ubonensis SL-4 Biodiesel Transesterification of Jatropha oil Pseudomonas aeruginosa AAU2 Ecodiesel 100 is produced from the partial 1,3-regiospecific alcoholysis using pig pancreatic lipase.
- 27. Bioremediation These enzymes are able to enhance the bioremediation of greasy effluents containing fats, oils and proteins discharged by the dairy industry. Application Process Details Species Bioremediation Potential application in the treatment of effluents laden with oil (degrading ayurvedic oil) Aspergillus awamori BTMFW032 The remediation of cooking oil wastes using orange lipase for transesterification reaction and proved less toxicity of treated waste oils compared to untreated oils.
- 28. Pharmaceutical Industry • Use of lipases in the preparation of optically active compounds, such as pure alcohols, amines and carboxylic acids. • Lipases can synthetize a chiral intermediate compound for the production of Polixatel (taxol 1), which has been applied as an anticancer drug, especially ovarian cancer. Application Process details Species Pharmaceutical industry Enantioselective esterification toward (R)-1-(4-methoxyphenyl)- ethanol (MOPE) Pseudomonas stutzeri Pharmaceutical industry Lipase-catalyzed degradation of polyhydroxyalkanoate (PHA) Bacillus subtilis
- 29. Detergent industry • Lipolase from Thermomyces lanuginosus represents the first industrial lipase to be introduced into detergent and was commercialized in 1988 by Novo Nordisk. • Other lipases including Lumafast (Pseudomonas mendocina) and Lipomax (Pseudomonas alcaligenes) were commercialized by Genencor.
- 31. REFERENCES • Adetunji AI, Olaniran AO. Production strategies and biotechnological relevance of microbial lipases: a review. Brazilian Journal of Microbiology. 2021 Sep;52(3):1257-69. • Melani NB, Tambourgi EB, Silveira E. Lipases: from production to applications. Separation & Purification Reviews. 2020 Apr 2;49(2):143-58. • Geoffry K, Achur RN. Screening and production of lipase from fungal organisms. Biocatalysis and agricultural biotechnology. 2018 Apr 1;14:241- 53. • Sarmah N, Revathi D, Sheelu G, Yamuna Rani K, Sridhar S, Mehtab V, Sumana C. Recent advances on sources and industrial applications of lipases. Biotechnology progress. 2018 Jan;34(1):5-28.