assignment lipase (1)

1. EMZYME KINETICS CHE7032 Submitted to: Dr. Muhammad Salman Submitted by: Umair Akram Roll No. 27433 Assignment topic: Production, characterization, immobilization and applications of Lipase enzyme (MS CHEMSITRY FALL 2023) RIPHAH INTERNATIONAL UNIVERSITY FAISLABAD CAMPUS

2. Production, characterization, immobilization and application of Lipase enzyme Enzymes: Due to their high catalytic efficiency and benign green reaction conditions, enzymes are widely used in the food, pharmaceutical, energy, and other industrial sectors. However, due to a variety of drawbacks such high cost, low operating stability, and challenges in recovery and reuse, continued industrial usage of free enzymes is constrained. Enzyme immobilisation can successfully overcome these difficulties. In the subject of enzyme research, there are several innovative technologies and techniques. Cellulose nanocrystals (CNCs): In recent years, interest in cellulose nanocrystals (CNCs) has grown as a result of their high surface-to-volume ratio, high aspect ratio, and good biocompatibility6. Due to the superior physicochemical properties of CNCs, glucose oxidase, peroxidase, papain, and lysozymeactivity and stability can be improved. However, the relatively steady dispersion of the CNCs makes it challenging to recycle them from the reaction system, which restricts their use. Lipase Introduction: Lipases: Lipases (EC 3.1.1.3) are known as triacylglycerol acylhydrolase which acts on carboxylic ester bonds is the part of hydrolases family. They do not require any cofactor and belongs to the class of serine hydrolases. Triglycerides hydrolyzed into diglycerides, monoglycerides, fatty acids, and glycerol by using the lipases naturally. The carboxylic esters bonds can be hydrolyzed by esterases in addition to lipases.

3. Many microorganisms, bacteria, fungus, yeast, animals, and plants all produce substantial amounts of lipase. In the dairy sector and oleochemical business, as well as in the production of structural triglycerides, lipase is widely utilized. Microorganism-produced extracellular lipase is being researched for its potential use in numerous industrial processes, including the production of detergents, oils, fatty acids, and dairy products, in addition to its immense medicinal applications. Recent developments have led to the discovery of numerous potential uses for lipases in the detergent, oleochemical, paper production, organic chemical processing, nutrition, cosmetics, pharmaceutical, and agrochemical industries. Conventional reducing in the leather business involves the use of kerosene, gasoline, and other solvents, or aqueous emulsification techniques utilizing detergents. There are hazards related to the use of solvents and detergents as well as environmental issues in numerous sectors. Enzymatic reducing of hides and skins is recommended as a workable solution to the difficulties with compact pollution brought on by the use of surfactants and solvents. Commercial manufacturing of microbial lipase is available; the bulk of these products are employed in the production of detergents, cosmetics, food technology, and chemicals. Due to their ability to function under benign conditions, high stability in organic solvents, broad substrate specificity, and typically high region and/or stereo selectivity in catalysis, lipases are considered to be biocatalysts. It has also been shown that extracellular lipase production increases when triglycerides and other lipids are present in the culture of microorganisms. Many significant bacterial genera that produce lipase include Bacillus, Pseudomonas, and Acnetobacter. The majority of bacterial lipases are extracellular and are produced through submerged fermentation Multi-faceted microbial lipases (tricyglycerol acylhydrolase, E.C.3.1.1.3) have emerged as key enzymes in swiftly growing modern biotechnology lipases are indispensable for the bioconversion lipids (triacylglycerols.) from one organism to another and within the organisms. In addition to their biological significance, lipase have tremendous potential in areas such as food technology, bio medical sciences and chemical industry.. Lipases are glycoproteins, but some extracellular bacterial lipases are lipoproteins reported that the enzyme production in most

4. of the bacteria is affected by certain polysaccharides. Many different bacterial species produce lipases, which hydrolyze the esters of glycerol with preferably long chain fatty acids. They act at the interface generated by a hydrophobic liquid substrate in a hydrophilic aqueous medium. In the rapidly expanding field of modern biotechnology, multifaceted microbial lipases (tricyglycerol acylhydrolase, E.C.3.1.1.3) have emerged as essential enzymes. Lipases are required for the bioconversion of lipids (triacylglycerols) from one organism to another and inside the organisms. Lipases offer enormous potential in fields including food technology, biomedical sciences, and the chemical industry .in addition to their biological significance. Lipases are glycoproteins, but certain extracellular bacterial lipases are lipoproteins. The specific polysaccharides have an impact on the production of enzymes in most bacteria. Lipases, which hydrolyze the esters of glycerol with ideally long chain fatty acids, are produced by a wide variety of bacterial species. They operate at the interface created by an aqueous hydrophilic medium and a hydrophobic liquid substrate. Historical Background: Enzymes are proteins that can catalyse different chemical and biochemical reactions inside or outside of cells. They are highly specific natural catalysts to the various types of substrates and operate under insignificant conditions of environmental factor such as temperature, pressure, pH, with high conversion. Lipase was first discovered in pancreatic juice as an enzyme by Claude Bernard in 1856, which hydrolysed unsolvable oil droplets and transformed them to soluble products. After that the productions of lipase have been observed in the bacteria Bacillus prodigiosus, B. pyocyaneus and B. fluorescens in 1901, and in the current scenario Serratia marcescens, Pseudomonas aeruginosa and Pseudomonas fluorescens species of bacteria have been detected for the production of lipases on large scale. Thermomycesl anugiwnosus was the source of the first commercially successful recombinant lipase, called lipolase, which was produced in Aspergillus oryzae in 1994.

5. Traditionally, lipase has been achieved from the animal pancreas and was made applicable as digestive supplements in the form of crude or in purified grade. It has been widely used in biocatalytic processes to create a number of unique chemical compounds. Lipase production: Culture and Media Conditions: The tributyrin agar medium was used to cultivate the Bacillus subtilis culture. In 100ml of distilled water, 2.5g of peptone, 3.0g of beef extract, 1.5g of agar, and 1ml of tributyrin were dissolved. The pH was changed by 7.2. Lipase-producing Colonies are Isolated from Tributyrin Agar Plates: The tributyrin agar medium was made, and it was autoclaved for 15 minutes at 1210 C. The medium was added to clean Petri plates, allowed to harden, and then monitored for 24 hours for contamination. Two labelled, 7.2-pH plates were inoculated with the acquired culture, and the plates were then incubated for 48 hours at 37 °C inverted. Medium for Lipase Production: In the current investigation, olive oil was used. The broth is made up of 100 millilitres of distilled water, 5 grammes of peptone, 3 grammes of yeast extract, 2 grammes of beef extract, and 5 millilitres of lipid substrates like olive oil, neem oil, castor oil, and gingelly oil. 7.2 was used to alter the pH.

6. By using different substrate sources, such as castor oil, neem oil, olive oil, and gingelly oil, Bacillus subtilis was able to produce the lipase enzyme. The substrates were purchased from the Perundurai local market, erode. Inoculum preparation and lipase production: The inoculum was prepared by placing a single colony of Bacillus subtilis in 10 ml of olive oil in a 250 ml conical flask. The flask was then incubated for 24 hours at 37 °C with agitation on a rotator shaker at 240 rpm. Lipase production process: 5 ml of inoculums were added to 100 ml of freshly prepared fermentation medium, which comprises 2 g of peptone, 2 g of glucose, 0.5 g of potassium dihydrogen phosphate, 0.1 g of diammonium sulphate, 0.1 g of diammonium carbonate, and 0.1 g of magnesium sulphate [21– 25]. The PH was adjusted to 7.2) in a 500 ml conical flask, which was then shaken at 100 rpm for 96 hours at 37 °C. Extraction of Lipase: Following submerged state fermentation, 5 ml of production medium was removed from the production flask and centrifuged at 10,000 rpm for 30 minutes at 40 c to extract the enzyme from the medium. The crude lipase source was the clear supernatant. Enzyme Assay: Lipase activity was measured using a titrimetric approach (olive oil-substrate emulsion) . The assay mixture was prepared by 1.0ml of substrate emulsion the composition of\ssubstrate

7. emulsion contains 70ml of emulsification, reagent (17.6g\sof sodium chloride, 0.4g of potassium dihydrogen phosphate, 5.4ml\sof glycerol, 10ml of gum Arabic was dissolved in 1000ml of distilled\swater) and 30ml of olive oil, 0.8ml of 0.2M potassium phosphate\sand 0.2ml of enzyme aliquot in 100ml of conical flask and incubated\sat 550\sc for 30minutes. 2.0 ml of acetone after the incubation. To stop the reaction, an ethanol mixture was added. Thereafter, the aforesaid aliquots were titrated with 0.1N NAOH using 1% phenolphthalein as an indicator, and the end point was the appearance of a pale pink colour. The quantity of enzyme needed to release 1 mg of fatty acid per ml of fluid per minute is referred to as one unit of lipase activity. Cleaning up Lipase: Salt Precipitations: Protein stability is dramatically increased when impacted by the rise in ionic strength. The process of "salting in" results in an increase in the protein's solubility. Yet beyond a certain amount, the solubility starts to decline; this is called salting out. Ammonium sulphate was utilised for the fractional precipitation of proteins, including lipase. It was offered in a highly pure form and has excellent solubility, enabling large changes in the ionic strength. the adjustments made to a solution's ammonium sulphate content by adding a known saturation solution to a crude enzyme extract. The supernatant from submerged state fermentation received a salt cut of 30%. 16.6 g of ammonium sulphate were added to this. In the instance of submerged fermentation, no precipitation was seen after the addition of the 70% salt cut (21.1g of ammonium sulphate). In cold circumstances, ammonium sulphate was added very gradually while the solution was continuously stirred on a magnetic stirrer.At 4 °C, the solution was centrifuged for 10 minutes at 10,000 rpm. These pellets were gathered and put into 10 ml of 50 mM Tris Hcl solution for dissolution.

8. Dialysis: The extract was centrifuged at 10,000 rpm for 10 minutes at 4 °C to separate the precipitate. Dialysis was performed on the precipitate after it had been dissolved in 10ml of 50mm tris Hcl. About 10cm size of dialysis bag was successively boiled in 100ml of distilled water, 2% sodium bicarbonate and IMM EDTA solution and once again 100 cc of distilled water at 100 °C for 10 minutes. The dialysis bag was then brought to room temperature and refrigerated for 30 minutes. On one end, a dialysis bag received the dissolved enzymes. The dialysis bag was hung using a glass rod and a tightly knotted bag in distilled water in a beaker. This setup was kept overnight in the refrigerator. Solvent Precipitation Using Acetone: Acetone was used as a solvent for solvent precipitation, which was done in a magnetic stirrer for 30 minutes at 60% saturation with the dialyzed enzyme solution. After centrifuging the contents of the tubes for 10 minutes at 10,000 rpm, the pellets were dissolved in 5 ml of 50 MM Tris HCL (PH 8.0) and dialyzed against the same buffer to measure the enzyme activity. Ethanol Precipitation: In the magnetic stirrer, the dialyzed enzyme acetone precipitation solution was treated to ethanol precipitation for 30 minutes at 705 saturation. Centrifuging the contents of the tubes for 10 minutes at 10,000 rpm, dissolving the pellets in 5 ml of 50 MM Tris Hcl buffer (PH-8.0), and measuring the enzyme activity were the next steps. The production of the lipase enzyme by the Bacillus subtilis was monitored by lipid hydrolysis in tributyrin agar media. All of the plates were used to create a clean zone around the Bacillus subtilis colonies. Due to the medium's lipid substrates' breakdown, a clean zone was seen around the colonies. Bacillus subtilis secreting the lipase enzyme, causing the breakdown of lipids. Impact of Lipase Production Substrates Bacillus\sSubtilis

9. Castor oil, Neem oil, and Gingelly oil were used as different substrates in an olive oil broth culture of B. subtilis. The current investigation found that using olive oil as a source of lipid substrate resulted in greater lipase synthesis. According to an assessment using an olive oil emulsion, the lipase enzyme activity was also high (32.67 l/ml/min) in substances containing olive oil, and it was lower in gingelly oil (28 u/ml/min), castor oil (25.67 u/ml/min), and neem oil (23.33 l/ml/min). According to Figures 1-3, olive oil produced the most enzymes, followed by gingelly oil, castor oil, and neem oil. However, as compared to other lipase substrates, olive oil degraded more quickly due to its high lipid content. Bacillus subtilis was used in the current investigation to manufacture the lipase enzyme using submerged state fermentation. Characterization of lipase enzyme The activity and stability are impacted by pH: The reaction mixture was incubated at different pH values between 4.0 and 11.0 for 30 minutes at a temperature of 50 2 °C in order to investigate the impact of pH on enzyme activity. Citrate phosphate buffer (pH 4.0 to 7.0), Tris HCl buffer (pH 8.0), and glycine-NaOH buffer were the buffers employed (pH 9.0 to 11.0) Optimal temperature and thermal stability: The assay was carried out at several temperatures ranging from 35 to 121 °C in order to determine the ideal temperature for the enzyme activity. The lipase was pre-incubated for 0 to 180 minutes at temperatures ranging from 30, 40, to 121 °C. Metal ions' impact: 2.5 ml of 20 mM in 2.5 ml of pure lipase Ca2+, Mg2+, Cu2+, Fe2+, Co2+, and Zn2+ were among the several metal ions (1 mM) that were incubated for 30 min in Tris HCl buffer (pH 8.0).

10. The lipase's shelf life: Pre-incubating lipase at 4 °C in a 20 mM Tris HCl buffer allowed researchers to establish the enzyme's shelf stability (pH 8.0). Up to nine days, enzyme activity was assessed every three days. Media additives' impact: Purified lipase was pre-incubated in 1 M phosphate buffer (pH 7.0) for 30 min at 50 2 °C to assess the effects of several additives, including SDS, EDTA, CTAB, Tween 20, Tween 80, Triton X 100, and glycerol, among others. Lipase immobilization: By adding a suspension of PD-MNPs in ultrapure water to a buffered enzyme solution, lipase immobilisation was accomplished. By dissolving the lyophilized enzyme in sodium phosphate buffer (10 mM, pH 7.0) solution, lipase (2 mg/ml) was created as an aqueous solution. The lipase solution (5 ml, 2 mg/ml) was mixed with a new solution of the previously discussed PD-MNPs at 4°C. As the two fluids combined, precipitation was seen to start happening right away. The lipase-loaded precipitates were gathered, washed three times with ultrapure water, and then stored at 4°C until use after being shaken at 180 rpm for 3 hours (at 4°C). The Bradford technique was used to compare the protein concentration in the lipase solution before and after immobilization. The loading of lipase onto the PD-MNPs was calculated using the variation in protein concentration. The stability of immobilised lipase was investigated under the identical circumstances as those mentioned in the activity assay section, which involved repeated magnetic isolation and reuse. After each enzyme run, the lipase-containing PD-MNPs were magnetically separated, and any residual substrate and product species were washed twice with hexane and ultrapure water in

11. preparation for the following experiment. The immobilised lipase's residual activity was normalised to its starting value after each cycle (the initial activity was set at 100%). Applications of Lipases Uses of Lipase Enzyme in Industry: As a result of its thermostability and wider pH range resistance, the lipase purified in the current study from Bacillus methylotrophicus PS3 can be considered potential candidates for the laundry detergent industry. Purified lipase was used to improve the cleaning of grease, butter, vegetable oil, olive oil, and grease (white) within 30 minutes as compared to the control (Plate 5). After washing with either water or water mixed with soup, stains were only partially removed; however, when purified lipase was added to the cloth to treat stains, stains were completely removed. Detergents with enzymes enhance the fabric's quality and maintain colour vibrancy. The grease contains a variety of lipids, including fatty acids, cholesterol, vitamins, and urging its use in the manufacturing sector. In the food sector Commercial lipases are used in the food sector to process other foods containing fat and flavour dairy products. Moreover, they can improve the cheese's distinctive flavour by acting on the milk lipids to release free fatty acids following hydrolysis. In the cosmetics sector Lipase is currently recognised as a crucial enzyme for biotechnological applications.

12. This enzyme serves as both a biocatalyst for the manufacture of particular cosmetic compounds a nd an active ingredient in the formulation of cosmetics in the cosmetics industry. In the fat and oil industries With the help of lipase, triglycerides are hydrolyzed to create glycerol and free fatty acids, which removes fats and grease from skin and hides. Palm oil is a tiny volume product compared to the volume of cocoa fat butter that is utilised in fo od and confections. Lipase Enzyme’s types and their fuctions: Stomach lipase Gastric juice, which is in charge of breaking down exogenous lipids, contains it. Lipase lingual It is found in saliva and facilitates the first breakdown of triglycerides. Lipoproteoprotein lipase These can be seen in fatty tissues on the surface of cells, muscles, and blood capillary walls. Triglycerides in the blood, delivered by lipoproteins from different organs, are hydrolyzed by it.

13. Lipase of lysosomal acid These enzymes can be found inside cells in lysosomes. They support the control of intracellular lipid reserves. Pantothenic lipase This lipase enzyme, which is necessary for pancreatic secretion, is present. Dietary fats are hydrolyzed into fatty acids and glycerol. Cholesterol acyltransferase lecithin (LCAT) This lipase enzyme's role in the transport of cholesterol is important. Phospholipase This enzyme participates in cell signaling pathways and hydrolyzes phospholipids. It aids in the recycling of the components of cell membranes. Economical Importance: Lipases are very versatile enzymes, and produced the attention of the several industrial processes. Lipase can be achieved from several sources, animal, vegetable, and microbiological. The uses of microbial lipase market is estimated to be USD 425.0 Million in 2018 and it is projected to reach USD 590.2 Million by 2023, growing at a CAGR of 6.8% from 2018. The hydrolysis of long chain triglycerides is catalysed by microbial lipases (EC 3.1.1.3). The microbial origins of lipase enzymes are logically dynamic and proficient also have an extensive range of industrial uses with the manufacturing of altered molecules.

14. The unique lipase (triacylglycerol acyl hydrolase) enzymes catalysed the hydrolysis, esterification and alcoholysis reactions.Immobilization has made the use of microbial lipases accomplish its best performance and hence suitable for several reactions and need to enhance aroma to the immobilisation processes. Immobilized enzymes depend on the immobilisation technique and the carrier type. The choice of the carrier concerns usually the biocompatibility, chemical and thermal stability, and insolubility under reaction conditions, capability of easy rejuvenation and reusability, as well as cost proficiency. Bacillus spp., Achromobacter spp., Alcaligenes spp., Arthrobacter spp., Pseudomonos spp., of bacteria and Penicillium spp., Fusarium spp., Aspergillus spp., of fungi are screened large scale for lipase production. Lipases as biological catalyst has given a favourable vision in meeting the needs for several industries such as biodiesel, foods and drinks, leather, textile, detergents, pharmaceuticals and medicals. References: 1. Isolation and Purification of Lipase by Aries Barros Mr, Taipa Ma, and Carbal Jms (1994). In: Petersen SB, Wooley P (Eds.). Cambridge University Press, Cambridge, UK: pp. 243–270. Lipases their structure, biology, and application. 2. Lipase induction in Mucor Hiemalis (1980) by Akhtar MW, Mirza AQ, and Chughtai MID. 40(2): 257-263 Appl Env Microbiol 3. Beloqui A., de Maria F.P.D., Golyshin P.N., Ferrer M. Recent trends in industrial microbiology. Curr. Opin. Microbiol. 2008;11:240–248. r]

15. 4. Kaur G., Singh A., Sharma R., Sharma V., Verma S., Sharma P.K. Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India, 3. Biotech. 2016;6:49. 5. Bradford M.: A quick and accurate approach that makes use of the dye-binding principle to quantify microgram amounts of protein. 1976; 72: 248–254; 10.1016/0003-2697(76)90527-3. Anal Biochem. 6.Javed, S., Azeem, F., Hussain, S., Rasul, I., Siddique, M.H., Riaz, Afzal, A., Kouser, and N. Bacterial lipases: a review on purification and characterisation. 132:23–34. Prog Biophys Mol Biol. 2018.