"Ansari Sufiya MS.MIC.3.03UID: 158602MARINE ENZYMES- PROTEASES AND LIPASESINTRODUCTION1 Enzymes are powerful and highly specific biological catalysts . Marine organisms arefound as symbionts (intracellular or extracellular) on their hosts (vertebrate and invertebratemarine animals). These microorganisms possess a large number of novel enzymes and pathways.Marine environment serves as a diverse source of various enzymes that can act oncarbohydrates, proteins and lipids. Marine microalgae also serve as sources of biocatalysts for2 biotransformations or biosysnthesis of secondary metabolites . The marine industry generates a large amount waste which is considered as a majorpollutant due to uncontrolled dumping. Since these compounds can be used for generating useful3 enzymes, this will be beneficial for the environment as well as for industries .Thus the marineeco-system have been explored in the past few decades, and various new applications of marineenzymes have come up i.e. in food industry, nutritional supplements, food additives,4 pharmaceuticals, cosmetics, molecular probes, agrochemicals and fine chemicals .MARINE PROTEASESProteases are the class of enzymes that catalyzes the hydrolysis of peptide bonds inproteins. According to their mode of action, proteases are classified into two major groups:endopeptidases and exopeptidases. Endopeptidases hydrolyze the peptide bond present withinpolypeptide chains and exopeptidases carry out the removal of terminal amino acids in thepolypeptide chain. Classification of proteases can also be based on the pH or active center (i.e.cysteine, serine, metallo or aspartyl proteases). Proteases isolated from marine environment haveunique advantage over those obtained from mammals due to the highly fluctuating and vast4 range of environmental conditions in the marine eco-system .Proteases have found great applications in detergent industries, leather industries andpharmaceutical industries (in digestive drugs and anti-inflammatory drugs). In 1960, the firstalkaline protease from Bacillus licheniformis was isolated, followed by a new type of alkalineproetease from marine Psychobacter. UV mutagenesis to obtain a protease with moreadvantages i.e. stability, high pH tolerance, temperature tolerance etc. have also been tried. Detergent stable alkaline serine proteases were isolated showing high stability and5 compatibility with a wide range of liquid and solid detergents .Proteases in marine bacteria and fungi: Microbial proteases have great demand due to theease of cultivation, and limited space and nutrient requirements of microbes. Fungus specieslike Rhizopus, Aspergillus and Mucor; bacterial species of Bacillus, Clostridium andPseudomonas serves as potential sources of marine proteases. The commercially availableextracellular protease used in detergent, surfactants, builders, bleaching agents, fabricsofteners, fillers and bleach activators is isolated from Bacillus species. These alkalineproteases have also found applications in bakeries, leathering, beer manufacturing, alcohol Ansari Sufiya MS.MIC.3.03UID: 158602production and oil manufacturing. Potent algicidal acitivity is possessed by the alkaline serineprotease isolated from a strain of Pseudomonas alteromonas. High activity protease ofArthobacter ramosus and Bacillus alcalophilus have found useful applications in formulationsfor blood stain removal from cotton cloth.Protease from thermophilic marine microoranisms: Hot springs, geothermal sedimentsserve as good source of heat stable proteases. These proteases find applications infermentations and composting protocols that operate at high temperature. Bacillus0 0 stearothermophilus(80 C), Pyrococcus furiosis(100 C) etc. are potential sources ofthermophilic proteases. These proteases are suitable for bioengineering and biotechnologicalapplications owing to their properties for high resistance to chemical denaturants and organicsolvents. These properties when introduced into a protease with good detergent or therapeuticactivity may increase its applications.Protease from Polychaeta: A lethal medical condition, thromboembolism i.e. obstruction ofblood vessels by blood clots need thrombolytic agents for treatment. Polychaeta contains anovel fibrinolytic serine protease in its coelomic fluid, possessing strong fibrinolytic activity.Plasmin is produced from plasminogen by thrombolytic agents, and serves as therapeutic forthromboembolism by destruction of blood clots. Presence of this novel enzyme in Polychaetasuggests its attractive use in therapeutics.Protease from crustaceans: Crustaceans are known to feed on animal tissues containingcollagen. However they possess collagenolytic protease enzyme that can be obtained from thehepato-pancreas of fiddler crab. This enzyme can be extracted in its active form, thuseliminating the need for enzyme activation before use. This enzyme had been reported todegrade collagen polypeptide backbone in conditions that do not denature the protein.Chymotrypsin and trypsin like activity is seen in collagenolytic protease enzyme, thus itserves an important role in biological digestive processes, as proenzyme or pro-hormone,4 making it useful in nutrition and pharmaceutical industries . An alkaline protease isolatedfrom marine shipworm found applications as a cleansing additive; this was found to be highly6 stable in sodium perborate, hydrogen peroxide and sodium hypochloride . Hepato-pamcreasand intestine of freshwater juvenile Crayfish Cherax albidus has been used for isolation of7 protease .Protocol for obtaining marine proteases: Samples are collected under sterile conditions andimmediately transferred to appropriate medium. Protease from sources other than micro- organisms can be directly subjected to purification. Microbes are grown and preserved mainly onSabourauds agar, subjected to fermentation and then recovered using ammonium sulphate9 precipitation and dialysis . Ansari Sufiya MS.MIC.3.03UID: 158602Screening of protease activity: Diluted enzyme can be mixed with casein and incubated for 30mins, followed by addition of tricloro-acetic acid to stop the reaction. If protease is present in theenzyme solution the amino acid tyrosine is liberated along with other amino acids and peptidefragments. Folin ciocalteau reagent primarily reacts with free tyrosine to produce a blue colored3 chromophore, which is estimated spectrophotometrically .10 Azocasein assay can be performed for screening proteolytic activity of the isolatedprotease. Azocasein is an orange coloured protein substrate that can be degraded to orangefragments into the solution by hydrolytic activity of protease. After 5-24 mins cold trichloro- acetic acid is added to precipitate the orange fragments from the solution, which is estimated11 spectrophotometrically .Casien agar, Skimmed milk agar or gelatin agar can be inoculated withthe isolated enzyme and checked for the presence of a transparent zone across the inoculated10,12 area . Another method employed, was the use of casein substrates tagged to BIODIPY dyes(flourogenic protease substrate). These dyes are quenched and do not release any fluorescence inabsence of protease. However in presence of protease activity, the fluorescent peptides arereleased are detected. This is a highly sensitive method which allows screening of nano amount13 of protease . Since a large number of different protease sequences are identified, the traditionalmethods fail at times due to the un-availability of specific substrate for detection. However a14 fluorous based peptide micro-array was developed for screening of protease .MARINE LIPASESLipases are ubiquitous enzymes belonging to the class of serine hydrolases that catalyzehydrolysis and synthesis of esters from long chain fatty acids and glycerol. Lipases are alsoefficient in carrying out various reactions like aminolysis, esterification and transesterification.Marine lipases have found great applications as additives for foods flavouring, for medicalassays, cosmetics, in leather industries, pharmaceuticals, dairy industry, as detergents, in15 manufacturing of paper, fine chemical synthesis and waste water treatment . However industrialapplications of lipases are limited due to high production costs; advances in techniques and gene16 manipulation may give high yield of lipase in future for industrial uses .Lipase in marine bacteria and fungi: In 1935, the first microbial lipase was isolated from17 Penicillium oxalicum and Aspergillus flavum . Several marine isolates have been screened forproduction of extracellular lipases. Filamentous fungi are however considered as the best source15 for large scale production e.g. Rhizopus, Mucor, Pennicillium, Aspergillus and Geotrichum .Lipase from thermophilic marine microoranisms: Lipase obtained from Thermomyceslaguginosa has a wide variety of application at industrial and environmental level e.g.modification and hydrolysis of oils and fats,production of structured lipid resembling humanmilk, biodiesel production, polymer degradation,waste water treatment from meat industry,18 pretreatment of wool and for cleansing of oils or grease . A large number of thermophilic Ansari Sufiya MS.MIC.3.03UID: 158602Bacillus strains were screened for high lipase activity and Bacillus thermocatelnulatus showedhigh , inducible extracellular lipase activity which was easily extracted using hexane solvent,followed by methanol precipitation and ion exchange chromatography. The lipase was highly0 19 stable at 50-60 C and pH 7.5-8.0 .Lipase from Polychaeta: Lipase isolated from the gut of Neanthes virens was analyzed andits distribution in the gut was studied; this would help better isolation techniques for lipase20 from this organism . Lipases from crustaceans: Digestive tract of freshwater juvenile Crayfish Cherax albidushas been used for isolation of lipase. A high amount of lipase activity was detected in the7 hepato-pancreas and intestine . The hepato-pancreas of prawn Macrobrachium borellii21 showed lipase activity .Protocol for obtaining marine lipases: Samples are taken from 3m depth under sterileconditions and immediately transferred to laboratory medium to support the growth of theisolate. Lipases from sources other than micro-organisms can be directly subjected topurification. For micro-organisms, appropriate agar medium is used for isolation e.g. malt extractfungal agar medium. A group of researchers used submerged fermentation for a fungal strain ofAspergillus owamori BTMFW032 in malt extract fungal medium and appropriate aeration and15 agitation was provided . Since fungal isolates give the advantage of using surface fermentation,22 Candida rugosa was grown on solid rice bran substrate for lipase production . The crudeenzyme obtained in the fermentation broth is subjected to ammonium sulphate precipitation,15 followed by dialysis and ion exchange chromatography .Screening of lipase activity: Gel diffusion assays using various lipid substrates like Tween 20,Tween 80, olive oil, tributyrin, triolein etc into the medium. Lipolytic activity results in a clearzone around the area inoculated with the enzyme. Gel diffusion assay can also be performedusing indicator dyes like victoria blue, blue sulfate, methylene blue, night blue, phenol red andthymopthalein. On lipolysis, fatty acids are released which results in change in pH leading tochange of colour of the indicator dye.Titrimetric estimation of the fatty acids released by lipolytic activity can also beperformed; however colorimetric methods are more sensitive and widely used. Copper soapmethod is a sensitive and widely used colorimetric method for lipase detection. The free fattyacids released due to enzymatic action forms blue color soaps of cupric complexes. These areextracted in an organic solvent and estimated spectrophotometrically.Fluorescent labelled fatty acids are supplemented into the medium and quenched. Due tothe action of lipases, fluorescent fatty acids are released and estimated. Since fluorescence isused, this method is highly sensitive and can detect nano amounts of lipase in the medium. Thisis of advantage when we cannot afford to use a concentrated solution of our highly purified23 enzyme solution ."