Microbial lipases constitute one of the most of import biocatalysts for assorted industrial applications due to their stableness and selectivity ( Griebeler et al. , 2009 ) . Lipases or triacylglycerolhydrolases ( E.C.18.104.22.168 ) are one of the most demanding lipolytic enzymes in industrial intent such as hydrolysis, acidolysis, alcoholysis, esterification, trans esterification, racemic solution, two-channel selective and chiral synthesis ( Reis et al. , 2009 ; Khare et al. , 2000 ) . Most of the commercial lipases are of bacterial or fungous beginning. The major fungous lipase bring forthing genera includesAspergillus,Pencillium,Mucor,Rhizopus,Fusariumetc. Bacterial genera that are major manufacturers of lipases includesPseudomonas,Bacillus,Achromobacter,Alcaligenes,BurkholderiaandStaphylococcus. AmongPseudomonas,Pseudomonas fragi,Phosphorus.fluroscenes,P. cepaciaandPhosphorus.aeruginosahave been commercially exploited for lipase production ( Alquati et al. , 2002 ; Hazaa et al. , 2009 ; Kaieda et al. , 2001 ; Cesarin et al. , 2014 ) .
Owing to the alone belongingss of lipases like stableness, specificity and their action over a broad scope of pH and temperature, the scientific community is now focused on the production of lipases on a big graduated table. Thus lipases can be employed in nutrient, pharmaceutical, cosmetics, leather, detergent, fabric industries etc. The major restrictions in the commercial usage of lipases owes to their high production cost and deficiency of effectual downstream processing. Hence ingestion of inexpensive agro-industrial residues as substrates for lipases could cut down the production cost to a considerable degree. Detailed treatment that comes in the wining portion of this reappraisal would pass on an ample penetration into the versatilities of microbic lipases.
Structure of Lipases and their mechanism of action
Lipases are serine hydrolases moving on the carboxyl ester bonds present in glycerides to let go of fatty acids and glycerin. Their active site consists of a Ser-His-Asp/Glu catalytic three consisting of a nucleophillic serine located in a extremely conserved Gly-X-Ser-X-Gly pentapeptide ( Jaeger et al 1999 ) . The three dimensional construction of lipases revealed a characteristic ?/? hydrolase crease ( Nardini, M. , & A ; Dijkstra, B. W. ( 1999 ) . The catalytic nucleus of lipase is composed of a cardinal ? sheet consisting of up to eight different ? strands connected to six ? spirals ( Jaeger and Reetz, 1998 ) ( Figure 1 ) .
Unlike esterases, lipases are activated merely when they are adsorbed to an oil-water interface ( Martinelle et al. , 1995 ) . Lipase enzyme starts ester hydrolysis, via an onslaught by the O atom of the hydroxyl group of the nucleophilic serine residue on the activated carbonyl C of the susceptible lipid ester bond. As a consequence a transeunt tetrahedral intermediate is formed with the oxyanion stabilized by two or three H bonds, the alleged oxyanion hole. The ester bond is cleaved and the intoxicant mediety leaves the enzyme. In a last measure, the acyl enzyme is hydrolyzed. The nucleophilic onslaught by the catalytic serine is mediated by the catalytic histidine and aspartic ( or glutamic ) acid ( Cygler et al. , 1994 ; Schrag et Al 1997 ) . A conventional illustration of general lipase action is given in figure 2.
By and large bacterial lipases are glycoproteins but some extracellular bacterial lipases are lipoproteins. The production of extracellular bacterial lipases have high commercial importance as their majority production is really easy. Microbial lipases have gained particular attending due to their biochemical belongingss, simple extraction processs and handiness ( Ghosh et al. , 1996 ; Macrae and Hammond et al. , 1985 ) . The natural home grounds of lipase bring forthing micro-organisms are chiefly vegetable oil processing mills, dairies, oil contaminated dirt, coal tips, disintegrating nutrient atoms, hot springs and compost tonss ( Wang et al 1995 ) ( Table 1 ) . Bacterial lipases are classified into 8 different households. Family I being the largest and consist of 6 subfamilies ( Arpigny and Jaeger 1999 ) , Families I.1 and I.2 contain lipase from the genusPseudomonas, a good known manufacturer of lipases.
Pseudomonas represents the heterogeneous group of Gram-negative bacteriums such asPseudomonas aeruginosa, Burkholderia multivorans, Burkholderia cepaciaetc that are good known manufacturers of lipase enzyme.Pseudomonaslipases exhibit interesting belongingss such as thermo opposition and activity at alkaline pH, which makes them possible campaigners for assorted biotechnological applications. Another of import character ofPseudomonaslipases is their enantio-/ stereoselective nature, in which they have the capableness to separate between the enantiomorphs of a racemic brace ( Reetz and Jaeger 1998 ) and this alone belongings is now widely exploited in pharmaceutical and agricultural sectors. ThermostablePseudomonaslipases were reported to defy 100OC or even beyond to 150OC with a short span of a few seconds ; ( Andersson et al. 1979 ; Swaisgood and Bozoglu 1984 ; Rathi et Al. 2001 ) . An alkalic lipase, fromP. alcaligenesM-1, which was found to extinguish the fatty discolorations from fabrics under conditions of a modern machine wash ( Gerritse et al. 1998 ) . Lipase fromPseudomonas cepaciaActs of the Apostless as a most effectual catalytic agent for the ethanolysis and methanolysis of lubricating oil ( Hsu et al 2002 ) .
Substrates for Lipase Production
Most of the microbic lipases are extracellular and their production is extremely influenced by composing of the medium besides physicochemical factors ie. , temperature, pH, and dissolved O. Lipases is considered as an inducible enzyme, so these enzymes are chiefly produced in the presence of a lipid based substrate such as hydrolysable esters, oil industry wastes, vegetable oils, wetting agents, fatty acids, triacylglycerols, gall salts, glycerin and tweens ( Gupta et al. 2004 ; Sharma et Al. 2001 ; Damaso et al. , 2008 ) . Several agro residues have been reported to be effectual for lipase production and these include brans, oil bars, bagasse cottonseed and soybean sludge. These residues are abundant and inexpensive substrates for microbic lipase production. Assorted nitrogen souces like proteose peptone, barm infusion, tryptone, meat peptone, ammonium sulphate, K nitrate etc was found enhance lipase production ( Lima et al. , 2003 ) .
Owing to high nutritionary content, the agro-industrial residues are considered as really all right substrates for enzyme production. In add-on these may assist to get the better of the agricultural waste direction and environmental pollution job. Numerous surveies have been successfully completed for lipase production from agro-industrial residues via solid province agitation. Vargaset Al. , 2008 reported the production of lipase byPencillium simplicissimumutilizing soybean repast as a substrate soya bean oil and effluent from a abattoir was used as auxiliary C and N beginnings which gave a maximal lipase activity of 30 U/gds. Alkan et Al. 2007 used assorted agro residues such as rice chaff, lentil chaff, banana residue, wheat bran and Citrullus vulgaris waste supplemented with NH4NO3as N beginning and 1 % olive oil as C beginning for lipase production utilizingB. coagulans.Candida rugosaproduced lipase when a assorted solid substrate incorporating wheat bran and coconut oil bar was used giving a maximal enzyme activity of 118.2 U/g after 72 H of agitation ( Benjamin and Pandey 1998 ) . Mahanta et al. , ( 2008 ) reported lipase production byP. aeruginosaPseA utilizingJatrophaseed bar along with NaNO3as nitrogen beginning giving an enzyme activity of 625 U soman?1at a pH of 7 after 120 H of incubation.
Lipase production by agitation
Submerged agitation ( SmF ) for lipase production
Submerged agitation ( SmF ) has been defined as agitation in presence of extra H2O and it is really easy to supervise. Teng and Xu ( 2008 ) has investigated lipase production byRhizopus chinensisand obtained an enzyme activity of 13875 U/ml at an olive oil concentration of 2.367 % . Lee et al. , 1999 reported the production of thermophilic lipase fromBacillus thermoleovoransID-1 utilizing 1.5 % olive oil as exclusive C beginning giving a maximal lipase activity of 520 U/l at a pH of 7.5 and temperature 70OC. Lipase fromPseudomonas aeruginosastrain Pse A was found to be tolerant to organic dissolvers and gave a maximal enzyme activity of 4580 IU/ml in a media incorporating gum Arabic as inducer.
Solid province agitation ( SSF ) for lipase production
Solid province agitation ( SSF ) is a microbic procedure in which a solid stuff is used as the substrate, by which micro-organism grow good and bring forth larger sums of excess cellular enzymes and other metabolites than they do in SmF. Mahanta et al. , 2008 reported the production of lipase byPseudomonas aeruginosaPse A utilizing deoiledJatrophaseed bar as C beginning and obtained an enzyme activity of 625 U/g. Shukla et al. , 2011 used different low cost oil bars like Jatropha oil bar, Teesi oil bar, Mustard oil bar and Ground nut oil bar as substrate for lipase production by Rhizopus oryzae KG-10 via SSF. Maximum enzyme activity of 170 IU was obtained with Mustard oil bar after 7 yearss of incubation.
Purification schemes for bacterial lipases
In nature, most of the lipase enzymes are extracellular and hence may hold residuary bacterial cells, medium constituents and different metabolites. So purification of lipase from the cultural medium is one hard undertaking faced by industries. In many instances, enzymes used in commercial applications need non necessitate high pureness ( eg. detergent industry ) , but in medical, decorative, nutrient and for analyzing protein construction the desirable enzyme must be needed in high pureness ( Taipa et al. 1992 ; Aires-Barros et Al. 1994 ; Saxena et Al. 2003 ) . Ideal purification schemes adopted in industries should be of low-priced, speedy, high-yielding and agreeable to large-scale operations. In add-on, it must hold the capableness for uninterrupted merchandise recovery, with a comparatively high selectivity for the coveted merchandise. About 80 % of the purification schemes attempted therefore far have used a precipitation measure( Table 2 ). This is followed by gel filtration, and ion exchange chromatography. Recently there are a figure of purification engineerings established for the purification of lipases. These include immune purification, aqueous two-phase systems, reversed micellar system, membrane procedures, hydrophobic interaction chromatography using an epoxy activated spacer arm as a ligand, column chromatography with PEG ( polyethylene ethanediol ) / Sepharose gel or poly ( vinyl intoxicant ) polymers as stationary stages and aqueous two-phase systems ( Saxena et al. 2003 ) .
Properties of bacterial lipases
Lipases from several micro-organisms have been studied extensively. Depends on the enzyme belongings have to make up one’s mind the choice of industrial application. Assorted belongingss of bacterial lipases such as pH, temperature, stableness, effectual of metal ions, substrate specificity are summarized under the below subdivisions.
pH and temperature dynamicss
Normally, bacterial lipases shows impersonal ( Dharmsthiti et al.1998 ; Lee et Al. 1999 ) or alkalic pH optima ( Schmidt-Dannert et Al. 1994 ; Sidhu et Al. 1998a, 1998b ; Sunna et al. 2002 ) , by the freedom ofP. fluorescensSIK W1 lipase, which has an acidic optimum at pH 4.8 ( Andersson et al. 1979 ) . Lipases fromBacillus stearothermophilusSB-1,B. atrophaeusSB-2 andB. licheniformisSB-3 show activity in wide pH scope ( Bradoo et al. , 1999 ) . Bacterial lipases exhibit stableness over a broad scope, from pH 4 to pH 11 ( Kojima et al. 1994 ; Wang et Al. 1995 ; Khyami-Horani, 1996 ; Dong et Al. 1999 ) . In by and large bacterial lipases posses temperature optima in the scope 30–60 °C ( Lesuisse et al. 1993 ; Wang et Al. 1995 ; Dharmsthiti et Al. 1998 ; Litthauer et Al. 2002 ) . But, reports suggest that bacterial lipases exhibit temperature optima in lower every bit good as higher scopes ( Dharmsthiti and Luchai 1999 ; Lee et Al. 1999 ; Oh et Al. 1999 ; Sunna et al. 2002 ) . Nawani and Kaur ( 2000 ) reported that the thermostability of the lipase enzyme byBacillussp was improved by the add-on of stabilizersi.e., glycerin, sorbitol, ethene ethanediol which retained enzyme activity at 70 °C even after 150 min. A few species formPseudomonashave been claimed that the enzyme are stable at 100 °C or even beyond to 150 °C with a really short life span of a few seconds ; ( Andersson et al. 1979 ; Swaisgood and Bozoglu 1984 ; Rathi et Al. 2001 ) .B. stearothermophiluslipase reported as a extremely thermo tolerant enzyme with a half life of 15–25 min at 100 °C ( Bradoo et al. 1999 ) .
Stability in organic dissolvers
Lipase enzyme stableness in organic dissolvers is desirable for utilizing them in assorted chemical reactions. Schmidt-Dannert et Al. ( 1994 ) reported that propanone, ethyl alcohol and methyl alcohol enhanced the activity ofB. thermocatenulatuslipase, but propanone and hexane inhibited the action ofP. aeruginosaYS-7 lipase andBacillussp. lipase ( Sugihara et al. 1991 ) . Lipase fromA. calcoaceticusLP009 showed extremely unstable nature with assorted organic dissolvers ( Dharmsthiti et al. 1998 ) .
Consequence of metal ions
Cofactors are usually non necessary for lipase activity, but Ca ( bivalent cations ) frequently enhances the enzyme activity. Metallic ions such as Co, Hg and Sn were found to suppress the activity of lipase enzyme ( Patkar and Bjorkling, 1994 ) . Calcium- activated lipase enzyme have been reported in figure of bacterial species such asP. aeruginosaEF2 ( Gilbert et al. 1991 ) ,B. thermoleovoransID- 1, ( Lee et al. 1999 ) ,B. subtilis168 ( Lesuisse et al. 1993 ) ,S. aureus226 ( Muraoka et al. 1982 ) . In contrast, lipase action was inhibited by the presence of Ca ions inP. aeruginosa10145 ( Finkelstein et al. 1970 ) . However, the lipase fromA. calcoaceticusLP009 was stimulated by the being of Fe3+( Dharmsthiti et al. 1998 ) .
Application of lipases
Lipases in detergent industry
The hydrolytic lipases, are chiefly used in industrial wash and family dish washers. To increase the action of detergence, a combination ofvarious enzymes such as lipase, amylase, peptidase and cellulase are used in modern types of heavy responsibility pulverization detergents and automatic dish washer detergents ( Ito et al 1998 ) .The chief advantages of these biological detergents are high biodegradability, deficiency of any harmful residues, have no negative impact on sewerage intervention procedures and do non do a hazard to aquatic life. Lipases have the capableness of taking the oil fatty contents in really promising mode owing to its suitableness under rinsing conditions. In a detergent industries, lipases should be thermophilic and alkalophilic nature. In add-on, it should be capable of executing in the presence of the assorted constituents of rinsing pulverization preparations ( Posorske 1984 ; Cheetham 1995 ; ) . In many instances lipases exhibit optimum activity at impersonal pH and in a temperature scope of 35–48 °C ( Cheetham 1995 ) .Pseudomonas alkaligeneslipase showed elevated activity at rinsing conditions, such as alkalic pH ( 7–11 ) and at a high temperature up to 60 °C ( Misset et al. , 1994 ) . Novo group has introduced aStreptomycesssp. lipase which is alkaline in nature and positionally non-specific so that it can be used in broad scope of applications like wash, dish-washing detergents and industrial cleaners intents.
Lipases in nutrient engineering
Fat and oil change is one of the major countries in nutrient processing industry that becomes a big possible market in future. Lipases have been added to nutrient to better gustatory sensation by production of esters in short concatenation fatty acids and intoxicants ( Macedo et al, 2003 ) . The lipases play a critical function in the agitation procedure of sausage production and to modulate the alterations in long-chain fatty acerb add-on in maturing. Lipases are used to hydrolyze oil from soyabean for doing Koji, a traditional Asiatic nutrient ( Aravindan et al. , 2007 ) . Lipase mediated nutrient merchandises in the market include staff of life, nutraceuticals, cocoas etc. Before, lipases of diverse microbic beginning have been used for cleansing rice spirit, changing soybean milk and for advancement the olfactory property and increase the agitation of apple vino ( Seitz1994 ) .
The preliminary grounds was found the antibiotic activity of lipases (Galleria mellonella) againstMycobacteria TB( MBT ) H37Rv. Lipases may be used as digestive AIDSs ( Gerhartz 1990 ) and the activators of Tumor Necrosis Factor accordingly can be used for the intervention of malignant tumours ( Kato et al 1989 ) . HGL ( Human gastric lipase ) was the most stable acerb lipase that comprised a all right campaigner tool for enzyme permutation therapy ( Ville et al 2002 ) . Lipases have been used for the intervention of cutaneal manifestations of digestive allergic reactions, GI perturbations, indigestion, etc ( Mauvernay et al 1970 ) . Lovastatin a drug synthesized fromCandida rugosalipase was used for the decrease of serum cholesterin degree in human organic structure ( Yang et al 1997 ) .
Pulp and paper industry
Microbial lipases have some important function in for mush production industries. Lipase enzyme was used for the fabrication of large-scale paper-making since early 1990s ( Bajpui 1999 ) . Lipases are widely used for increasing the pulping rate of mush, to augment the whiteness and strength and deinking of wastepaper. So it can assist the lessening of chemical use, prolong equipment life, diminish the hazard of pollution degree in H2O and cut down composite cost. Lipases are used to take hydrophobic constituents of wood known as ‘pitch’ ( Irie et al. , 1993 ) .
Use of lipase in fabric industry
Lipases in fabrics industries is chiefly used for the remotion of size lubricators and to give a cloth with better absorbency for enhanced levelness in dyeing. In add-on it reduces the opportunity for frequence of line and interruption in denim scraping systems. Lipase enzymes commercially used for the readying of the desizing of jean and other cotton cloths ( Cortez 2000 ) . JP 5344897 A by Amano Pharmaceutical KK is a commercial lipase which is dissolved in solution with an aliphatic polyester which improved fabric texture without losing its strength ( Dyson et al. , 2006 ) . Moisture recovering ability of polythene terephthalate cloths was found to be improved upon utilizing lipases fromP. cepaciaandP. fluorescens. Lipase fromPseudomonasspp. can degrade the polymers of aliphatic polythene. Genencor International, Inc. has submitted a patent ( PCT Publication No. 97/33001 ) sing a new techniquei.e., handling polyester cloths with lipase for bettering the wetting ability and optical density ( hypertext transfer protocol: //www.wipo.int ) .
Therefore the latest tendency in lipase research is the development of new and improved lipases which have broad industrial applications and they play a important function in the turnover of H2O indissoluble compounds.