catalysis of heterogeneous reactions in water-soluble as well as insoluble systems. Further, lipases have the properties like chemo-specificity, region-specificity, and stereo-specificity [111]. When classification is made based on region-specificity, there come three classes of lipases: 1) non-specific lipases, 2) 1,3-specific lipases, and 3) fatty acid-specific lipases. Non-specific lipases have ability to attach with all the possible positions of triglycerides to give FFAs and glycerol. The intermediates of the reaction, diglycerides, and monoglycerides do not accumulate in the reaction as they are instantly hydrolysed into fatty acids and glycerol [112]. 1,3-specific lipases are specific for the 1 and 3 positions of triglycerides and remove fatty acids from these positions. 1,3-specific lipases carry out the conversion of triglyceride to diglycerides much faster than diglyceride to monoglyceride [113]. Fatty acid-specific lipases carry out hydrolysis of a specific type of esters which have double bonded long chains of fatty acids in cis position between C-9 and C-1. Hydrolysis of esters with unsaturated fatty acids occur slowly and such class of lipases is not much common [114]. All the hydrolytic enzymes including lipases have common folding pattern involve in a hydrolytic activity called α/β hydrolase fold which is made up of a β sheet of eight strands (one of which is antiparallel while remaining seven strands are parallel) connected by α helices. Histidine residue, catalytic acid residue and Nucleophilic residue are present in α/β hydrolase fold. Pentapeptide sequence (Gly-X-Ser-X-Gly) which is a highly conserved in most of the lipases involved in the construction of ‘nucleophilic elbow’ which is a typical β-turn-α motif having active nucleophilic serine residue between a β strand and an α-helix. Catalytic triad made up of amino acids like histidine, serine, and aspartic acid or glutamic acid build the active site of lipases. The same catalytic triad is seen in serine proteases predicting common catalytic mechanism in them. Amphiphilic α helix peptide sequence forms a lid or flap which covers the active site of lipase and has a structural variability depending upon the lipase source organism. Changes in the structure of the lid are responsible for the activation/inactivation of lipases [114]. Changes in the conformation of lipase structure as well as the quality and quantity of interface being used in the reaction are responsible for the activation of lipase. When the lipase enzyme meets the oil/water interface there occur some changes in lipase structure that results in its activation. For the activation of lipase first, the lid opens to uncover the active site of lipase upon its contact with the ordered interface [115]. Due to this restructuring of lipase, electrophilic region is created around serine residue present in active site, lid hydrophilic side which was exposed in native form now partly buried inside the polar cavity and hydrophobic side of lid completely exposed, thus creating a non-polar surface around the active site for efficient attachment of lipid interface with it [115].
1.7.1 Mechanisms of Lipase Action
Lipases interact with ester bonds of their substrate like acylglycerols to catalyze the reactions of hydrolysis, synthesis, and transesterification. Triglycerides, which are insoluble and long chained fatty acids, are precisely catalyzed by lipases [113]. Lipase carries out triglyceride oil transesterification with methanol in three reversible steps with the first step for conversion of triglycerides to diglycerides followed by the second step of diglycerides to monoglycerides conversion, and finally, monoglycerides convert into glycerol molecules. Here, each conversion step produces one FAME molecule; hence, a total of three FAME molecule are produced from one triglyceride [116]. Two models are mainly under discussion to describe the kinetics mechanism for esterification reactions, Michaelis-Menten kinetics and Ping Pong Bi Bi model. Lipase catalyzed esterification mainly elaborated by Ping Pong Bi Bi mechanism which is a bi-substrate reaction that releases two products. It involves following steps: 1) acyl-donor donate their acyl group to the enzyme resulting in the formation of acyl-enzyme complex, 2) release of the water molecule as a product, 3) binding of acyl acceptor with the enzyme complex, and 4) release of ester [117, 118]. Many researchers made some modifications in this model depending upon inhibiting factors [118]. The catalytic activity begins with the transient tetrahedral intermediate formation with a negatively charged carbonyl oxygen atom. The reaction between the hydroxyl group oxygen present in nucleophilic serine residue of lipase enzyme and activated carbonyl carbon of the substrate involved building this transient tetrahedral intermediate. The intermediate thus formed is stabilized by its interaction with two peptide NH groups. After that nucleophilic hydroxyl group of water react with the carbonyl carbon of acyl-enzyme complex resulting in the formation of acyl product and enzyme is released for further catalysis [119].
1.7.2 Efficient Lipase Sources for Biodiesel Producing Biocatalyst
Lipases can be obtained from plants, animals, and microorganisms and based on that lipases can be classified on it as plant, animal, and microbial lipases depending on their origin respectively. Lipases from different sources with different structure has different properties and catalytic activities. We can use this to counter lipase problems in biodiesel production like lipase cost and methanol inhibition. We need to optimize reaction conditions according to chosen substrate and lipase from specific source [107]. Microbial lipases are widely used at industrial and commercial level as biocatalysts for biodiesel production because microbial lipases are more stable and can be produced in bulk amount from microorganism [23]. Microbial lipases can be manipulated genetically with ease, seasonal changes have nothing to do with lipase production, and rapid growth of microbes makes them the ideal candidates as lipase source [120, 121]. Use of microbial lipase is increasing day after day and currently 5% of the world enzyme market is being shared with microbial lipases [121]. Microbial lipase weighs around 30–50 kDa and their optimum pH to work at is 7.5. Based on temperature tolerance, microbial lipases can be categorized as mesophilic and thermophilic lipases. Mesophilic lipases normally work at 35°C–50°C and they become denature above 70°C while thermophilic or thermostable lipases normally work at 60°C–80°C, but some also work at 100°C under specific conditions [122, 123]. According to Hotta et al. [123], lipases from Pyrobaculum calidifonti (hyperthermophilic archae) showed its activity at 90°C. Similarly, thermostable lipases from Caldanaerobacter subterraneus and Thermoanaerobacter thermohydrosulfuricus (highly thermophilic bacteria) performed well in the range of 40°C–90°C. They not only performed well at high temperatures but also were resistant toward organics solvents [124]. Lipase producing microorganisms can be isolated from soil, waste water, marine water, and industrial wastes. Isolates of Mucor, Sclerotina, Candida, and Aspergillus strains have been reported, which were isolated from soil. Similarly, various strains of microbes such as P. alcaligenes, Bacillus acidophilus, Enterobacter intermedium, P. fluorescens, and Geotrichum asteroids are also reported as lipase producing strains when isolated from vegetable oil processing plants [141]. Screening of microbes is done by checking their lipolytic. Generally, lipases are screened using batch cultures having agar as substrate but this is time consuming. So, the two mostly used methods for lipase production are solid state fermentation and using submerged culture [142]. Purified form of lipases is used in biochemical reaction to get maximum benefit from it, so purification is required. Purification of lipase requires several techniques including ammonium sulfate precipitation or ultrafiltration and after that more sensitive and advanced techniques are utilized like gel filtration, ion exchange chromatography, and affinity chromatography [143]. Moreover, some other novel techniques can also be applied to purify lipases such as immunopurification, column chromatography, hydrophobic interaction chromatography, and membrane process. Generally, the strategy used for purification lipases starts with the removal of lipase producing cells from their growth culture to get extracellular lipases after fermentation. Then, the extract without cells is concentrated by organic solvents, ultrafiltration, and precipitation using ammonium sulfate. Ammonium sulfate precipitation is used in the first stages of purification and it crudely separate out things from mixture. After that advanced techniques of chromatography are used to finely purify lipases [144]. According to Javed et al. [112], diverse data from various research work suggested that purification had been done from 2.4 to 500 folds with an increase in yield from 10.3% to 36%. Effective production and purification strategies of lipases are being designed to get maximum yield at a very small expense. Among microbial lipases, the most commonly used lipase sources are bacteria, fungi, yeast, and algae; see Table 1.1 for some bacterial