Biochimie 182 (2021) 23e36

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Biochimie

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Current perspectives for microbial lipases from extremophiles and metagenomics

** * Swati Verma a, Gautam Kumar Meghwanshi a, , Rajender Kumar b, a Department of Microbiology, Maharaja Ganga Singh University, Bikaner, 334004, India b Department of Clinical Microbiology, Umeå University, SE-90185, Umeå, Sweden article info abstract

Article history: Microbial lipases are most broadly used biocatalysts for environmental and industrial applications. Li- Received 21 July 2020 pases catalyze the hydrolysis and synthesis of long acyl chain esters and have a characteristic folding Received in revised form pattern of a/b hydrolase with highly conserved catalytic triad (Serine, Aspartic/Glutamic acid and His- 21 December 2020 tidine). Mesophilic lipases (optimal activity in neutral pH range, mesophilic temperature range, atmo- Accepted 31 December 2020 spheric pressure, normal salinity, non-radio-resistant, and instability in organic solvents) have been in Available online 7 January 2021 use for many industrial biotransformation reactions. However, lipases from extremophiles can be used to design biotransformation reactions with higher yields, less byproducts or useful side products and have Keywords: Microbial lipases been predicted to catalyze those reactions also, which otherwise are not possible with the mesophilic Extremophilic lipases. The extremophile lipase perform activity at extremes of temperature, pH, salinity, and pressure Metagenomics which can be screened from metagenome and de novo lipase design using computational approaches. Classification Despite structural similarity, they exhibit great diversity at the sequence level. This diversity is broader Structural information when lipases from the bacterial, archaeal, plant, and animal domains/kingdoms are compared. Furthermore, a great diversity of novel lipases exists and can be discovered from the analysis of the dark matter - the unexplored nucleotide/metagenomic databases. This review is an update on extremophilic microbial lipases, their diversity, structure, and classification. An overview on novel lipases which have been detected through analysis of the genomic dark matter (metagenome) has also been presented. © 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Contents

1. Microbial lipases ...... 24 2. Types and sources of extremophilic microbial lipases ...... 24 2.1. Archaeal lipases ...... 24 2.2. Extremophilic lipases ...... 25 2.2.1. Thermophilic lipases ...... 25 2.2.2. Psychrophilic lipases ...... 26 2.2.3. Halophilic lipases ...... 26 2.2.4. Acidophilic lipases ...... 26 2.2.5. Alkaliphilic lipases ...... 26 2.3. Lipase from microbial metagenomics “dark matter” ...... 27 2.3.1. Hot springs metagenomics ...... 27 2.3.2. Soil metagenomics ...... 27 2.3.3. Marine environments metagenomics ...... 27 3. Classification of microbial lipases ...... 28 3.1. Based on protein sequences and physiological properties ...... 28

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (G.K. Meghwanshi), rajender.kumar@ umu.se, [email protected] (R. Kumar). https://doi.org/10.1016/j.biochi.2020.12.027 0300-9084/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36

3.2. Types of microbial lipases based on function ...... 28 3.2.1. Regioselective (regiospecific) lipases ...... 28 3.2.2. Substrate specific lipases ...... 29 3.2.3. Non-specific lipases ...... 29 3.2.4. Enantioselective lipases ...... 29 4. Current status of microbial lipase structure ...... 29 5. Lipase engineering ...... 31 6. Conclusion ...... 32 Authors contributions ...... 32 Funding...... 32 Declaration of competing interest ...... 32 References ...... 32

1. Microbial lipases physico-chemical properties of the lipase [9]. Most of the commercially accessible lipases are immobilized on different Enzymes or biocatalysts are backbone of the biotechnology- matrices [17]. Commonly used immobilized lipases are: Novozym based industries. They are produced by all living beings, but ma- 435, Lipozyme TL IM and Lipozyme RM IM which are secreted from jority of the industrial enzymes come from microbial sources. At Candida antarctica, Thermomyces lanuginosus and Rhizomucor mie- present, more than 200 microbial enzymes are used commercially hei, respectively [17]. and approximately 20 types are produced on industrial scales [1,2]. Lipases have been reported from a huge number of microbes but Most of the industrially significant enzymes are hydrolytic in na- only a limited number of microbial lipases have been fully char- ture; of which lipolytic enzymes play key roles in the conversion of acterized and purified, and used for industrial purposes [18]. There hydrophobic lipids, that constitute the abundant biomass on earth are some most imperative lipase producing bacterial genera for [3,4]. Lipases are catalytically active in organic solvents, which example Bacillus [19], Staphylococcus [20], Arthrobacter [21], Ach- make them ideal tools for organic chemists [4,5]. Lipases are romobacter [22] and Pseudomonas [23]. Many microbial lipases defined as carboxylesterases, which catalyze the hydrolysis and have been found which are thermostable (optimally active at synthesis of esters of glycerol and long chain fatty acids [6]. temperatures >70 C) and alkali stable (optimally active at alkaline The first microbial lipase was obtained from Aspergillus flavus pH of >9) [24e27]. Only a few microbial lipases have been studied and Penicillium oxalicum in 1935 [7]. The natural substrates of lipase from thermoalkaliphilic bacteria [26]. A potential lipase was iso- are long chain acylglycerols, (10 C-atoms) whereas the other lated and characterized from bacterium Acinetobacter sp. strain lipolytic enzymes grouped under esterases act on short chain SU15. The activity of this lipase was not affected by organic solvents acylglycerols. Besides, an important characteristic of lipolytic en- and metal ions. The lipase activity was partially inhibited by ethyl þ zymes is the exclusive physico-chemical characteristic of the re- acetate, butanol and metal ion-Ni2 [28]. actions they catalyze at lipid-water interface [8]. A number of The extremophilic bacterial lipases are more economical and studies have been carried out to evaluate the activities of microbial stable and several extremophilic bacterial species such as Bacillus lipases under different physical, chemical and environmental con- thermoleoverans, B. stearothermophilus, thermoacidophilic bacte- ditions [3,6,9e11]. Some recent publications have comprehensively rium, B. acidocaldarius and alkaliphilic bacterium, Bacillus sp. are discussed the research on lipases obtained from thermophilic and commonly used for different applications [6,16,29e31]. In the alkaliphilic microbes [12e14]. Further information on a huge public domain database UniProt (www.uniprot.org), more than number of bacterial but only a few archaeal lipolytic enzymes is 75000 true lipase protein sequences have been reported at present available with online databases like Lipase Engineering Database (2020), out of which only 182 are reviewed and more than 4700 are (LED), the Microbial Esterase and Lipase Database (MELDB, http:// reported only from bacterial domain. Only a few lipase proteins www.gem.re.kr/meldb), ESTHER database (http://bioweb.ensam. sequences have been reported from Archaea such as from the inra.fr/esther) and Uniport (https://www.uniprot.org/). These da- Euryarchaeota and TACK group, and Candidatus Heimdallarchaeota. tabases provide considerable information on lipase classification Microbial lipases have huge diversity at sequence level as well as in and recent functional and biochemical studies on them. Many 3D their physiological and chemical properties. The present investi- crystal structures of lipases have been resolved originating from gation shows that the archaeal lipase sequences have homology to bacteria, fungi, and higher organisms with natural substrate and bacterial and fungal lipase sequences (Fig. 1). some with metal ion and inhibitors. 2.1. Archaeal lipases 2. Types and sources of extremophilic microbial lipases Archaea are excellent source for extremozymes (enzymes active Microbial lipolytic enzymes are distinct biocatalysts for envi- under extreme environmental conditions) for biotechnological and ronmental and industrial applications. They have been in more industrial processes due to their unique stability at high tempera- demand and have become indispensable for biotechnological ap- tures, extreme pHs, in organic solvents, and in highly salty envi- plications over plant and animal lipases due to their wide-ranging ronments. Owing to these salient features, enzymes from archaea properties [6,15,16]. The lipases produced by microorganisms can are suitable for a broad range of biocatalytic applications in various be extensively used for different activities/applications in various industries and environmental management [33]. The archaeal en- forms, such as extracellular, intracellular, immobilized/dry or liquid zymes can act as super biocatalysts and catalyze bio- [17]. Microbial lipases are mostly extracellular which can be pro- transformations under extreme reaction conditions normally duced by submerged or solid-state fermentation. Purification of required for industrial biochemical processes. Many thermophilic lipases is a difficult process and it depends on the origin, and and hyperthermophilic lipases/esterases have been reported from

24 S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36

Fig. 1. Evolutionary analysis of the microbial lipases. The diverse lipase sequences from- Fungi, Algae, Bacteria and Archaea were considered for this analysis. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances, estimated using a JTT model, and then selecting the topology with superior log likelihood value using MEGA X [32]. The Archaeal sequences are marked in red. Phylogenic tree is based on the representative set of sequences, which have been selected based on 50% sequence identity among the reviewed entries.

Archea, e.g. Pyrococcus furiosus (lipase with optimal activity at 2.2.1. Thermophilic lipases 80 C) [34], Archaeoglobus fulgidus (lipase with optimal activity at Microorganisms living at high temperatures are called ther- 90 C) [35], Pyrobaculum sp. 1860 (Esterase with optimal activity at mophiles. They can be divided into three types (i) Moderate ther- 80 C) [36] and Sulfolobus tokodaii strain 7 (esterase with optimal mophiles (optimal growth between 45 and 65 C) (ii) Thermophiles activity at 70 C) [37]. Archeal lipases/esterases have been reported (optimal growth between 65 and 85 C) and Hyperthermophiles from Sulfolobus, Aeropyrum, Archaeoglobus, Haloarcula and Hal- (optimal growth at ˃ 85 C) (Demirjian et al., 2001). Many lipases ococcus [33]. Ozcan et al. (2009) reported lipolytic enzymes from which are obtained from mesophilic microorganisms have wide pH halophilic isolates, which exhibited optimal activity at moderately tolerance but most of them are not thermostable above 70 C. A high temperatures (60e65 C) and at high pHs (pH 8e8.5) [38]. In number of research findings on moderately thermostable lipases addition, the isolates showed more of esterase activity compared to from mesophilic microbes have been reported [27,40]. Mainly lipase activity based on kinetic parameters. Verma et al. (2019) thermoactive lipases have been reported from Bacillus sp. (optimal screened some lipases from archaea via bioinformatics approach, activity at 60 C) [41,42], Bacillus thermoleovorans ID-1 (optimal most of them were alkaliphilic from the Haloarchaea [14]. These activity from 70 to 75C)[43], Bacillus thermocatenulatus (optimal newly explored lipases are potentially useful for biotechnological activity from 60 to 70 C) [44], Geobacillus stearothermophilus applications and have broadened knowledge on the diversity of (formerly Bacillus stearothermophilus) (optimal activity from 68 C) lipases and future lipase engineering prospects. [42,45], Bacillus sp. J33 [46] and several Pseudomonas spp [47]. and some Lactobacillus spp. [48]. A highly thermostable (up to 100 C) 2.2. Extremophilic lipases lipase has been reported from Geobacillus stearothermophilus [49]. The structural characterization of lipase from G. stearothermophilus The categorization of extreme environments refers to a wide showed that it contains a unique zinc-binding site, which may be variety of normally non-inhabitable environments to which mi- accounted for its thermal stability [50]. A few strains of B. subtilis croorganisms have adapted. Extremophilic organisms are evolved [51] and B. coagulans [52] also produce lipases with thermo- to exist in a diversity of extreme environments and they are cate- tolerant property. A thermostable lipase mutant was generated gorized into different classes that comprise thermophiles, alka- and characterized by Sharma et al. (2012, 2014), that mutant lipase liphiles, acidophiles, halophiles and psychrophiles [39]. showed 144-fold enhanced thermostability over the wild type

25 S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36 enzyme at 60 C[24,53]. bacteria capable of producing industrial hydrolases. They identified A thermostable lipase was purified and characterized from some halophilic bacteria such as Marinobacter, Virgibacillus, Chro- Geobacillus thermodenitrificans which showed highest activity at mohalobacter, Halobacillus, Bacillus and Halomonas genera on the 60 C[54]. Avila-Cisneros et al. (2014) reported fungal thermophilic basis of morphological, biochemical and 16S rRNA analysis. Ozcan lipases, with a notable example of Thermomyces lanuginosus [55]. et al. (2009) reported lipolytic enzymes from five different halo- The maximum activity of this lipase was obtained between 60 and philic archaeal strains which were active at high temperatures 80 C. Devi et al. (2015) reported a thermophilic lipase from Pseu- (60e65 C), alkaline pHs (pH 8e8.5) and high salt concentration domonas sp. VITSDVM1 isolated from bovine milk [56], which (3.4e5 M NaCl) [38]. An extremely halophilc lipase producing exhibited optimum activity at 40 C. A thermostable lipase (LipSm) bacterium Halomonas sp. LM1C was able to grow and produce li- from Stenotrophomonas maltophilia Psi-1 from Psittaleia, Greece has pases in hypersaline environments (up to 20% of NaCl) [74]. Lipase been assigned as the first member of the recently proposed lipase production has been reported from marine bacterial species like family X1X [57]. Interestingly, this lipase has the high percentage of proteobacterium Alteromonas isolated from Bay of Marseille, France alanine residues along with the occurrence of the AXXXA motif [75] and Alcanivorax borkumensis [76]. Psychrobacter okhotskensis nine times, suggesting that it is a thermostable lipase. Recently, a isolated from the Okhotsk Sea, have been shown to exhibit lipolytic novel thermostable lipase from marine Geobacillus sp. 12AMOR1 activity on Tween 20e80 in 0e10% NaCl [77]. Lipase has been re- has been characterized by Wei Tang et al. (2019), which exhibited ported from an extremely halophilic archaeon, Natronococcus sp. optimal activity at 60 C[58]. Many thermophilic and hyperther- strain TC6, which exhibited optimal activity at 4 M NaCl, 50 C and mophilic lipases/esterases have been reported from Archaea (dis- pH 7. Besides halophilic lipases have been reported from Halomonas cussed in Archaeal lipases section). sp. LM1C, Oceanobacillus, and Exiguobacterium species [74], Fusa- rium solani strain NFCCL 4084 [78] and Marinobacter sp. EMB5 [79]. 2.2.2. Psychrophilic lipases Psychrophilic enzymes have become important for industrial 2.2.4. Acidophilic lipases applications, as they can be employed to design the processes/ The microorganisms of acidic environments have great di- technologies with decreased energy demand. There is a great de- versity, are mainly autotrophic and/or mixotrophic [80]. These mand for cold active enzymes in the detergent industry. The use of microbial communities produce stable extremozymes in conditions cold-active hydrolytic enzyme such as lipases in the preparation of of extreme temperature and pH and are tolerant to solvents and detergents would be a significant improvement for cold washing as detergents [80,81]. Therefore, the search for extremozymes pro- it will decrease the energy demand and will result in longer cloth duced by acidophilic microorganisms represents a major research life due to reduced wear and tear. Paper manufacturing industries focus. The acidophilic microorganisms have also been investigated also require low temperature active enzymes used for polymer- for particular physical, structural, and molecular properties that degradation and pitch removal for enhancing the quality of the allow them to live in acidic environments [82]. An extremely paper. Few bacterial species have been isolated and characterized acidophilic lipase with optimum activity at pH 1 has been reported from deep-sea sediments where temperature is below 3e4 C[59]. from B. pumilus [83]. An extremely acidophilic esterase with pH Psychrophilic lipases have been reported from Photobacterium lip- optimum of 1.5 has been reported from Ferroplasma acidiphilum olyticum (optimum activity at 25 C; temperature tolerance from 5 [84]. Choi et al. (2016) have reported a moderately acidophilic to 25 C) [60], Aeromonas sp. (optimal activity at 5 C) [61], Pseu- lipolytic enzyme from thermoacidophilic archaeon Sulfolobus sol- doalteromonas sp. (optimal activity at 20e30 C) and Psychrobacter fataricus P1 having pH optimum of 6.0 [85]. Conversely, the pH sp. (optimal activity at 20e30 C) [62,63]. Maximum lipase pro- optimum of esterase from acidophilic bacterium Bacillus acid- duction by these bacteria occurred in the temperature range of ocaldarius was obtained at pH 7.1 [86]. Similarly, lipase from ther- 10e20 C; however, their lipases exhibited optimal activity in the moacidophilic archaeon Sulfolobus tokodaii strain 7 exhibited temperature range of 20e30 C. Cold regions such as glaciers and optimal activity at pH 7.5e8.0. Similarly, Merone et al. (2005) has mountains are another habitats for psychrophilic lipase producing reported phosphotriesterase from acidophilic archaeon Sulfolobus microorganisms [63]. Cold active lipases from Psychrophilic mi- solfataricus having optimal activity in the pH range of 7e9[87]. croorganisms of Alpine regions have also been studied [59]. Only a few lipolytic fungi are reported to produce psychrophilic lipases. 2.2.5. Alkaliphilic lipases The cold active lipase from Candida antarctica is extensively studied At present, alkaliphilic and alkali tolerant lipases have gained [64,65]. Other species such as Candida lipolytica, C. albicans,C. zey- enormous interest as biocatalyst due to their stability and robust- lanoides, Geotrichum candidum and Penicillium roqueforti have also ness under alkaline conditions. Alkaliphilic and alkali tolerant li- been reported to produce cold active lipases [66]. pases have many industrial applications such as in the production of biodiesel and biodetergents, application in bioremediation of 2.2.3. Halophilic lipases wastes, in biodegreasing, and other types of bioconversions [88]. In general, halophiles grow in hypersaline environments by Numerous alkaliphilic lipases of microbial origin have been their ability to maintain osmotic balance with varying concentra- discovered so far. A recombinant moderately alkaliphilic lipase tions of different salts such as NaCl, KCl, etc. A number of halophilic from Bacillus subtilis DR8806 has been reported, exhibiting pH bacteria which produce extracellular hydrolases have been re- optimum of 8.0 [89]. A highly salt-tolerant and moderately alka- ported by Sanchez-Porro et al. (2003) [67]. Lipases from the halo- liphilic esterase with pH optimum of 8.0 and high stability in the pH philes show activity at very high salt concentrations [44,68,69]. range of 5.0e11.0 has been reported from Bacillus licheniformis [90]. Archaeal domain is the main source of extremely halophilic en- A thermoalkaliphilic lipase has been reported from Aeribacillus zymes. Many potential halophiles and their haloenzymes have pallidus [91] with pH optimum of 10.0. This lipase was highly stable, previously been reviewed [70,71]. The halophilic bacteria have it maintained 100% of its original activity in the pH range of 9e11 been isolated and characterized from different hypersaline habi- after incubation at 40 C during 1h. Lipase from Bacillus sp. LBN2 tats, such as salt lakes, solar salterns, saline deserts and saline de- had optimal activity at pH 10 and it was stable in the pH range of posits [72]. Kumar et al. (2012) studied the diverse halophilic 8e11 [92]. The recombinant lipase of Cohnella sp. A01 exhibited bacteria producing enzymes of industrial importance [73]. They maximum activity at pH 8.5 and was stable in the pH range of screened different saline habitats across India to isolate halophilic 8.5e10.0 for 180 min [93]. Lipase of Bacillus sp. RSJ-1 also exhibited

26 S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36 maximal activity at pH 8.5 and was stable in a pH range of 8.0e9.0 metagenomic lipase is OSTL28 was isolated from an oil contami- for 120 min [94]. The lipase Trichoderma harzianum IDM14D was nated site in China [100]. This lipase was highly stable in organic stable in the pH range of 8.0e10.0 with an optimum enzymatic solvents and glycerol, a feature required for catalyzing the sus- activity at pH 8.5 [95]. Another alkaliphilic fungal lipase has been tainable reactions under micro-aqueous conditions, like the pro- reported from Aspergillus fumigatus, exhibiting optimum activity at duction of biodiesel, flavors, and fragrance compounds. pH 9.0 [96]. The diversity of HSL lipases from glacier soils was carried out using the metagenomic approach [101]. Bio-prospecting for new 2.3. Lipase from microbial metagenomics “dark matter” lipases/esterases from Arctic soils had been carried out through metagenomics, which resulted in isolation of two esterases e.g., Microbial dark matter encompasses the vast majority of mi- EstM-N1 and EstM-N2. EstM-N1 and EstM-N2 gave optimal ac- croorganisms (usually bacteria and archaea), which cannot be tivities at 20 C and 30 C, respectively, and were highly homolo- cultured in lab conditions due to lack of knowledge about their gous to b-lactamases. They also exhibited unusually high b- actual growth conditions or inability to supply them with the lactamase activity similar to esterases of family VIII [102]. CHA2 an required growth nutrients and environmental conditions. The ge- extremely alkaliphilic esterase, was isolated from Antarctica soil netic information from metagenomics or single cell genomics has [103]. This esterase showed activity in the temperature range of 5- provided insights into microbial metabolic cooperation and 40 C. Its temperature optimum was 20 C, and its pH optimum dependence, which engendered new avenues for cultivation of was 11. Such extremely alkaliphilic esterases are rare, and there is normally unculturable microorganisms. Nowadays, the only one more report on a psychrophilic and extremely alkaliphilic metagenome-based sequencing technologies are very successful esterase which was isolated from deep-sea sediment metagenome for the identification of novel biocatalysts and for exploring the [103]. A number of lipases/esterases adapted to extreme temper- dark matter to find other novel natural beneficial products. To atures have been revealed by soil metagenomics. In this context, a identify the novel lipases from the unexplored extremophiles the moderately thermophilic and thermostable lipase (80% residual metagenomic approach is the best preference. Various studies on activity at 50 C after 1 h) was obtained from metagenome of the screening of lipases and esterases in metagenomes have been Atlantic Rain Forest Soil and a cold-active esterase was isolated reported in the last decade [97]. Some of these lipases showed from a mountain soil metagenome that retained 60% of its maximal remarkable activity at high pH and temperatures. More recently, we activity at 1 C[104]. have screened some lipolytic enzymes from metagenomics data- base using advanced bioinformatics approach such as the “Recon- 2.3.3. Marine environments metagenomics struction of Ancestral Sequences” [14]. The variety of marine microbial enzymes is enormous, and they The PCR-based method is a common approach with degenerate have great potential for bioindustries. The marine ecosystem har- primers designed based on the conserved regions of already- bors a broad range of microorganisms such as bacteria, fungi, known lipolytic enzymes. In recent years, some microbial lipo- archaea, viruses, and protists. Enzymes derived from marine lytic enzymes (lipases or esterases) have been isolated from ecosystem are usually thermostable, with broad range of pH extreme environments using the metagenomic approaches, which tolerance and are resistant to other harsh conditions encountered have brought out new information on them such as enzymes with in most of the industrial processes [97]. Their sturdy structure and dual function, novel lipase families, engineered lipases (derived characteristics adapted to adverse environment has raised their from metagenomes) and some very extreme features like enhanced scope in academic research and industrial R&D. thermostability, stability in polar organic solvents, stability at A study on isolation of lipase from metagenomics of tidal flat extreme pHs etc. sediments resulted in a new lipase -LipG of bacterial origin. LipG and six other putative lipases constitute a family of novel bacterial 2.3.1. Hot springs metagenomics lipases. This family is related to fungal lipases as its members show Recently, thermostable esterases have been discovered through a signature sequence (Arg-Gly oxyanion hole sequence) which is metagenomics of hot spring areas, e.g. EstE1 was obtained via distinctive of filamentous fungal lipases [105]. screening of metagenomic libraries of high temperature regions of In the second study, a psychro-alkaliphilic lipase, LipEH166, was Indonesia, and Est 1 was isolated from a hot spring in Thailand. recognized as a member of a new family of lipolytic enzyme, (This EstE1 and Est1 exhibited characteristic thermophilic features, family includes three putative open reading frames as well). showing very high stability at 80 C and 70 C in the absence of any LipEH166 exhibits low temperature activity with optimum at 30 C; stabilizer, with optimal activity at 95 C and 70 C, respectively [97]. however, it shows considerable activity (47% of its maximal activ- These enzymes maintained very high activities at lower tempera- ity) at very low temperature of 5 C and is active over a broad pH tures, for instance in case of EstE1, 20% and 30% of its optimal ac- range (5-11) (maintaining 80% of the maximal activity) [106]. Other tivity were retained at 30 C and 40 C, respectively [98].Recently, lipolytic enzymes have been isolated from sea sediments obtained another lipase was discovered from metagenomic DNA obtained from coastal sites: two esterases from the Arctic seashore and one from the hot springs in Manikaran (Himachal Pradesh) in India [41]. lipase from the Baltic Sea [107]. A number of psychrotolerant li- pases/esterase have been obtained from deep-sea sediments, e.g. 2.3.2. Soil metagenomics lipase EML1 obtained from Edison Seamount (South West Pacific), Soil metagenomics is another source to get novel lipases/ester- belongs to the family of lipolytic enzymes belonging to meta- ases. LipCE, a low temperature-active lipase, obtained from the genomic library from tidal flat sediments as proposed by Lee and metagenomics of soil samples from oil contaminated sites of north collaborators [105]. Thorough screening of a metagenomic library Germany, exhibited optimal activity at 30 C. It showed consider- from deep sediments of South China Sea, gave 15 new lipases, most ably high activities even at low temperatures, i.e. 28% and 16% re- of them showed activity in the temperature range of 40-50 C. sidual activities were maintained at 0 C and -5 C, respectively [97]. Two esterases were isolated from the South China Sea, one [99]. This lipase showed high specificity and enantioselectivity to- was very stable in high concentrations of divalent ions and NaCl, wards esters of primary alcohols, enabling the LipCE as a potential and second was highly stable in organic solvents such as dime- biocatalyst for different organic synthetic reaction associated with thylformamide, dimethyl sulfoxide, 30% methanol, and ethanol agrochemical, pharmaceutical and other industries. Another [108].

27 S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36

3. Classification of microbial lipases 3.2. Types of microbial lipases based on function

3.1. Based on protein sequences and physiological properties Lipases can be divided into different types based on their specificity, which is a key factor in determination of their industrial Initially, lipolytic enzymes were classified into eight families applications. Based on specificity lipase can be grouped into four based on similarity of amino acid sequences and physiological major types: (i) regioselective (ii) substrate-specific (iii) non- properties [109]. The conserved features of sequences and struc- specific and (iv) enantioselective (Fig. 2). tures are described for all families in order to simplify the assign- ment of newly discovered lipolytic enzymes to the respective family. Numerous lipolytic enzymes have been identified and 3.2.1. Regioselective (regiospecific) lipases characterized in the last one decade. Lipolytic enzymes’ classifica- The regioselective lipases tend to guide the reaction in a favor- tion was further extended into nineteen families based on phylo- able direction over other side reactions. Such a property of lipases is genetic criteria, conserved sequence motifs and biological of tremendous importance to chemical and pharmaceutical in- functions. Recently, lipase enzymes were classified into I-XXXV dustries especially in the production of structured glycerides such families; however, the more detailed description of these families as Cocoa-butter substitutes (CBS) and Medium Long Medium chain was given by Hausmann and Jaeger (2010) [110]. triglycerides (MLM’s). Regiospecific lipases are further divided into: In the year 1999, Arpigny and Jaeger proposed the classification of bacterial lipolytic enzymes, in which bacterial lipolytic enzymes (a) 1,3 regiospecific lipases- They catalyze the hydrolysis of tri- were systematically grouped into eight families based on similar- acylglycerols at C1 and C3 positions of the glycerol backbone ities of their primary protein sequences and physiological proper- of the triglycerides, producing free fatty acids, 2- ties [109]. This classification brought out the following monoacylglycerols and 1,2 or 2,3 diacylglycerols. Recently, characteristic features of microbial lipase such as (1) important 1,3-specific immobilized lipases from R. delemar (PPRhDL) structural features and the catalytic site residues or the presence of and R. miehei (Lipozyme) were utilized in acidolysis of wal- disulphide bonds, (2) mechanisms of lipase secretion and lipase- nut oil with caprylic acid to synthesize long-chain fatty acid specific foldases, and (3) the significant relationship to other esters. Immobilized 1,3-specific R. oryzae lipase expressed in lipase families. Later on, Hausmann and Jaeger in 2010, classified Pichia pastoris has been used for biodiesel production lipolytic enzymes into nineteen families based on (i) phylogenetic [112,113]. Recently, a 1, 3-regiospecifc lipase has been re- distances (ii) conserved sequence motifs and (iii) biological func- ported from Streptomyces violascens (ATCC 27968), which tions [110]. Furthermore, Kovacic et al. (2019) presented a preferentially hydrolyzed EPA from the sn-1/3 position of comprehensive overview as an extension of the original Arpigny codfish oil compared to DHA which was located at the sn-2 and Jaeger classification of lipolytic enzymes, which classifies li- position, enriching the final (hydrolyzed product) with pases into 35 families and 11 true lipase subfamilies [111]. The 3.24-fold of EPA as against 1.98-fold of DHA compared to the conserved sequences and structural features were described for all initial levels [114]. families in order to simplify the assignment of newly discovered (b) 2-Regiospecific lipases- They catalyze the selective removal of lipolytic enzymes to the respective family. In this extended classi- fatty acid from the 2nd (carbon) position of the glycerol fication, they correlated the biochemical properties of some en- backbone of a triacylglycerol molecule, producing specif- zymes with the corresponding enzyme from a single extremophilic ically 1,3-diacylglycerol. The sn-2 specificity is very rare, and microorganism. In this extended classification, several new families it had been ascribed to a lipase from Geotrichum candidum, are represented by only one experimentally studied enzyme, that has an ability to hydrolyze oleic and linoleic acids from mostly from extremophilic organisms. the sn-2 position of triglycerides [115,116].

Fig. 2. Microbial lipase Types [31].

28 S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36

3.2.2. Substrate specific lipases industry to biodiesel production. They generally catalyze the hy- Substrate-specific lipases can be effectively used in reactions, drolysis of triacylglycerols into free fatty acids and glycerol with where they selectively act on a specific substrate in a mixture of mono and diacylglycerols as intermediates [129]. They are also different materials, facilitating the desired product synthesis. In capable of reversing the reaction under microaqueous conditions general, substrates that can be acted upon by substrate-specific li- leading to the formation of glycerides from glycerol and fatty acids. pases include fatty acids and alcohol. A recent study reports the importance of substrate specificity along with enzyme stability in 3.2.4. Enantioselective lipases order to exploit lipases in various industrially relevant processes These enantioselective lipases specifically hydrolyze one of the [30]. These lipases have specificity for the fatty acid or alcohol isomers of a racemate. These lipases can differentiate enantiomers moiety of a lipase substrate. They can be broadly classified into the in a racemic mixture. Some of the processes catalyzed by enan- following categories: tiospecific lipases include transesterification of secondary alcohols to pharmaceutical products, hydrolysis of menthol benzoate to (a) Fatty acid specific lipases- These lipases are specific for the cosmetic/food products, and hydrolysis of glycidic acid methyl ester fatty acids with respect to their chain length. Some lipases to medical/health care products [31]. Examples of applications of preferably act on short chain fatty acids, some on medium such lipase include synthesis of isomeric compounds that exhibit chain while others on long chain [4]. The lipase from Bacillus optimal function only under specific configuration. Some of the cereus showed specificity for short (C4:0) to medium chain recent findings of regioselective lipases include acylation of quer- fatty acids (C12:0)[117]. The lipase from Pseudomonas sp. cetin with ferulic acid using Rhizopus (R.) oryzae lipase to synthesize showed specificity for fatty acids related to position of dou- flavonoid derivatives, synthesis of acacetin and resveratrol 3,5-di- ble bond and the order of preference was as follows a-lino- O-beta-glucopyranoside using C. antarctica lipase B (Novozym 435) leic acid > stearic acid > oleic acid > b-linoleic and Burkholderia cepacia lipase (Amano PS-IM), etc. [130]. Li et al. acid > conjugated linoleic acid [118]. The lipases from S3 (2018) has reported chiral resolution of recemic 1-dodecanol in Penicillium citrinum, MJ1 A. niger, MJ2 A. oryzae, YM Bacillus esterification reaction with lauric acid using immobilized B. cepecia coughing, S9 Geotrichum candidum, and S11 C. lypolytica were lipase [131]. Recently, lipase from Rhizopus microsporus has been found to exhibit the strongest specificities for short-chain reported to exhibit s-enantiopreference in the resolution of sec- fatty acid esters [119]. A recombinant lipase of Streptomyces ondary alcohols [132]. violascens (OUC-Lipase 6) has been shown to carry out preferential hydrolysis of EPA from codfish oil glyceride 4. Current status of microbial lipase structure backbone as compared to DHA [114]. (b) Alcohol specific lipases- This is another form of substrate Several lipase crystal-structures have been determined during specificity of lipases [120]. Alcohol moiety of a substrate is an the last few years and are available with Protein Data Bank (PDB, important factor for determining the affinity of lipase for that http://www.rcsb.org). At present (November 2020), a total of 220 substrate. Microbial lipases have wide substrate specificity PDB IDs are reported for true lipases (Keyword: EC 3.1.1.3), in which allowing them to catalyze the synthesis of esters of primary, 122 were Eukaryotic, 93 were Bacterial 4 were Archaeal and one secondary and tertiary alcohols with aromatic, aliphatic and was Metagenomes. There representative 3D structures at 95% allylic compounds [121,122]. Esters of primary and secondary sequence identity of microbial (Fungal, Bacterial and Archaeal) true alcohols have been successfully synthesized using lipases lipases are given in Table 1. The first 3D-crystal structures of lipases [123] while biotransformation of tertiary alcohols is still from the fungi Rhizomucor miehei and Geotrichum candidum were challenging due to the steric hindrance of tertiary structure resolved in 1990 and 1991, respectively [133e135]. The first crystal [124]. Krishna et al. (2002) reported that the lipase from structure of a bacterial lipase from Pseudomonas glumae was solved Candida antarctica catalyzed enantioselective trans- in 1993 by Lang. This bacterial lipase belongs to lipase family I.2 and esterification of a tertiary alcohol at low rate [125]. A novel was explored for the first time for catalytic triad sequence identity. halophilic, alkalithermostable lipase LipR2 from Alkalispir- In 1993, fungal lipase structure i.e., the Candida rugosa lipase (CRL) illum sp. NM-R002 has been used for the synthesis of levu- structure was determined, that allowed a comparison with its ho- linic acid esters of different alcohols; the lipase showed molog from Geotrichum candidum and new insights on interfacial preference for short chain alcohols, giving higher ester yields activation were figured out [136]. In 1994, structural conforma- of 48.8% and 45.9% for ethanol and 1-butanol, respectively; tional changes corresponding to the closed and open state of the whereas, for 1-dodecanol (long chain alcohol), the yield was lipase active site were investigated [136]. In the open conformation, 26.2% only [126]. Candida rugosa lipase has been used for the a large hydrophobic area near the active site of the enzyme is synthesis of biolubricants from products of soybean oil and exposed increasing its total hydrophobic surface. alcohols like neopently glycol (NPG) and trimethylol propane The crystal structure of the Pseudomonas aeruginosa bacterial (TMP), wherein higher productivity was obtained for NPG lipase from the family I.1 was resolved in the year 2000 [137]. This based biolubricant (11.75 mol h 1L 1) than TMP based bio- lipase showed a variant of the canonical a/b hydrolase fold in that it lubricant (8.38 mol h 1L 1)[127]. Rodrigues et al. (2019) does not have the first two b-strands and one a-helix. A stabilizing have improved the rate of lipase catalyzed biotransformation intramolecular disulfide bridge is formed between residues Cys183 of tertiary alcohols through lipase engineering using the and Cys235. The active site loop containing the catalytic histidine is semi-rational design approach [124]. Mutated and designed stabilized by the metal ion calcium. The crystal structure of lipase lipases have also been used to enhance the methanol toler- from Bacillus subtilis was solved in 2001 [138]. This bacterial lipase ance in the biodiesel/biofuel production [128]. belongs to family I.4 and it showed a compact minimal a/b-hy- drolase fold with a six-stranded parallel b-sheet and five a-helices. Tyndall et al. (2002) resolved the first crystallographic structure of a 3.2.3. Non-specific lipases lipase from a thermophilic Bacillus stearothermophilus P1 [50]. This This class of lipases is very robust and capable of acting on lipase shared >20% amino acid sequence similarity with any other multiple substrates, as demonstrated by Mucor meihei lipases in previously reported lipase structure. Interestingly, its structure carrying out a wide variety of applications ranging from cosmetic contains a zinc binding site and significant insertions in the

29 S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36

Table 1 The reported representative 3D structures at 95% sequence identity of microbial (Fungal, Bacterial and Archaeal) lipases.

PDB ID Structure/paper title R.F. (Å) Source Microbes type Primary Citations

3TGL Structure and molecular model refinement of Rhizomucor miehei triacylglyceride 1.9 Rhizomucor miehei Fungus [135] lipase: a case study of the use of simulated annealing in partial model refinement 1CRL Insights into interfacial activation from an ‘open’ structure of Candida rugosa lipase 2.06 Diutina rugosa Fungus [136] 1THG 1.8 Å refined structure of the lipase from Geotrichum candidum 1.8 Geotrichum candidum Fungus [139] 5CH8 Crystal structure of MDLA N225Q mutant form Penicillium cyclopium 2.1 Penicillium camemberti Fungus [140] 1CVL Crystal structure of bacterial lipase from Chromobacterium viscosumatcc 6918 1.6 Chromobacterium viscosum Bacteria [141] 1LGY Lipase ii from Rhizopus niveus 2.2 Rhizopus niveus Fungus [142] 1QGE New crystal form of Pseudomonas glumae (formerly Chromobacterium viscosum ATCC 1.7 Burkholderia glumae Bacteria [141] 6918) lipase 1EX9 Crystal structure of the Pseudomonas aeruginosa lipase complexed with rc-(rp,sp)- 2.54 Pseudomonas aeruginosa Bacteria [137] 1,2-dioctylcarbamoyl-glycero-3-o-octylphosphonate 1LLF Cholesterol Esterase (Candida cylindracea) Crystal Structure at 1.4A resolution 1.4 Candida cylindracea Fungus [143] 1GZ7 Crystal structure of the closed state of lipase 2 from Candida rugosa 1.97 Diutina rugosa Fungus [144] 1YS1 Burkholderia cepacia lipase complexed with hexylphosphonic acid (R)-2-methyl-3- 1.1 Burkholderia cepacia Bacteria [145] phenylpropyl ester 2HIH Crystal structure of Staphylococcus hyicus lipase 2.86 Staphylococcus hyicus Bacteria [146] 2QUB Crystal structure of extracellular lipase LipA from Serratia marcescens 1.8 Serratia marcescens Bacteria [147] 2Z8X Crystal structure of extracellular lipase from Pseudomonas sp. MIS38 1.48 Pseudomonas sp. MIS38 Bacteria [148] 2ORY Crystal structure of M37 lipase 2.2 Photobacterium sp. M37 Bacteria [149] 2ZYR A. fulgidus lipase with fatty acid fragment and magnesium 1.77 Archaeoglobus fulgidus Archaea [35] 3ICV Structural consequences of a circular permutation on lipase B from Candida antartica 1.49 Moesziomyces antarcticus Fungus [150] 3G7N Crystal Structure of a Lipase from Penicillium expansum at 1.3 1.3 Penicillium expansum Fungus [151] 3GUU X-ray structure of Candida antarctica lipase A 2.1 Moesziomyces antarcticus Fungus [64] 3NGM Crystal structure of lipase from Gibberellazeae 2.8 Fusarium graminearum Fungus [152] 3O0D Crystal structure of Lip2 lipase from Yarrowia lipolytica at 1.7 A resolution 1.7 Yarrowia lipolytica Fungus [153] 3ZPX Ustilago maydis lipase um03410, short form without flap 1.99 Ustilago maydis Fungus e 4FKB An Organic solvent tolerant lipase 42 1.22 Bacillus sp. 42 Bacteria 4L3W Crystal structure of lipase from Rhizopus microsporus var. chinensis 2 Rhizopus microspores Fungus [154] 4OPM Crystal structure of a putative lipase (lip1) from Acinetobacter baumannii AYE at 1.70 1.7 Acinetobacter baumannii Bacteria e A resolution 5A71 Open and closed conformations and protonation states of Candida antarctica Lipase 0.91 Moesziomyces antarcticus Fungus [65] B: atomic resolution native 5CTA G158E/K44E/R57E/Y49E Bacillus subtilis lipase A with 10% [BMIM][Cl] 1.24 Bacillus subtilis Bacteria [155] 5AH1 Structure of estA from Clostridium botulinum 1.2 Clostridium botulinum Bacteria [156] 5AP9 Controlled lid-opening in Thermomyces lanuginosus lipase - a switch for activity and 1.8 Thermomyces lanuginosus Fungus [157] binding 5H6B Crystal structure of a thermostable lipase from Marine Streptomyces 2.3 Streptomyces sp. W007 Bacteria [158] 5MAL Crystal structure of extracellular lipase from Streptomyces rimosus at 1.7A resolution 1.71 Streptomyces rimosus Bacteria [159] 6CL4 Lipc12 - Lipase from metagenomics 2.64 uncultured bacterium [160] 6JD9 Proteus mirabilis lipase mutant - I118V/E130G 1.58 Proteus mirabilis Bacteria [161] 6QPR Rhizomucor miehei lipase propeptide complex, Ser95/Ile96 deletion mutant 1.45 Rhizomucor miehei Fungus [162] 6S3G Crystal Structure of lipase from Geobacillus stearothermophilus T6 variant A187C/ 1.90 Geobacillus stearothermophilus Bacteria [163] F291C 6J1P Crystal structure of Candida Antarctica Lipase B mutant - SR 1.76 Moesziomyces antarcticus Fungus [164] 6KSM Staphylococcus aureus lipase -Orlistat complex 2.23 Staphylococcus aureus Bacteria [165] 6ZL7 Crystal structure of c173s mutation in the pmgl2 esterase from permafrost 1.50 - e metagenomic library 6XOK X-ray structure of the rhombohedral form of the lipase from Thermomyces 1.30 Thermomyces lanuginosus Fungus [166] lanuginosa at 1.3 A resolution

canonical a/b hydrolase fold that may be important for thermal pH, temperature, solvent and enzymatic activities. In some cases, stability. In addition, this lipase has more salt bridges and a-helical different structural topology has been reported for microbial lipase content, as well as proline and aromatic residues than any other however, they are very similar at sequence level. For example, an previously reported lipase. extracellular lipase (PDB ID: 5MAL) isolated from Streptomyces The structure of alkalohyperthermophilic Archaeoglobus fulgidus rimosus belonging to SGNH hydrolase superfamily. SGNH hydrolase (AFL) lipase has been resolved in 2009 [35]. This lipase contains a family has an alpha/beta/alpha fold structure, where the beta- unique C-terminal domain that is essential for long fatty acid chain sheets are arranged in the parallel strands. The SGNH hydrolase substrate binding. This AFL enzyme has optimal activity at 70- enzymes have sequence homology to true lipases but act as ester- 90 C and pH 10e11. The AFL lipase comprises of an N-terminal ases and lipases [159]. The microbial lipases structural similarities alpha/beta-hydrolase fold. In last few years, various crystal struc- and difference are shown in separated groups (Fig. 3). Mainly mi- tures and some mutated lipases have been reported as mentioned crobial lipase has been clustered into two groups based on struc- in Table 1. ture similarity. This unique cluster of structural homologs The comparative analysis at sequence and structural level of maintains enzymatic activities under specific environment condi- microbial lipase explored their diversity and conserved structural tions. The thermostability of lipases can be attributed to several topology during evolution. Structurally minimum a/b hydrolase features such as presence of larger lid domain, non-covalent elec- fold is highly conserved in microbial lipase while sequence di- trostatic and hydrogen bonding interaction, disulfide bonds, higher versity has been observed [167]. Specific structural adoption via content of proline residues, polar surface area, etc. [168]. For engineering/directed evolution reflect their stabilities at different example, increasing the content of a-helices in lid domain and

30 S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36

Fig. 3. The microbial lipases (a total 39 PDBs) are compared to get an overview of the position of representative’s members of microbial lipases in protein structure space (a) comparative structural analysis based phylogenetic tree and (b) heatmap. Dali sever was used for protein structure comparison (http://ekhidna2.biocenter.helsinki.fi/dali/)[171] presence of conserved tryptophan residues in the lid domain play design approach, specific sites in the gene are targeted to make an importance role in the thermostability as well as the activity of specific changes in the catalyst based on information obtained from lipases at high temperatures, which comes from new intermolec- protein sequence, structure, and function. Nowadays, advanced ular interactions [169,170]. computational approaches are used for rational design of proteins with novel functionalities. The important conserved functional 5. Lipase engineering domains, active sites, and key structure points are the focus of the rational design for a specific target protein/enzyme. The active sites fi Lipases are used as biocatalysts in many chemical reactions. The or functionally important regions of the proteins are suf cient for fi desired applications are generally different from the natural func- engineering in order to obtain signi cant results in the desired tion of the enzyme. The development of more potent lipases is phenotype. There are many successful engineering/designing ap- fi limited by the characteristics of the biocatalyst, its stability, selec- proaches for the alteration of the substrate speci city, stereo- tivity, and other properties. The sequence and structural knowledge selectivity, and stability of lipases. The thermostability of Candida are very important to design the desirable enzymes. Advanced antarctica Lipase B (a widely used industrial enzyme) has been structural biology techniques such as X-ray crystallography, NMR enhanced through directed evolution and rational design [174,175]. spectroscopy, Cryo-electron microscopy and computational struc- Liebeton et al. (2000) used directed evolution to enhance the tural modeling allowed a better understanding of protein sequence, enantioselectivity of the extracellular lipase from Pseudomonas structure, and function relationships [172,173]. Enzyme engineer- aeruginosa [176]. ing is a way to improve enzyme activity by making suitable changes Structural and modeling studies of CalB indorsed a great range in its amino acid sequence. The recombinant DNA technology is of experiments to improve its thermal stability and selectivity. Site used to introduce the desired changes in amino acid sequences of directed mutationS74A exhibited a higher enantioselectivity to- enzymes and production level of an enzyme can be increased by wards 1-chloro-2-octanol [177]. Replacement of a threonine near introducing more copies of the desired gene into the concerned the active site by valine caused the loss of lipase activity, which is organism or by incorporating a strong promoter. Improvement of restored when 2-hydroxy-propanoate is used as a substrate [178]. kinetic properties, enhanced substrate specificity, increased ther- This mutant also showed improved enantioselectivity. By ’ mostability, alteration in optimal pH and increased/decreased decreasing mutant s propensity to aggregate in the unfolded state, optimal temperature are main objectives of enzyme engineering a 20-fold increase in half-life at 70 C over the wild-type CalB was that is more useful for industrial and other applications. achieved in two mutants i.e. mutants 23G5 (V210I and A281E) and Mainly, two different approaches i.e. the directed evolution and 195F1. Mutations A281E and V221D were important for lipase ’ rational design are used for protein engineering. The directed stability, while V210I had only a negligible effect. The mutant s fi evolution approach consists in making a series of random muta- catalytic ef ciencies against p-Nitrophenyl butyrate and 6,8- tions in the gene that code for the target protein. There are various diuoro-4-methylumbelliferyl octanoate were higher than that for techniques to obtain the core steps of directed evolution such as wild-type CalB [177]. mutation, recombination, and screening or selection. In the rational Kamal et al. (2012) obtained a mutant of Bacillus subtilis named

31 S. Verma, G.K. Meghwanshi and R. Kumar Biochimie 182 (2021) 23e36

6B. It was manipulated to poses twelve stabilizing mutations (A15S, Funding F17S, A20E, N89Y, G111D, L114P, A132D, M134E, M137P, I157 M, S163P, and N166Y) by performing multiple rounds of directed This research did not receive any specific grant from funding evolution and mutation by recombination in the lipase A gene agencies in the public, commercial, or not-for-profit sectors. [179]. These mutations may have caused better anchoring of loops or probably have increased their rigidity through substitution of Declaration of competing interest any amino acid by a proline. More importantly, three of the stabi- lizing mutations (A132D, M134E, and I157 M) are adjacent to two All authors have no conflicts of interest to disclose. residues of the catalytic triad (D133 and H156), causing increased firmness of the active site. Interestingly, this increased sturdiness may have resulted in a higher activity of the lipase. More recently, References two mutants (D311E and K344R) of T1 lipase isolated from Geo- [1] S. Li, X. Yang, S. Yang, M. Zhu, X. Wang, Technology prospecting on enzymes: bacillus zalihae were constructed to introduce an additional ion pair application, marketing and engineering, Comput. Struct. Biotechnol. J. 2 at the inter- and the intra loop, [180]. The T(m) for wild-type lipase (2012) e201209017. and mutants K344R and D311E were approximately 68.52 C, [2] G.K. Meghwanshi, N. Kaur, S. Verma, N.K. Dabi, A. Vashishtha, P. Charan, P. Purohit, H. Bhandari, N. Bhojak, R. Kumar, Enzymes for pharmaceutical and 68.54 C and 70.59 C, respectively. So, the thermostability of the therapeutic applications, Biotechnol. Appl. Biochem. 67 (4) (2020) 586e601, mutant D311E was increased. The crystal structure of mutated https://doi.org/10.1002/bab.1919. D311E lipase revealed an additional ion pair around amino acid [3] N. Gurung, S. Ray, S. Bose, V. Rai, A broader view: microbial enzymes and their relevance in industries, medicine, and beyond, BioMed Res. Int. 2013 E311 that may be responsible for the improved stability at high (2013) 329121. temperatures [180]. Geobacillus zalihae HT1 is an engineered lipase [4] G.K. Meghwanshi, A. Vashishtha, Biotechnology of Fungal Lipases, Fungi and with mutation D43E, T118 N, E226D, E250L, and N304E, which Their Role in Sustainable Development: Current Perspectives, Springer, 2018, pp. 383e411. showed higher stability in organic solvents such as DMSO, n-hex- [5] S. Raveendran, B. Parameswaran, S.B. Ummalyma, A. Abraham, A.K. Mathew, ane, and n-heptane [181]. A. Madhavan, S. Rebello, A. Pandey, Applications of microbial enzymes in Korman et al. (2013) engineered a methanol tolerant lipase from food industry, Food Technol. Biotechnol. 56 (2018) 16e30. Proteus mirabilis called Dieselzymes for biodiesel production by [6] K.E. Jaeger, T. Eggert, Lipases for biotechnology, Curr. Opin. Biotechnol. 13 (2002) 390e397. directed evolution. This designed lipase with 13 mutations showed [7] D. Kirsh, Lipase production by Penicillium oxalicum and Aspergillus flavus, greatly improved thermal stability and dramatically increased Bot. Gaz. 97 (1935) 321e333. methanol tolerance, (exhibiting a 50-fold increase in half- [8] L. Sarda, P. Desnuelle, Actions of pancreatic lipase on esters in emulsions, Biochim. Biophys. Acta 30 (1958) 513e521. inactivation time in 50% aqueous methanol) [129]. More recently, [9] R.K. Saxena, A. Sheoran, B. Giri, W.S. Davidson, Purification strategies for Soni and co-workers (2019) engineered a true thermostable lipase microbial lipases, J. Microbiol. Methods 52 (2003) 1e18. from an archaeal esterase via N-terminal domain swapping strat- [10] D. Sharma, B. Sharma, A.K. Shukla, Biotechnological approach of microbial lipase: a review,, Biotechnology 10 (2011) 23e40. egy [182]. The triacylglycerol lipase from archaeon Thermococcus [11] H. Treichel, D. Oliveira, M. Mazutti, M. Luccio, J. Oliveira, A review on mi- onnurineus was designed by replacing its 61 residues at N-terminus crobial lipases production, Food Bioprocess Technol. 3 (2010) 182e196. € € with 118 residues bearing lid domain of a thermophilic fungal [12] M. Royter, M. Schmidt, C. Elend, H. Hobenreich, T. Schafer, U. Bornscheuer, G. Antranikian, Thermostable lipases from the extreme thermophilic lipase from Thermomyces lanuginosus. Another fungal lipase from anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Pichia pastoris was engineered by directed mutagenesis of a specific Caldanaerobacter subterraneus subsp. tengcongensis, Extremophiles, life residue in the lid region to increase its hydrophobicity [183]. under extreme conditions 13 (2009) 769e783. [13] M. Rabbani, M.R. Bagherinejad, H.M. Sadeghi, Z.S. Shariat, Z. Etemadifar, F. Moazen, M. Rahbari, L. Mafakher, S. Zaghian, Isolation and characterization 6. Conclusion of novel thermophilic lipase-secreting bacteria, Braz. J. Microbiol. 44 (2013) 1113e1119. fi pThe present review has presented the insights on extermo- [14] S. Verma, R. Kumar, G.K. Meghwanshi, Identi cation of new members of alkaliphilic lipases in archaea and metagenome database using reconstruc- philic lipases, their sources, structure and types. Among sources tion of ancestral sequences, 3 Biotech 9 (2019) 165. lipases from extremophilic bacteria, archaea and metagenomic [15] C.C. Akoh, S.W. Chang, G.C. Lee, J.F. Shaw, Enzymatic approach to biodiesel e ‘dark matter’ have specifically been discussed with a view to add a production, J. Agric. Food Chem. 55 (2007) 8995 9005. [16] I.P. Sarethy, Y. Saxena, A. Kapoor, M. Sharma, S.K. Sharma, V. Gupta, S. Gupta, new dimension on this aspect; however other lipase sources have Alkaliphilic bacteria: applications in industrial biotechnology, J. Ind. Micro- also been discussed in brief. Based on enzyme specificity different biol. 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Finally, the role lipase engi- strains to alleviate oil pollution, BioMed Res. Int. 2015 (2015) 820575. [20] W. Xie, V. Khosasih, A. Suwanto, H.K. Kim, Characterization of lipases from neering in improving its properties like thermostability, stability in Staphylococcus aureus and Staphylococcus epidermidis isolated from human organic solvents etc. have been discussed with a number of ex- facial sebaceous skin, J. Microbiol. Biotechnol. 22 (2012) 84e91. amples. Advanced genome sequencing, structural biology and [21] A. Chaubey, R. Parshad, S. Koul, S.C. Taneja, G.N. Qazi, Arthrobacter sp. lipase computational modelling approaches, and de novo designing pro- immobilization for improvement in stability and enantioselectivity, Appl. Microbiol. Biotechnol. 73 (2006) 598e606. vide the tools for designing more potential extremophilic lipases [22] I.M. Khan, C.W. Dill, R.C. Chandan, K.M. 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