A Review on Geotrichum Lipases : Production , Purification , Immobilization and Applications

R. R. Maldonado,a,* D. B. Lopes,b E. Aguiar-Oliveira,c E. S. Kamimura,d and G. A. Macedob aUniversity of Campinas, Food Department, Technical College of Campinas (COTUCA), University of Campinas, R. Jorge de Figueiredo Corrêa, 735, Parque Taquaral, Postal Code: 13.087-261, Campinas, São Paulo, Brazil bUniversity of Campinas, Laboratory of Bioactive Compounds, Food and Nutrition Department, Food Engineering Faculty, Cidade Universitária ZeferinoVaz, s.n., Postal Code: 13083-862, Campinas, São Paulo, Brazil cExact Science and Technology Department, Santa Cruz State University, R. Jorge Amado, km 16, Salobrinho, Postal Code: 45.662-900, Ilhéus, Bahia, Brazil dUniversity of São Paulo, Laboratory of Bioprocess, Food Engineering Department, Animal Husbrandy and Food Engineering Faculty, Av. Duque de Caxias Norte, 225, Campus USP, Postal Code: 13635-900, Pirassununga, São Paulo, Brazil


Introduction
Enzymes are known due the extreme specificity that they present on their substrates.This characteristic results in high productivity, low energy costs, and low quantity of undesirable byproducts when enzymes are applied as biocatalysts in industrial processes.An ecofriendly appeal is obtained in the industrial process utilizing enzymes 1,2 .
Lipases can replace alkaline catalyzers in biodiesel production facilitating the glycerol recovery and the purification of fatty methyl esters.How ever, high cost and low stability of the enzyme in the presence of alcohol are the largest disadvantages in this case.Whole cell, recombinant methods, protein and metabolic engineering are promising options to increase lipase applications in the biofuel processes 6,7 .In the food industry, lipases can be utilized for oil enrichment, flavor synthesis, quality improve-ment in bread, dough and dairy products, fragrance development, structured lipids and antioxidants production, etc. [8][9] .Lipases are capable of hydrolyzing triacylglicerol which facilitates the removal of greasy material from wastewaters, reducing the damage to the environment [14][15] .Furthermore, some lipases have high stability and activity in alkaline mediums, which allows the application of this biocatalyst in different types of detergent [16][17] .
Lipases can be produced by different organisms, including animals, plants, and microorganisms, but only microbial lipases are commercially significant due to their higher production and greater variety.Filamentous fungi and yeasts are the most preferred sources for lipase production, since this enzyme is mostly described as extracellular, which facilitates its recovery from the fermented medium.Approximately 50 % of commercial lipases are produced from yeast and filamentous fungi, and some genus are frequently cited as good lipase producers, such as: Aspergillus, Candida, Fusarium, Geotrichum, Mucor, Penicilium, Pseudomonas and Rhizopus, Rhodotorula, etc. 4,7,[19][20][21][22][23] .
Geotrichum sp. is a group of dimorphic yeasts that can occur in cream-colored yeast-like and in white mould-like colonies.The genus is composed of 22 species (including 10 sp.nov.) and the ecology shows a rather unexpected degree of consistency given the large phylogenetic distances between the species, including a high degree of rDNA polymorphism [24][25] .
Geotrichum is commonly related to the cheese ripening process, biodegrading of dyes, and lipase production [24][25][26] .It was undesirable in traditional cheese making until the 1970s because it can cause unstable slim or 'toad skin'.However, since the 1980s it has been used to reduce bitterness as well as to develop flavors (especially in Camembert cheese).In some cases, it shows an antagonist effect against Mucor spp.and Listeria monocytogenes, and it can contribute to the cheese ripening process via action of its proteases and lipases.These benefits depend on the morphotype and strain used 24 .
Geotrichum consumes lactate, but it rarely consumes sugars and has a lower salt-tolerance during its growth.It is known for proteolytic, peptiodolytic and lipolytic activities, and for the production of long-chain free fatty acids and fatty acids esters, which have a minor flavor importance in different cheeses.It grows well in pH of 2.5 to 8.1 and with low level of oxygen.Its excess can inhibit other microorganisms during the cheese ripening process; however, the low quantity of its spores causes a lower flavor complexity 25 .
Among them, Geotrichum candidum and Galactomyces geotrichum (the teleomorph state or sexual form) are the most common and studied spe-cies for lipase production.Lipases from Geotrichum candidum are known for their high specificity for unsaturated long chain fatty acids and esters, while lipases from Galactomyces geotrichum present specificity for other esters of long chain fatty acids and good stability regarding temperature, pH and organic solvents.These properties allow their utilization in different reactions utilizing various vegetable oils 22,25,[27][28][29] .These microorganisms have been isolated from soil and plants 29,30 , dairy products 31,32 , rotten vegetables and fruits 33 , different infections 34 , and many other sources 35 .
This review is focused on Geotrichum lipases and their biotechnological applicability.The aim is to discuss the latest aspects related to their production, purification, characterization, immobilization and application.The databases utilized for this purpose were mainly: Science Direct, Wiley Online Library, Springer, Scielo and Taylor & Francis Online, between January, 2014 and October, 2016.

Production of Geotrichum spp. lipases
Geotrichum sp. can be obtained from different natural sources and its cultivation can occur under the most variable conditions of: nutritional requirements (carbon, nitrogen and lipid sources), growth parameters (pH, temperature, agitation and aeration), inoculum conditions, and bioreactor system.Table 1 presents a list of a few recently published papers about lipases produced by Geotrichum species.G. candidum is the most studied amongst the genus in relation to lipase production, but other strains also are related to different processes such as G. fragrans, G. siamensis sp.nov., G. phurueaensis and G. klebahnii [36][37][38] .
Different substrates have been studied lately in search for better lipase productivity and/or lower process costs  . Organc and inorganic nitrogen sources are frequently investigated for lipase production from G. candidum (Table 1).Peptone, yeast extracts and urea as organic nitrogen sources, and ammonium chloride, ammonium nitrate and sodium nitrate as inorganic nitrogen sources, are the most cited.The concentrations used vary greatly, but the range from 0.1 to 5.0 % w/w is more commonly used, and organic sources are added in higher quantities than inorganic sources  .Concentrations (% w/w) of: 5.0 of peptone; 2.0 of yeast extract and 0.1 of NaNO 3 and 0.5 of NH 4 NO 3 are commonly applied in the lipase production [43][44][45][46]49,57 ; however, optimized studies about medium composition reduced or eliminated some nitrogen sources in the process.strated that sodium nitrate did not have a significant effect on the lipase activity 52,61 .Ginalska et al.
showed that 0.4 % w/w of urea was the best condition of nitrogen source for lipase production compared to other organic sources.They also showed that the use of only inorganic nitrogen sources had an inhibitory effect on lipase activity 57 .The cost of these substrates is relatively high, and several studies have been conducted to find ways to replace these nitrogen sources with other cheaper sources.Corn steep liquor, yeast hydrolysate and soybean molasses are cited as good nitrogen sources due to their high concentration of nitrogen and amino acids.They have been used to replace the traditional nitrogen sources, maintaining or increasing lipase activity from Geotrichum.The used quantity of these sources varies greatly (3.0 -15.0 % w/w) according to the substrate and fermentation conditions 53,54,56,60,62,64 .These agro-industrial residues present a certain quantity of salts and their use eliminated the necessity of supplementing the medium with inorganic salts.
The addition of lipids substrates is a known strategy to increase the lipase activity for different microorganisms.Long-chain fatty acids have been used as inductors for lipase production from Geotrichum.Shimada et al., 1992, demonstrated that the use of unsaturated fatty acids (especially C 18:1, 18:2 and 18:3) results in high level of lipase activity in comparison to other fatty acids 43 .Several other studies had successful results utilizing vegetable lipids sources as inductors for lipase production from G. candidum.Some examples are babassu, cottonseed, fish, olive, soybean oils; almond, coconut, ginger, groundnut Jatropha, niger seed oil cakes, palm and sunflower effluents, and many others  . Oliveoil contains a high concentration of C18:1 unsaturated fatty acid, and innumerous studies indicate the use of this oil as an inductor for lipase production 44,45,[47][48][49]51,57,56 .Soybean oil also shows similar results due to its similar composition to olive oil, and its low cost is a strong appeal for its application.Concentrations of 1.0 % w/w are frequently utilized, but optimized studies showed that lower concentrations (0.5 to 0.7 % w/w) are enough to induce the lipase production, and concentrations above 1.0 % w/w do not have significant effects on the increase in lipase activity [52][53][54]56,[60][61][62] .
Glucose is the most used carbon source for the growth of G. candidum and the utilized concentration is usually 1.0 % w/w.Other carbon sources can be used, such as sucrose, galactose, lactose or lactic acid 44 .A lipid source can be a carbon source and inductor simultaneously.In fact, glucose contributes to cellular growth but it can also reduce the lipase activity, as verified by Burkert et al. 56 .
The inoculum is another important variable in the lipase production from Geotrichum, and the liquid form 45,46 , used at 10 % (v/v) 47 or 5 % (v/v) 48 , is frequently applied.However, this type of inoculum can be an obstacle to the homogenization and standardization of inoculum, as shown by Resende-Maldonado et al. 49 The authors proposed the utilization of an initial solid inoculum (with a specific area) followed by an intermediate liquid inoculum that reduced the variability of the lipase activity by 33 % during the fermentation.In another study, the use of intermediate liquid inoculum doubled the level of lipase activity compared to the use of the spore solution.The reduction in size of the inoculum by 10 to 2 % v/v also increased the maximum lipase activity 59 .This technique was applied with success in other studies with G. candidum and G. sp [52][53][54][60][61][62] and it was verified that the lower quantity of solid inoculum increases the lipase activity. The redtion in the quantity of inoculum probably reduced the crowding effect (a typical mycological phenomenon that causes self-inhibition in spore fungus germination under crowded conditions).A similar technique for sampling solid cultures of G. candidum and P. camembertii was applied by Aldarf et al.50 In relation to the bioreactor choice, several studies have been conducted in shaken flasks (Table 1), but other types of bioreactors have also been applied successfully for Geotrichum cultivation; in general, the scale up from shaken flasks to a bioreactor system requires optimized cultivation conditions that can be achieved with the factorial designs methods 49,[51][52][53][54]65 .The air lift bioreactor was successfully applied for lipase production from Geotrichum because the absence of mechanical agitation reduces the damage in the mycelium and provides better conditions for obtaining high lipase activity 52,53,60 .The use of a low rate of aeration also contributes to increasing lipase activity since G. candidum grows well with low levels of oxygen 25 .Studies in stirred tank bioreactor indicated that aeration of 1.0 vvm or lower are the best conditions for lipase production from Geotrichum candidum [51][52][53]60 .The low level of oxygen is also efficient in shaken flasks; several studies have indicated agitation conditions below 200 rpm for lipase production from Geotrichum, which reduces the quantity of dissolved oxygen 46,47,54,56,[62][63][64] .
Geotrichum is a mesophilic yeast and most studies have shown that temperatures near 30 °C are more appropriate for its growth and lipase production.The optimum pH for lipase production for this genus is around 7.0, but some species are able to produce lipases in more acidic 62,64 or in more alkyne mediums 42 .In general, the pH increases during the fermentation time and at the end of fermentation it is possible to observe a great decrease in lipase levels, mostly due to the pH increase (above 8.0) or proteases activities [53][54][55][56][60][61][62] .
Lipases can be produced intra-(cell-bound) and/or extracellularly, as described by several authors 41,44,46,48,66 .The level of lipase activity and selectivity varies according to the location, strain and fermentation conditions.Hlavsová et al. 41 and Loo et al. 46 used a similar fermentation medium to produce cell-bound lipases, but in the first study the cell-bound lipase showed high selectivity for saturated fatty acids, while in the second study the highest selectivity was observed for polyunsaturated fatty acids, probably due to the difference in the strains used.In general, intracellular lipase production takes place in the initial hours of cultivation, followed by its release into the culture medium during the stationary phase when the highest levels of lipase are obtained 41,66 , although the duration of the stationary phase can vary greatly and the maximum lipase activity can occur from 24 to 96 hours, depending on fermentation conditions 44,59 .
Genetic engineering is very powerful and helpful in improving the enzyme expression.There are two ways to achieve this goal: Geotrichum species can be mutated to produce higher levels of lipases or Geotrichum genes for lipases can be expressed in bacteria.According to Cao et al. 67 an increase of 53 % in biomass and an increase of 158 % in lipid yield were obtained with the cultivation of a mutated strain of G. robustum.Mughal et al. 65 obtained a lipase activity almost 24 times higher after inducing the mutagenesis of G. candidum.Yan et al. 68 expressed G. candidum Y162 lipase genes in Pichia pastoris and obtained 55 U mL -1 of activity, and Pan et al. 69 , by expressing Geotrichum sp.lipase genes also in P. pastoris, obtained (273 ± 2.4) μmol FFA min -1 g -1 of dry cells.In addition, Galactomyces geotrichum lipases have also been successfully expressed, mostly in Picchia pastoris 28,70 .In all these examples, the lipase activity or lipid contents in the clones or mutants of Geotrichum were much higher compared to original strains, indicating that these techniques can used successfully in industrial applications that need a high level of lipases activity.Furthermore, the clones or mutants were able to produce lipases that are more stable in high pH (above 8.0), high temperature (above 45 °C), and in different low polarity organic solvents, which normally does not occur with original strains of Geotrichum 28 .
Lipase activity can be measured differently.The most common definition of U (unit of lipase activity) is the amount of enzyme capable of releasing 1 μmol of free fatty acids (FFA) per minute 71 and its variations, such as, the amount of enzyme capable of releasing 1 mmol of FFA per gram of dry cells 46 or the amount of enzyme that releases 1 μeq of FFA per min per mg of dry cell 72,73 .Another defi-nition applied is the amount of enzyme capable of releasing 1 μmol of p-nitrophenol (p-NP) per minute 45 .When comparing results from different authors, it is crucial to pay attention to these peculiarities.According to Table 1, the obtained range of lipase activities is wide, since the methods for cultivation and the methods for lipase activity determination also widely vary.

Purification and characterization of Geotrichum spp. lipases
Crude lipases preparations from different Geotrichum strains have been studied and characterized throughout the years.Different degrees of specificity, in relation to type and positions of fatty acids in triacylglycerols and fatty esters, different molecular weights, stability, and biochemical properties have been observed [74][75][76][77] .Innumerous methods for purification and characterization of these enzymes have also been applied, as presented in Table 2.The simple concentration (partial purification) applied mostly for extracellular lipases has been conducted by precipitation with acetone 72 , ethanol 77 and ammonium sulphate 79 , ultrafiltration 83 , extraction by aqueous two-phase system 84 and many other methods.
Among the precipitating agents, ammonium sulphate has been the most used 45,58,78,79,86,88 .Partial purification with this salt is easy to apply and results in high purification factors (in average 87 % of recovery factor 88 ); including situations with enzyme hyperactivation 78 , which is very interesting for improving the recovery factors in the subsequent purification process.On the other hand, the total time of this method is long, since a long time is required for complete enzyme precipitation, and a dialysis step is necessary to remove the excess salt from the precipitate 78 .
In order to reduce the pre-purification time, other substances such as acetone 45,63 , ethanol 78 and trichloroacetic acid 80 were also applied.In these cases, pre-purifications were conducted with cold solvent to prevent denaturation of the lipases since most of these enzymes were not stable in the presence of organic solvents.Acetone has been cited as a good option to wash and activate whole cell lipases 41,45 , improving the catalytic activity.Ethanol resulted in a high concentration factor (64 -66) with short time processes (1-2 h) 77,78 , but the recovery factor was lower compared to ammonium sulphate precipitation because the lipases (in general) have low stability in contact with ethanol 78 .
Ion exchange is the most applied technique in purifying enzymes, and it is also applied to lipase purification as mentioned before.This type of purification is fast, easy to perform, requires low volume of sample solution, and is used for many other compounds.For lipase purification, ion exchange presents varied results for recovery factors (5 -80 %), and for purification factor (1.1 -33 fold) 58,63,70,72,75,77,79,83,86 .
Hydrophobic interaction chromatography is another interesting method for lipase purification since the lipases have good affinity to hydrophobic compounds.The recovery factors are normally high (48 -96 %), and under optimized conditions high purification factors can be obtained, such as 86.7-fold, which was achieved under optimized conditions for purification lipase produced using yeast hydrolysate 97 .
Several studies have shown the use of more than one purification technique in order to obtain high levels of purify.Ammonium sulphate precipitation combined with ion exchange and gel filtration resulted in a 7.76-fold purification 58 and 11.2 % of recovery factor 79 .A purification factor of 11.3 and 48 % recovery were achieved with combined purification using acetone precipitation, ion exchange and hydrophobic interaction chromatography 63 .In another study, it was possible to obtain relevant data regarding a large-scale purification of a 61.6 kDa lipase from G. candidum with two consecutive chromatographic steps: ion exchange and hydrophobic interactions, resulting in a purification factor of 13.2 and a specific activity of 1.052 μmol FFA min -1 mg -1 of protein 89 .Examples of combined methods have shown that a high level of purity can be obtained, but the recovery factors reduce with the increase in the number of purification steps due to the application of sequential methods improving enzymatic denaturation.The choice of the type and sequence of purification steps depends greatly on the properties of lipases and the potential applications for these enzymes.
Aspects related to optimum pH and temperature, stability, molecular weight, etc., vary according to the strain investigated and the medium composition applied.The characteristics are quite different from one lipase to another, but a few similarities can be observed among them.Literature cites that most of the values for optimum pH and temperature for lipase from Geotrichum vary between 6.0-7.0 and 20-40 °C, respectively 28 .Some studies indicate the optimum pH and/or optimum temperature is within these ranges 58,72,77,78,86,90,95,97 , however different results were obtained with modi-fications in several relevant factors for lipase production and purification, as shown in Table 2.
Genetic modifications, lipase location (cellbound or extracellular lipase), use of new strains and modifications in fermentation medium, etc., can cause drastic changes in lipase properties.A clone containing genes from Galactomyces geotrichum (a species less studied within the genus Geotrichum) produced high levels of lipase activity with high optimum pH (8.0), high stability pH (until pH = 10.0), and high optimum temperature (50 °C) 28 .The cellbound lipase showed higher activity and higher stability than the extracellular lipase from the same strain in pH = 8.5 41 and the range of pH stability was wider (5.2-9.2) for mycelium lipase than extracellular lipase (7.2-9.2) 90 .A new strain of Geotrichum sp.produced two new cold-adapted lipases, which showed optimum temperatures between 15-20 °C despite the cultivation conditions at 30 °C79 .A lipase with high optimum temperature (47 °C) was obtained from Geotrichum candidum NRR-LY-552 when clarified corn steep liquor was used as nitrogen source in fermentation medium 97 .The same strain exhibited an optimum temperature 10 °C lower when the microorganism was cultivated with yeast hydrolysate 97 or peptone 52,61 .
According to Sharma et al. 4 , most lipases obey Michaelis-Menten kinetics, and that is also valid for Geotrichum lipases as demonstrated in other studies 45,82,97 for different strains and cultivation conditions.It has also been stated that Geotrichum lipases can present even four types of enzymes with different molecular weight and substrate specificities 91 , but Baillargeon and McCarthy 92 , working with G. candidum NRRL Y-553, were able to obtain five glycosylated lipases with pI from 4.88 to 4.78 and with molecular weight also varying from 64 to 57 kDa.The isoelectric points of lipase from Geotrichum does not change much as do other parameters, and the results are normally in the range from 4.0 to 5.0 77,83,86,89,92 .
Geotrichum lipases are frequently cited by their substrate specificity for fatty acids having at least one cis-Δ9 double bond 70,74,89,90,92,94 , but different catalytic specificities are found in different lipases from this genus 93 .A few examples are shown in Table 2, and it is important to highlight that some lipases from Geotrichum showed high specificity for saturated fatty acids 41,74,76,90 .This characteristic can be related to one of the most important habitats of this genus, which is dairy products containing a significant quantity of saturated fatty acids.Differences in the specificity were mentioned for the position of the chain in the tryacilglycerol.There are three types of lipases -1,3-position 72,76 , 2-position 91 and non-position specifics 72,75,90 .Sometimes the same microorganism produces different isolipases with different position specificities 72 .This is a great advantage because it allows application on different substrates.
Other examples can be cited to illustrate different possibilities regarding lipase properties: two lipases from G. candidum ATCC 66592 94 presented different velocities of hydrolysis of palmitic acid methyl ester, the 61 kDa lipase revealed a higher initial velocity compared to the 57 kDa lipase.An extracellular Galactomyces geotrichum lipase, with 57 kDa in the unglycosylate state and 62 kDa as glycosylated, showed a similar specificity to G. candidum lipase but a different amino acid composition 95 .
Expressed Galactomyces geotrichum lipases have been also characterized, although less frequently than Geotrichum lipases.For example, Fernández et al. 70 purified 59 kDa lipase from G. geotrichum BT107 expressed in P. pastoris LF163 by ultrafiltration and anion exchange chromatography, which yielded 97.5 % and 42 %, respectively.Also, Yan et al. 28 , by expressing a 64 kDa lipase from G. geotrichum Y05 in P. pastoris GS115 and pPIC9K, obtained 1.22-fold (ammonium sulphate precipitation), 2.8-fold (anion exchange chromatography), and 3.2-fold (gel filtration chromatography) purification.Additionally, Bertolini et al. 96 expressed G. candidum lipases I and II in S. cerevisiae, and significant differences between them were observed as lipase I presented much more affinity to long fatty acyl chains substrates than lipase II.

Immobilization of Geotrichum spp. lipases
Lipase has been successfully immobilized with different techniques [98][99][100][101] and the same rules for general enzyme immobilization are also applied for Geotrichum lipase immobilization.It is common sense among researchers that there is no 100 % perfect technique for it.All techniques will always present some disadvantages as well as advantages.
A few examples of how Geotrichum lipases have been immobilized over the last years are presented in Table 3, and a few others examples will also be cited.
The entrapment of cells, instead of enzymes, seems to be a simple technique with good results for Geotrichum lipases.Pan et al. 106 immobilized Geotrichum sp.G38 cells in silicon granules and obtained the same pH profile for both forms, free and immobilized, but an increase of 5 °C in optimum temperature after immobilization was observed.In addition, the glucose supplementation into the medium reaction -for the cells maintenance -contributed to increasing cycles of use from 23 to 28 with 90 % activity.Bleve et al. 107 immobilized G. candidum cells in Ca-alginate beads, and Carballeira et al. 108 tested 6 different polymeric matrixes.In both cases, they were focused on other enzymes not lipases; Nakamura et al. 109 also immobilized G. candidum IFO 4597 cells, but by adsorption onto the surface of a water-absorbing polymer (BL-100 ® ), and applied it for stereo-selective oxidation and reduction in organic solvent.
Immobilization by adsorption is a simple technique with good results and is considered the most applied enzymatic immobilization technique 110 even in combination with other techniques 111 .Two purified Geotrichum CMICC 335426 lipases, for example, were successfully adsorbed on hydrophobic macroporous polypropylene particles (Accurel EPI00) precoated with ovalbumin by Charton and Macrae 112 and, according to their results, immobilization and the use of an organic media (tri-n-butylphosphate) did not have a major effect on the specificities of lipases A and B. However, for the immobilized lipase B, the hydrolysis of 20 % of triolein presented a v max value 357 times lower than the free lipase.The authors attributed this fact, among other reasons, to inactivation by the solvent.
Entrapment and adsorption can be simple techniques but they can also result in an undesirable leakage.For that purpose, the use of a stronger force, such as an ionic or covalent bond, can be of great help.As a brief example, Matsuda et al. 113 immobilized cells of G. candidum NBRC 5767 onto an ion exchange resin in order to use its alcohol dehydrogenase to reduce ketones.
Silica gel is a very common support for enzyme immobilization.It was used by Sonnet et al. 114 to adsorb a commercial G. candidum lipase preparation.Bhattacharyya et al. 115 also adsorbed carbonyl reductase from G. candidum NCIM 980 in silica gel, followed by cross-linking with glutaraldehyde for the immobilization.Several other materials, such as agriculture wastes 116 , membranes 117 and ceramic materials 118 have been proposed as a support for lipase immobilization in general, and the decision for the right support and technique should only be based on the process characteristics.
When an enzyme is produced attached to the cell membrane it is possible to consider that it is already immobilized, using the cell as its natural support 44,48,69,76,80,119 .In cases like these, the biomass can be easily removed from the reaction medium and used again in a new medium, avoiding several enzyme purification downstream steps.

Applications of Geotrichum lipases
In general, Geotrichum lipases have the same broad applicability like any other lipase, since this group of enzymes is the most investigated and in-dustrially applied.Apparently, the majority of the Geotrichum lipases have a special characteristic that allows their utilization in different vegetable oil.They are very selective towards unsaturated long-chain fatty acids with cis-9 double bonds, such as oleic and linoleic acids, as mentioned before.Other applications can also be cited for these enzymes, as follows.

Polyunsaturated fatty acids enrichment
Polyunsaturated fatty acids (PUFA) are very important for different physiological functions 120 , thus, their enrichment in medium is of great interest and Geotrichum lipases can be perfectly applied for that purpose.Pan et al. 69 , when studying the enrichment of eicosapentaenoic (EPA) and docosahexae- The purified lipase powder aggregates were immobilized by cross-linking with glutaraldehyde with organic solvents (CLEA) and with polyethylenimine (PEI-CLEA).
Both techniques, CLEA and PEI-CLEA, avoided the pellet agglomeration and resulted in 33 and 42 % of hydrolysis degree, respectively, contrasting to the 12 % degree with the free enzyme.The PEI-CLEA biocatalyst also presented more thermal stability, better tolerance towards strong polar and non-polar organic solvents, no leakage of enzyme and better reusability after retaining 72 % of its relative hydrolysis degree after 5 cycles of use.
-PCMC methodology: lipase was dehydrated (in organic medium) on the surface of micro-crystals of K 2 SO 4 , coating them; -CLPCMC methodology: the obtained crystals described above were crosslinked with glutaraldehyde.
PCMC and CLPCMC presented a biodiesel yield of 69 and 72 % respectively; free lipase resulted in 29 %.CLPCMC also presented higher maintenance of activity after 5 successive batch reactions (80 %), better stability in pH from 4.0 to 6.0 and in temperatures from 45 to 50 °C.
The biocatalyst obtained presented a 18.4-fold enhancement in its sterification activity towards metyloleate synthesis, a highly improved operational stability compared to the crude form and after 10 cycles of use it was capable of retaining 90 % of its initial activity.

105
*FFA = free fatty acid noic (DHA) acids contents from fish oil, using a Geotrichum sp.lipase displayed on the P. pastoris wall, obtained an increase of about 1.2-fold for each acid, reaching 46.62 % of total yield, for both.The best hydrolysis degree in fish oil for producing EPA and DHA was 40 % in both cases, using immobilized lipase from Geotrichum sp. 102These results were about 2 and 4 times higher than with the use of free enzyme.In addition, γ-linolenic acid (GLA) was successfully enriched up to 40 % in the acylglycerol fraction, when Fregolente et al. 29 applied an enzymatic extract of G. candidum in borage oil, a 1.9-fold enrichment.Fogliaand Sonnet 121 , with silica immobilized and free lipases from G. candidum, resulted in up to 70 % recovery of the same acid, GLA.

Conjugated linoleic acid enrichment
Conjugated linoleic acid (CLA) are C18 fatty acids mainly composed of cis-9, trans-11 isomers and are considered anti-carcinogenic, among other benefits, and are naturally found in many natural food sources, especially animal (ruminant) products 122 .A commercial G. candidum lipase preparation was applied by McNeil et al. 123 , with safflower oil, in order to enrich the composition of the cis-9, trans-11 and also the trans-10, cis-12 isomers.According to the authors, obtained was a maximum fraction of about 98 % (between 13-35 % of conversion) of the cis-9, trans-11 in a much higher velocity.The same commercial enzyme preparation was also applied by Haas et al. 124 besides the recombinant P. pastoris with the cis-9 selective B lipase gene of G. candidum CMICC 335426 expressed; both enzymes demonstrated a highly selective production of the cis-9 isomer with compositions of 77 and 94 %, respectively.

Lipid hydrolysis
Lipases are capable of catalyzing both hydrolysis and synthesis of esters, and their specificities vary from strain to strain as much as their hydrolytic yield in di-and monoacylglycerol, glycerol and free fatty acids.The hydrolysis of triacylglycerols of black currant oil was evaluated for three different strains of G. candidum and one of G. ludwigii by Stránský et al. 76 and it was observed that the medium in which they were induced presented a great influence on the profile of the obtained hydrolyzed fatty acids.Diks and Lee 125 evaluated the selective hydrolysis of sunflower oil by the wild G. candidum CMICC 335426 lipase B and the one expressed by P. pastoris aiming at the production of a very slightly saturated fatty acid oil in which obtained degrees of hydrolysis of around 60 % (w/w) with more than 99 % (w/w) of unsaturated compounds in free fatty acid fraction, using sunflower oil for the hydrolysis.Other examples of Geotrichum lipases application in hydrolysis are cited briefly in Table 2 as Brabcová et al. 45,81 and Cai et al. 79

Synthesis of aroma compounds
Lipids are an important source of aroma compounds 126 and several Geotrichum strains are responsible, for example, for the characteristic aroma/ flavor of different cheese, not exclusively because of their lipases but also other enzymes produced during the ripening of cheeses [127][128][129] .In addition, according to Neto et al. 130 , hydrolyzed castor oil and its fatty acid derivatives proved to be effective precursors of γ-decalactone, responsible for a fruit/ chocolate aroma by the lipases from G. fragrans and Geotrichum sp. with the first lipase resulting in a better production (600 mg L -1 ).Macedo and Pastore 131 evaluated, among others, a lipase from Geotrichum sp. in order to produce aromatic esters, and this lipase was able to esterify all the substrates tested, resulting in, for example, 80 % of esterification with butanol and acetic acid.Recently, Vong and Liu 132 evaluated different strains of yeasts to reduce the undesirable flavor of okara (a soybean residue obtained during the production of soymilk).During the fermentation of okara using the G. candidum, these authors observed a significant reduction in hexanal and trans-2-hexane, which were converted mainly into 2-propanol and pentanoic acid.

Conclusions
According to this review, it is possible to see that there are different aspects regarding production, purification, immobilization, and application of lipase from Geotrichum genus.Production is carried out to obtain extracellular lipases (mainly) and cell bond lipases using different substrates, operating conditions and bioreactors.Most studies produce lipases using submerged fermentation, and in recent years many alternative nitrogen sources have been explored in order to reduce the cost of production.Organic nitrogen sources, lipids for the induction and as carbon source, and glucose for growth are the most typically applied substrates for lipase production from Geotrichum.The application of different substrates and conditions allow production of lipases with different biochemical characteristics.In addition, genetic modifications have been evaluated to increase the lipase production using different species of Geotrichum or by expressing their genes in other species like P. pastoris and S. cerevisiae, for example.
Purification has been studied through different techniques, such as precipitation using salt solutions or organic solvents, ultrafiltration, and different chromatographic techniques, especially employing hydrophobic systems.A great number of techniques have also been used to immobilize lipases from Geotrichum genus, especially adsorption in hydrophobic supports, entrapment using different supports, and in its own cell (natural support).Production, purification and immobilization processes can change the characteristics of lipases from Geotrichum drastically, however, most of the studies have shown that the lipases from this genus are good biocatalysts in pH near 7.0 and temperature near 30 °C.These characteristics can be changed with genetic modifications, use of different strain, and different fermentation conditions.
Finally, different applications have been mentioned in the literature about the use of lipases from Geotrichum.The most important characteristic of lipases from this genus is the specificity from unsaturated long-chain fatty acids with cis-9 double bonds, extensively studied for a long time.Furthermore, other applications are frequently cited, such as enrichment of both polyunsatured fatty acids (PUFA) and conjugated linoleic acids, hydrolysis of lipids, and synthesis of aromas.All this information is of great value to increase the potential for Geotrichum lipases applicability.