Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013

Abstract

The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell-bound). Both enzymes were tested for their hydrolytic ability to pnitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium-(C12) and long-(C18) chain fatty acids were used as substrates. Cell-bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p-nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p-nitrophenyl decanoate > p-nitrophenyl caprylate > p-nitrophenyl laurate > p-nitrophenyl palmitate > p-nitrophenyl stearate. The cell-bound lipase indicates preference for p-nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell-bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K m ) and maximum reaction rate (V max ) for p-nitrophenyl palmitate hydrolysis of cell-bound lipase were significantly higher (K m 2.462 mM and V max 0.210 U/g/min) than those of extracellular lipase (K m 0.406 mM and V max 0.006 U/g/min).

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

“…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. Burket et al and Maldonado et al showed that 3.58 % w/w of peptone is enough to obtain a high level of lipase activity 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 . Olive oil 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.…”

“…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.…”

“…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 definition 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.…”

“…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.…”