‘Synthetic lipase’ production from a newly isolated Sporidiobolus pararoseus strain by submerged fermentation

The lipase produced by a newly isolate Sporidiobolus pararoseus strain has potential catalysis ability for esterification reactions. In order to improve its synthetic activity, this work aimed at optimizing ‘synthetic lipase’ production by submerged fermentation of a conventional media based on peptone, yeast extract, NaCl and olive oil using experimental design technique. According to the results obtained in the first experimental design (24–1), yeast extract and NaCl concentrations were tested to further optimization by response surface methodology. The maximum ‘synthetic lipase’ activity obtained was 26.9 U/mL in the optimized media (5.0, 6.8, 7.0 and 1.0% (wt/v) of peptone, yeast extract, NaCl and olive oil, respectively), representing a 6.36-fold increase compared to the initial medium. The time course of ‘synthetic lipase’ production in the optimized condition was evaluated in terms of synthetic activity, protease activity, biomass and total carbon and the maximum synthetic activity was observed during the stationary phase of growth.

'Synthetic lipase' production from S. pararoseus biocatalysts, as they present high synthesis rates, high-yield conversion of substrate to product, great versatility and simplicity in environmental and genetic manipulation of its productive capacity and improved recovery from fermentation broth (3, 20).
Microbial lipases are produced mainly by submerged culture (31). A wide variety of culture conditions that stimulate or suppress the production of microbial lipases are described in the literature, and there is no general procedure to improve its production (15,31). The expression of lipolytic proteins is often induced and can be modulated by several parameters (20). Among them, sources of carbon and nitrogen supplied during fermentation are of particular importance, as well as the addition of compounds which can act as inducers, such as lipid carbon sources (31,37). It has been reported that some eukaryotic microorganisms can produce different forms of lipases with different catalytic properties, and production of these biocatalysts were influenced by culture conditions (39).
Hydrolytic and synthetic activities are often used to characterize a lipase catalytic ability, and the former is mostly preferred. However, it has been proposed that synthetic activities of lipases in organic solvents do not correspond to their hydrolytic activities, and not all the lipases were capable of catalyzing synthetic reactions in organic solvents. In order to improve the lipase catalytic ability in organic solvent, the effects on the enzyme production must be studied systematically and the culture condition needs to be optimized (35,36,39).
Being an indicator of catalytic ability in organic solvent, synthetic activity of lipase attracts more and more researchers' eyes (39). The production of lipases for use in organic media has advantages such as higher solubility in hydrophobic substrates, shift of thermodynamic equilibrium in favor of synthesis instead of hydrolysis and increased thermo-stability of the enzyme. However, catalytic activities are generally lower than those expressed in water, due to difficulties in homogenization of the catalytic system (22).
'Synthetic lipases' are gaining more attention nowadays because of their potential to catalyze the production of biodiesel, reducing the operational cost associated with the conventional process, as well overcoming problems related to chemical catalysis. As the lipase cost of producing is a major obstacle to the commercialization of the lipase catalyzed process, various attempts have been made to develop cheaper systems (28,38). Besides biodiesel production, many other applications of lipase with synthetic activity have been proposed, such as enantioselective reactions (6, 24), synthesis of various esters (5, 21,26) and kinetic resolutions of chiral compounds (2).
Till date yeast lipases from Candida rugosa and Candida antarctica stand out as the sources of most commercially available lipase preparations (17,37). At the same time, continuous demand for highly active enzymes with appropriate properties encourages the research for new enzyme sources (17). In a previous work developed by our research group, a number of microorganisms potentially producers of synthetic and hydrolytic lipases were isolated from various sources (13).
Here, we report media optimization for maximum 'synthetic lipase' production from a newly isolated strain of Sporidiobolus pararoseus by submerged fermentation of a conventional media.

Yeast identification
Firstly, the strain was incubated in PD medium at 28 0 C for 24 hours. The yeast DNA was extracted according to the methodology proposed by Stearling (32) and quantified in a spectrophotometer model NanoDrop, ND-1000 (NanoDrop Technologies).
The domain D1/D2 of the 26S rDNA, that provided a similar identification when compared with DNA reassociation, is a quickly and easy methodology for yeast species (18).Using primers NL1 and NL4 (10,19) (11), in which 1000 bootstrapped trees were generated from the re-sampled data.

Cell production
The yeast used in the present study was previously isolated from soybean meal (13). This microorganism was

Optimization of lipase production
The concentration of media components, which could influence 'synthetic lipase' production by Sporidiobolus pararoseus under submerged fermentation, was evaluated.
Before optimization, a time course experiment was conducted using the conventional media optimized in a previous work (25). Lipase synthetic activity in a media constituted by 2.0, 0.5, 0.5 and 1.0 % wt/v of peptone, yeast extract, NaCl and olive oil, respectively, was monitored within 168 hours of fermentation at 30 ºC and 150 rpm. The media optimization for the production of lipase by S. pararoseus was performed at 48 hours of fermentation using the strategy of sequential experimental designs. In the first experimental design, the effects of media components were evaluated using a 2 4-1 fractional design. After statistical analysis, a second experimental design (full 2 2 ) was performed keeping constant peptone and olive oil concentrations (5.0 and 1.0%wt/v, respectively). In order to optimize the lipase production, three concentrations of NaCl (7.0, 8.0 and 9.0% wt/v) were evaluated in triplicate assays, keeping constant the peptone, yeast extract and olive oil concentrations (5.0, 6.8 and 1.0%wt/v, respectively). Table 1

Time course of lipase production
Lipase synthetic activity, protease activity, biomass (dry cellular weight) and total carbon concentration were monitored within 120 hours of fermentation in the optimized condition.

Analytical assays
Lipase synthetic activity was assayed by alkali titration

Yeast identification
The isolate was identified as Sporidiobolus pararoseus,  'Synthetic lipase' production from S. pararoseus

Lipase production optimization
Before optimization process was carried out, a time course experiment was conducted in a media optimized previously (25). Fig. 1 presents the time course of 'synthetic lipase' production within a 168 hours incubation period. It can be observed that the highest lipase activity (4.23 U/mL) was obtained after 48 hours of fermentation. In this way, this fermentation time was chosen to perform the sequential experimental design.   According to these authors, the enhanced synthetic activity with whole-cell lipase is probably ascribed to some co-factors or amino-acids contained in peptone which accidentally match R. chinensis physiological requirements for WCSL biosynthesis (33). In the optimization of submerged fermentation media for the production of synthetic membranebound lipase from Rhizopus chinensis, the authors found that peptone concentration was the most important factor influencing lipase production, followed by olive oil concentration (39).
Many researchers have published their results on effects of oils in enhancing lipase synthetic and hydrolytic activities (33).
However, a few authors have produced high yields of hydrolytic lipases in the absence of fats and oils (31). In a work evaluating the influence of the presence of different lipid compounds on the production of different lipases (intracellular, extracellular and membrane-bound) by Thermus thermophillus in submerged fermentation, it has been shown that the presence of olive oil improved the production of intracellular and membrane-bound lipases, but not extracellular lipases (7).
The salinity was found to be a critical factor on the production of hydrolytic lipase by a marine isolated Pseudomonas sp. strain. In the absence of sodium chloride, only traces of enzyme were produced and no considerable microorganism growth was observed. The lipase production reached the highest value when the production media contained 1.5% NaCl (16).      (36).

Time course of lipase production
Lipase synthetic activity, biomass (dry cellular weight) and total carbon concentration profile for lipase production in the optimized media is given in Fig. 3. From this profile it can be observed that the highest lipase synthetic activity was obtained in 72 hours of fermentation using the optimized media. It was not observed protease activity during the fermentation according to the measurements. It can be also observed that there is a tendency of stabilization of the microorganism growth while total carbon keeps slightly decreasing. The maximum rate of carbon consumption in lower fermentation time coincides with the maximum rate of cellular growth, which means that most of the carbon present in the media was consumed for the microorganism growth.

CONCLUSION
The isolate Sporidiobolus pararoseus strain yielded good results for the production of 'synthetic lipase' by submerged fermentation by a conventional medium. The optimized condition for 'synthetic lipase' production was determined as 5.0, 6.8, 7.0 and 1.0 % (w/v) of peptone, yeast extract, NaCl and olive oil, respectively, at 30 ºC and 150 rpm and 10% of inoculum. The optimized media yielded a lipase synthetic activity of 26.9 U/mL in 72 hours of fermentation, a 6.36-fold increase compared to the non-optimized media.