Agroindustrial Wastes as Alternative for Lipase Production by Candida viswanathii under Solid-State Cultivation: Purification, Biochemical Properties, and Its Potential for Poultry Fat Hydrolysis

Abstract

The aims of this work were to establish improved conditions for lipase production by Candida viswanathii using agroindustrial wastes in solid-state cultivation and to purify and evaluate the application of this enzyme for poultry fat hydrolysis. Mixed wheat bran plus spent barley grain (1 : 1, w/w) supplemented with 25.0% (w/w) olive oil increased the lipase production to 322.4%, compared to the initial conditions. When olive oil was replaced by poultry fat, the highest lipase production found at 40% (w/w) was 31.43 U/gds. By selecting, yeast extract supplementation (3.5%, w/w), cultivation temperature (30°C), and substrate moisture (40%, w/v), lipase production reached 157.33 U/gds. Lipase was purified by hydrophobic interaction chromatography, presenting a molecular weight of 18.5 kDa as determined by SDS-PAGE. The crude and purified enzyme showed optimum activity at pH 5.0 and 50°C and at pH 5.5 and 45°C, respectively. The estimated half-life at 50°C was of 23.5 h for crude lipase and 6.7 h at 40°C for purified lipase. Lipase presented high activity and stability in many organic solvents. Poultry fat hydrolysis was maximum at pH 4.0, reaching initial hydrolysis rate of 33.17 mmol/L/min. Thus, C. viswanathii lipase can be successfully produced by an economic and sustainable process and advantageously applied for poultry fat hydrolysis without an additional acidification step to recover the released fatty acids.

Figure 1

Time course of lipase production by C. viswanathii in solid-state cultivation using wheat bran plus barley spent grain 1 : 1 (w/w) supplemented with 25% (w/w) olive oil. Cultures were carried out without nitrogen source supplementation and 50% (w/v) initial moisture provided by Vogel salts solution at 28°C for 120 hours. ■: lipase production (U/gds); ∘: specific activity (U/mg of protein).

Figure 2

Effects of different triacylglycerol sources (a) and poultry fat concentration (b) on lipase production by C. viswanathii in solid-state cultivation. Cultures were carried out with wheat bran plus barley spent grain supplemented with 25% (w/w) of each triacylglycerol source without nitrogen source supplementation and 50% (w/v) initial moisture provided by Vogel salts solution, at 28°C for 5 days (a). Cultures were carried out in the same conditions with only poultry fat (b). ■: lipase production (U/gds); □: specific activity (U/mg of protein).

Figure 3

Effect of yeast extract concentration on lipase production by C. viswanathii in solid-state cultivation. Cultures were carried out with wheat bran plus barley spent grain supplemented with 25% (w/w) of each triacylglycerol source and 50% (w/v) initial moisture provided by Vogel salts solution, at 28°C for 5 days. ■: lipase activity (U/gds); □: specific activity (U/mg of protein).

Figure 4

Profile of hydrophobic interaction chromatography of C. viswanathii lipase produced in solid-state cultivation under the best conditions for enzyme production. Chromatograph conditions: 0.02 M ammonium acetate buffer pH 6.9; 2.0 mL/min flow rate; 3.0 mL fractions, at 4°C; elution with Triton X-100 0–1.0% (w/v).

Figure 5

SDS-PAGE of purified lipase from C. viswanathii produced in solid-state cultivation. Column 1: standards: phosphorylase b (97 kDa), albumin bovine serum (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and α-lactalbumin (14.2 kDa). Column 2: purified lipase.

Figure 6

Optimum pH (a) and pH stability (b) and optimum temperature (c) and thermal stability (d) of crude and purified C. viswanathii lipase. Assay conditions: 0.05 M glycine-HCl buffer pH from 2.0 to 3.0, McIlvaine buffer pH from 3.0 to 8.0, and 0.05 M glycine-NaOH pH from 8.6 to 10 at 37°C (a); the crude enzyme was incubated in the same buffers for 24 h at 10°C and lipase activity was assayed in McIlvaine buffer pH 5.0 for crude enzyme and 5.5 for purified enzyme at 37°C (b); lipase activity assays were assayed in McIlvaine buffer pH 5.0 for crude enzyme and 5.5 for purified enzyme (c); ■: crude enzyme; ●: purified enzyme.

Figure 7

Activity of crude and purified C. viswanathii lipase on p-nitrophenyl esters. Activity was determined in McIlvaine buffer pH 5.0 at 50°C for crude enzyme and 5.5 at 45°C for purified enzyme. ■: crude lipase; □: purified lipase. C2 acetate, C4 butyrate, C8 caproate, C10 decanoate, C12 laurate, C14 myristate, C16 palmitate, and C18 stearate.

Figure 8

Poultry fat hydrolysis profiles by crude (a) and purified (b) C. viswanathii lipase. Assay conditions: hydrolysis was carried out using enzyme concentration 10 U/mL, S ₀ = 100 g·L⁻¹, 200 rpm at 40°C. ■: pH 4.0; ●: pH 6.0; ▲: pH 8.0.