ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2014, том 50, № 3, с. 311-317
UDC 577.156
BIODEGRADATION OF FEATHER WASTES AND THE PURIFICATION AND CHARACTERIZATION OF A CONCOMITANT KERATINASE
FROM Paecilomyces lilacinus © 2014 Q. Y. Wang and M. D. Liao
Key Lab of Natural Pesticide and Chemical Biology of Ministry of Education, College of Resources and Environment, South China Agricultural University, Guangzhou, Guangdong, 510642 China e-mail: meideliaotg@163.com; qywtg66@163.com Received April 21, 2013
Paecilomyces lilacinus strain PL-HN-16 was found to have the ability to degrade feathers. During the degradation process, the broth initially turned as sticky as gelatin and then turned into fluid that means the feathers can be hydrolyzed completely. Keratinolytic protein (Ker) of aforementioned strain was purified using ammonium sulphate precipitation, HiTrap Butyl FF chromatography and Sephacryl S-200 gel filtration. The Ker of P. lilacinus PL-HN-16 had molecular mass of 33 kDa, the optimum pH 8.0 and temperature optimum at 40° C. It used the soluble keratin as substrate. The enzyme showed high activity and stability over a wide range of pH (6.0 to 10.0) and temperature (30°C to 60°C) values but was completely inhibited by PMSF. Ker of P. lilacinus PL-HN-16 exhibited stability toward SDS. These promising properties make the enzyme a potential candidate for future applications in biotechnological processes as keratin hydrolysis and dehairing during leather processing.
DOI: 10.7868/S0555109914030313
Millions of tons of keratin-containing wastes are discharged every year by the commercial poultry, leather, and meat processing industries. Feather waste, which contains over 90 and 70% crude protein and amino acids, respectively, represents a potential protein alternative to more expensive dietary ingredients for animal feeds [1—2]. Thus, a significant amount of these low-cost keratin-containing wastes is available for biotransformation into value-added products, especially in the form of non-polluting proteolytic enzymes process [3].
Keratinases (EC. 3.4.99.11) constitute a group of enzymes that are regarded as the best enzymes for bioconversion of keratinous residues. These enzymes could hydrolyze the highly stable and insoluble keratin structure consisting of disulfide, hydrogen, and hydrophobic bonds in the form of a-helices and P-sheets [3]. Bioconversion of keratinous residues is attracting increasing biotechnological interest, as it could represent an alternative waste management method that could be combined with the production of valuable products [4].
Proteases play a significant role in the detergents since they work as additives. It is necessary to enhance the performance of the prevailed enzyme-based detergents by adding newer enzymes equipped with various properties [5]. There are some basic requirements for a proteolytic enzymes, for example, they should be active and keep functional at alkaline pH values and at relatively high temperatures and can be mixed up with other detergent components like surfactants, per-
fumes, bleaches, etc. [6]. On the other hand, the majority of commercially available enzymes can't perform well when they work at a relative high temperatures or detergents appearing.
Various biotechnological applications of microbial proteases have been described in the feeds, leather and detergents industries. Thus, we can only meet the growing demand for these enzymes in various fields by screening for novel enzyme-producing microorganisms with potential applications [7].
Paecilomyces lilacinus has been used as biocontrol agent against plant parasitic nematodes in the agriculture field all over the word. The aim of this study was to test P. lilacinus strain PL-HN-16 isolated from the soil for degradation of feathers, to purify and characterize keratinolytic protein (Ker), to evaluate its stability against such factors as pH, temperature, reducing agents, surfactants, and finally to find some properties better suitable for commercial exploitations.
MATERIALS AND METHODS
Microorganism, culture medium. P. lilacinus strain PL-HN-16 was obtained from soil and saved in Key Lab of Natural Pesticide and Chemical Biology of Ministry of Education, South China Agricultural University, Guangzhou, Guangdong. The medium for cultivation of microorganism and keratinase production was composed of (g/L): KCl - 0.5; K2HPO4 - 1.0; MgSO4 • 7H2O - 0.5; FeSO4 • 7H2O - 0.01; sucrose -30 and feathers - 4.0.
Effect of cultural conditions on enzyme production.
To optimize the conditions for enzyme production, initial pH was changed from 6.0 to 9.0 with space 0.5 while the temperature was kept at 29°C. The temperature was kept at 20, 25, 29, 34 and 38° C while initial pH was set at 7.0. Aliquots of 200 mL were distributed into 1000 mL Erlenmayer flasks and autoclaved. To test the effect ofgrowth substrate concentration on enzyme production the media containing 1, 4, 10, 15 and 20 g/L of feathers were prepared. After inoculation with a spore suspension of P. lilacinus strain PL-HN-16 (105—107 spores/mL of medium), the Erlenmayer flasks were put in an orbital shaker at 160 rpm for 20 days. After incubation, culture supernatants obtained after centrifugation at 7000 g for 20 min were used as crude enzyme.
Determination of the degradation rate of feathers (DRF). The DRF was calculated using equation 1:
DRF (%) = (TF - RF) x 100%/TF,
(1)
where TF is the total feather dry weight of control (without inoculation) and RF is the residual feather dry weight of experimental group.
Assay of keratinolytic activity. Ker activity was determined by using soluble keratin as a substrate. It was obtained by the method described by Wawrzkiewicz et al. [8]. The soluble keratin was suspended in 50 mM Tris-HCl buffer (pH 8.0).
The reaction mixture contained 1.0 mL of appropriately diluted enzyme of P. lilacinus strain PL-HN-16, 1.0 mL of keratin suspension and was incubated at 40°C in a water bath for 10 min. The reaction was terminated with 2 mL of 10% (w/v) trichloroacetic acid (TCA) and incubation mixture was kept at 4°C for 30 min. After centrifugation at 1800 g for 10 min, the OD280 of the supernatant was measured spectrophoto-metrically. The enzyme control was treated the same way except that TCA was added before the incubation. One unit of keratinolytic activity was defined as an increase of corrected OD280 for 0.01 under the conditions described [9].
Protein determination. Protein concentration was measured by the method of Bradford [10], using BSA (Sigma, USA) as a standard.
Ammonium sulphate fractionation. Non-kerati-nolytic proteins in the supernatant of P. lilacinus strain PL-HN-16 were precipitated by adding solid ammonium sulphate at 40% saturation. Ker in the supernatant after centrifugation at 7000 g was precipitated at 80% saturation. The precipitate was collected by cen-trifugation at 7000 g for 20 min. Target precipitate was dissolved in 50 mM Tris-HCl buffer (pH 8.0) before further purification.
Chromatography of Ker. The preparation enzyme fluid (contained 1.0 M (NH4)2SO4) was applied to a HiTrap™ Butyl FF column (GE Healthcare Bio-Sciences AB, Sweden), which was processed at a flow rate of 2 mL/min with 50 mM Tris-HCl buffer (pH 8.0)
containing 1.0 M (NH4)2SO4. After washing the column with the same buffer to remove unbound proteins, the bound enzyme was eluted by stepwise decreasing salt concentrations from 1 M to 0 M (NH4)2SO4 in 50 mM Tris-HCl buffer (pH 8.0). The keratinolytic active fractions were collected, pooled and concentrated by ultrafiltration through a 10 kDa cut-off membrane (Millipore, USA). The most active fractions were loaded onto Sephacryl S-200 column (Amersham Pharmacia Biotech. AB, Sweden) previously equilibrated with 50 mM Tris-HCl buffer (pH 8.0) and then Ker was eluted with the same buffer at a flow rate of 0.25 mL/min. The resulting active fraction was collected and used as the purified Ker of P. lilacinus PL-HN-16.
Gel electrophoresis and molecular weight determination. Protein purity and molecular mass of the enzyme were evaluated by SDS-PAGE as described by Laemmli [11]. The gels were then stained with Coo-massie brilliant blue R-250, destained, and the elec-trophoretic migration of the protein was compared with that of low-molecular mass protein markers (Dingguo, China). Zymography was performed according to the method described by Koshikawa et al. [12], Pillai and Archana [7], and Bressollier et al. [13]. After electrophoresis, the gel was washed with 2.5% (v/v) Triton X-100 for 30 min and then with 50 mM Tris-HCl buffer pH 8.0 for 60 min. Soluble keratin prepared in 50 mM Tris-HCl (pH 8.0) was poured on the gel slab. After 4 h of incubation at 40°C, the gel was washed with distilled water, stained with Coomassie brilliant blue R-250 and then destained.
Effect of pH and temperature on Ker activity. The optimal pH for the activity of Ker from P. lilacinus PL-HN-16 was determined at 40°C at pH region from 3.0 to 10.0. Na2HPO4-citric acid, Tris-HCl and gly-cine-NaOH were used for pH pH 3.0-7.0, 7.0-9.0 and 9.0-10.0, respectively For pH stability, residual activity was measured at pH 8.0 after 2 h of incubation at 40°C in the pH buffers being tested.
The effect of temperature on enzyme activity was examined at temperatures ranging from 30 to 90°C in 50 mM Tris-HCl buffer (pH 8.0). Thermal stability was determined by incubating Ker of P. lilacinus PL-HN-16 at 30-80°C and pH 8.0 for 0.5, 1.0, 2.0, 3.0, 4.0 h.
Effects of metal ions, protease inhibitors, reducing agents and surfactants on Ker activity. One, ten and one hundred mM of K+, Na+, Ca2+, Mg2+, Cu2+, Mn2+, Co2+, Ba2+, Zn2+, Fe3+, PMSF, EDTA, DTT and SDS; 0.1, 1, 10% (v/v) of Tween 80, Triton X-100, and ß-mercaptoethanol were used to test the action of different reagents on Ker of P. lilacinus PL-HN-16. The residual enzyme activity was measured after the pre-incubation ofKer with various chemicals at 30°C for 1 h. K
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.