ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2013, том 49, № 4, с. 396-401
UDC 577.154
PURIFICATION AND CHARACTERIZATION OF PECTIN LYASE SECRETED
BY Aspergillus flavus MTCC 10938
© 2013 S. Yadav, A. K. Dubey, G. Anand, and D. Yadav
Department of Biotechnology, D.D.U. Gorakhpur University, Gorakhpur, 273009, India e-mail: sangeeta_rahul@rediffmail.com Received October 29, 2012
An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the Km and kcat values of the enzyme lyase were obtained as 1.7 mg/mL and 66 s , respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55°C and it was completely stable up to 40°C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres.
DOI: 10.7868/S0555109913040156
Pectic substances are ubiquitous in the plant kingdom representing the major component of middle lamella of a plant cell wall conferring firmness and structure to the plant cells [1]. They are mainly degraded by a group of enzymes referred as pectinases which have been classified according to their modes of action and substrates used viz. pectinesterases (EC 3.1.1.11); polygalacturonases (EC 3.2.1.15); pectate lyases (EC 4.2.2.2) and pectin lyases (EC 4.2.2.10). Based on the potential industrial applications of pectinases in clarification of fruit juices, retting of fibers, treatment of pectic waste water, coffee and tea leaf fermentation, oil extraction, virus purifications, these enzymes have been extensively studied [2-5].
Pectin lyases (PNL) are of particular interest as they degrade pectin polymers directly by P-elimina-tion mechanism resulting in the formation of 4,5-un-saturated oligogalacturonides while other pectinases act sequentially to degrade pectin molecule totally. Further, PNL have biotechnological potential in fruit juice industries due to the fact that they degrade pectin without disturbing the ester group which is responsible for specific aroma of the juice and also do not form methanol which is highly toxic [6]. The production, purification, biochemical characterization, molecular biology and applications of PNL have recently been reviewed [7]. These enzymes are relatively less explored enzyme as compared to other pectinases. PNL with different properties were used for different applications, e.g enzymes with pH optima in the acidic range were suitable for clarification of fruit juice [8],
while PNL with pH optima in alkaline range were effective in retting of natural fibres [9]. Thus, there is a scientific need to search new PNL source with properties suitable for different applications.
The aim of the study was to purify and characterize an alkaline PNL from a new fungal isolate Aspergillus flavus MTCC 10938 showing efficient retting of sunn hemp (Crotolaria juncea) fibres.
MATERIALS AND METHODS
Chemicals. Citrus pectin, DEAE cellulose and Sephadex G-100 were purchased from Sigma (USA). Rests of the chemicals were procured either from Merck (Germany), Navi Mumbai (India) or S.D. Fine Chem. Ltd. (India) and were used without further purification.
PNL assay. Activity of PNL was detected by the method of Albersheim [10]. Assay was performed by monitoring the increase in optical density at 235 nm due to formation of unsaturated uronide product using spectrophotometer Hitachi (Japan) model U-2000. Enzyme solution (0.2 mL) was added to the reaction mixture containing 0.8 mL citrus pectin (1% w/v) and 2.0 mL of the 100 mM phosphate buffer (pH 8.0) maintained at 37°C. Optical density was measured at zero time and after 20 min of incubation and the steady state velocity was calculated as absorbance change per min. PNL activity was defined as ^mole of unsaturated product released per min, based on the molar extinction coefficient value of 5500 M-1 cm-1.
Protein estimation. Protein was determined by the Lowry's method taking bovine serum albumin as the standard.
Organism and growth conditions. The fungal strain was isolated from the contaminated tissue culture media in the Plant Tissue Culture laboratory of the Department of Biotechnology, DDU Gorakhpur University (India), using a standard serial dilution technique [11]. The isolated and purified fungal strain was screened for the secretion of pectinases by plate assay method [12]. The PNL producing fungal strain was identified and deposited as Aspergillus flavus MTCC 10938 by Microbial Type Culture Collection Centre and Gene Bank, Chandigarh (India). The culture was maintained by cultivation on Czapek-Dox agar slants at 25°C fortnightly. Based on prominent halo zone formation the culture was further screened for the PNL secretion in liquid culture medium containing 0.3% KH2PO4, 0.05% MgSO4 • 7H2O, 0.2% L-Asp and 0.5% pectin. The initial pH of the medium was adjusted to 4.5. Once the culture conditions for maximum secretion of the pectin lyase were optimized, the fungal strain Aspergillus flavus MTCC10938 was grown for purification of extracellular PNL in submerged fermentation medium in 25 x 100 mL culture flasks. One mL of spore suspension (spore density 5 x 106 spores/mL) from agar slant was inoculated aseptically into 25 mL of sterilized liquid culture medium, contained in 100 mL culture flasks. The flasks were incubated at 25°C in BOD incubator and cultures were allowed to grow under stationary conditions. On the seventh day (day for maximum production) culture was filtered through Whatman no 41 filter paper, this cell free filtrate (CFF) was used as crude for further purification.
Purification of the enzyme. Ultrafiltration by Amicon. The clear culture filtrate was centrifuged at 14000 x g for 10min and 400 mL supernatant was concentrated to 2.0 mL with the help of Amicon Concentration Cell (USA) using PM 10 ultrafiltration membrane having 10 kDa molecular mass cut off value.
DEAE cellulose column chromatography. The concentrated crude PNL was dialyzed against 2.0 L of 10 mM citrate phosphate buffer (pH 7.0) with 3 changes at the intervals of 8 h. The sample was then centrifuged for 10 min at 14000 x g and the supernatant was loaded on DEAE cellulose column (6.5 x 2.0 cm) equilibrated with 10 mM citrate phosphate buffer (pH 4.0). The adsorbed protein was then washed twice by bed volume of the same buffer. The protein was eluted stepwise using 10 mL of NaCl (0.2—1.0 M) in the same buffer at the flow rate of 18 mL/h. Fractions of 3.0 mL were collected and analyzed for activity for the PNL and protein.
Sephadex G-100. The pooled fractions from DEAE cellulose showing maximum activity were concentrated with sucrose and dialyzed against 2.0 l of 100 mM citrate phosphate (pH 7.0) buffer. The dialysed PNL was centrifuged at 14000 x g and the supernatant was
loaded on a Sephadex G-100 column (1.0 x 30.0 cm), pre-equilibrated with the same buffer. The flow rate was maintained at 9.0 mL/h and fractions of 1.5 mL were collected and analyzed for protein and PNL activity. All the active fractions were pooled, concentrated and tested for homogeneity by electrophoresis.
SDS-PAGE. The enzyme purity and the molecular mass were estimated by the 10% SDS-PAGE method, using phosphorylase B (97.4 kDa), BSA (66.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (29.0 kDa), soyabean trypsin inhibitor (20.1 kDa) and lysozyme (14.3 kDa) as standard protein markers [13]. Silver staining was done for locating the protein bands [14].
Characterization of the A. flavus MTCC 10938 PNL. The Km and kcat values of the purified A. flavus MTCC 10938 PNL were determined by measuring steady state velocities of the enzyme catalyzed reaction at different concentrations of citrus pectin and drawing double reciprocal plots [15]. Calculations were made using linear regression analysis of the data points of double reciprocal plot. The pH optimum was determined by measuring steady state velocity in the buffered reaction solution using different 100 mM buffers at in the pH range 1.0—12.0. The buffers used were: HCl-KCl (1.0-2.0), citrate-phosphate (3.07.0), sodium-phosphate (8.0), glycine-NaOH (9.010.0) and sodium phosphate-NaOH (11.0-12.0).
Steady state velocity of the PNL reaction against pH of the reaction medium was plotted. The pH stability of the enzyme was studied by exposing the enzyme to buffers of different pH for 24 h at 4°C. The residual activities were assayed and plotted in the form of percent residual activity versus pH. The optimum temperature for the A. flavus MTCC 10938 enzyme activity was determined by assaying the activity of the enzyme at different temperatures in the range 5-100°C and plotting a graph of the enzyme activity versus temperature of the reaction. Thermal stability of the enzyme was tested by incubating an enzyme aliquot at a particular temperature (10-100°C) for 30 min, assaying its residual activity and plotting the percent residual activity against temperature.
Effect of metal ions and protein inhibitors on the A. flavus MTCC 10938 PNL activity. The effect of metal ions like Ca++, Mg++, Mn++, Co++, Zn++, Hg++, K+, Na+, Ag+ and protein inhibitors like sodium azide, potassium permanganate, potassium ferro-cyanide and EDTA were studied by measuring the steady state velocity in the reaction solutions containing 1mM of the metal ions or protein inhibitors and comparing it with the value in the absence of these ions or inhibitors.
Retting
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