научная статья по теме KINETIC STUDY OF PARTIALLY PURIFIED CELLULASE ENZYME PRODUCED BY TRICHODERMA VIRIDE FCBP-142 AND ITS HYPERACTIVE MUTANTS Биология

Текст научной статьи на тему «KINETIC STUDY OF PARTIALLY PURIFIED CELLULASE ENZYME PRODUCED BY TRICHODERMA VIRIDE FCBP-142 AND ITS HYPERACTIVE MUTANTS»

МИКРОБИОЛОГИЯ, 2011, том 80, № 3, с. 356-365

= ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

KINETIC STUDY OF PARTIALLY PURIFIED CELLULASE ENZYME PRODUCED BY TRICHODERMA VIRIDE FCBP-142 AND ITS HYPERACTIVE MUTANTS

© 2011 Shazia Shafique*, 1 and Sobiya Shafique

*Institute of Mycology and Plant Pathology, University of the Punjab, Quaid-e-Azam Campus Lahore 54590, Pakistan

Received 27.05.2010

Cellulases are the enzymes that cleave P-1,4 linkages of cellulose, and carbohydrate that is main part of plants' cell walls. Presently, cellulase isolation and partial purification was executed through ammonium sulfate precipitation. The isolated protein of parental and derived mutants conferred molecular weights of 30, 45 and 55 kDa. The optimum temperature for maximal cellulase activity was 50°C with Ea for substrate hydrolysis of 77.73, 83.97 and 83.14 kJ mol-1 and temperature quotient of1.0020, 1.0022 and 1.0022 by Trichoderma viride FCBP-142, Tv-UV-5.6 and Tv-Ch-4.3, respectively. The enzyme was stable at 50°C for about 60 min but rapid denaturation occurred above 55°C. The enzyme showed optimum activity at pH 4.0 and involved two types of acidic and basic limbs with pKaj and pKa2. The pKaj of active site presented a significant shift from 2.55 to 2.9 and 3.1 by Tv-UV-5.6 and Tv-Ch-4.3, respectively in comparison to parental strain. Likewise, pKa2 moved from 6.05 to 6.5 and 6.4. Enzyme kinetics displayed Michaelis-Menten constant Km 0.6, 0.5 and 0.28 mg mL-1 and Vmax value of 8.33, 10 and 9.09 Units mL-1 for parental, Tv-UV-5.6 and Tv-Ch-4.3, respectively.

Keywords: cellulase enzyme, partial purification, Trichoderma viride FCBP-142, enzyme kinetics.

Cellulases are the enzymes which occur in multiple forms and catalyze reactions to participate in the degradation of insoluble cellulose to soluble carbohydrates. These can be used for enhancing drainage rates, deigning, and disposing of cellulosic fines, also used in industries; in the preparation of medicines, resins, perfumes, paper and pulp, in feed treatment, brewing, cereal processing, fruit and juice processing, food fermentation, textile and laundry, detergents, in starch production, waste treatment and baking etc. [1-4]. Keeping in view the great significance of enzymes in wide area of potential applications in various sectors requires purity of this enzyme. Thus, it is essential requisite to develop fiscal processes for the purification of active enzyme [5, 6]. Previously, Becerra et al. [7] compared different enzyme purification techniques to investigate their proficiency, requirement of small amounts of crude extracts as well as the one that allowed enough purified enzyme for several uses. Nagy et al. [8] purified p-galactosidase from Penicillium chrysogenum Thom and reported that it is a multimeric enzyme of about 270 kDa with a molecular mass of 66 kDa. The optimum pH and temperature of enzyme activity was 4.0 and 30°C, respectively. Similarly Immanuel et al. [9] worked on the partial purification of the cellulase enzyme from Aspergillus niger (Van Tieghem) and A. fumigatus (Fres.) by SDS-PAGE which revealed that A niger showed two protein bands with the molecular weight of36 and 23 kDa, respectively. Similarly A. fumigatus also had

1 Corresponding author (e-mail: drshazi81@hotmail.com).

two protein bands with molecular weight of 32 and 21 kDa, respectively. Many other investigators have reported the purification of enzyme from different sources as well as characterization of enzyme [10-15].

Thus the objective of present study was the extraction, partial purification and characterization of cellulase enzyme produced by indigenous strain of Trichoderma viride FCBP-142 and its hyperactive mutants Tv-UV-5.6 and Tv-Ch-4.3 to meet the industrial sector demand.

MATERIALS AND METHODS

Isolation and Partial Purification of Enzyme. The selected strains (Trichoderma viride FCBP-142 and its hyperactive mutants Tv-UV-5.6 and Tv-Ch-4.3, obtained after UV and chemical mutation, respectively) were grown in Mandel's fermentation medium (Urea 0.3 gL-1, (NH4)2SO4 1.4 gL-1, KH2PO4 2.0 gL-1, CaCl2 0.3 gL-1, MgSO4 0.3 gL-1, yeast extract 0.25 gL-1 and proteose peptone 0.75 gL-1 with 10 gL-1 of carboxymethyl cellulose) for 5 days on a rotary shaker at 100 rpm and 30 ± 2°C. After 5 days, the broth (crude cellulase) was collected, centrifuged at 7000 rpm for 15 min at 25°C and filtered through a sterilized 0.2 ^m Millipore filter to obtain a cell free filtrate (CFF). This supernatant or cell free filtrate with enzyme was chilled and precipitated from supernatant with the addition of solid ammonium sulfate from 20% to 100% (W/V) saturation. All the subsequent steps were carried out at 0°C under constant stirring and left for

10—15 min. The aqueous phase was kept overnight at 4°C. The precipitates were formed by binding the protein fraction with ammonium sulfate [16, 17]. The precipitates hence produced were amassed by centrifugation at 7000 rpm for 20 min at 4°C and obtained pallet was dissolved in distilled deionized water and dialyzed against distilled water at room temperature for 24 hours. Diffusion bioassays were performed for the detection of cellu-lolytic activity.

Quantification and Molecular Weight determination by SDS-PAGE. Total proteins were estimated according to Bradford method [18]. The OD of different samples was determined at 595 nm wavelength for protein quantification. Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol [17] was adopted for the estimation of molecular weight of partially purified enzyme of selected strains. A protein marker of 11— 170 kDa molecular weights was used to pinpoint the desired purified protein. The gel was run at 120 Vfolts for 14 hours at room temperature. At the same time a native gel was run in the absence ofSDS to scrutinize the activity pattern. Entire gel columns of parental as well as mutant derivatives from native gel were separately placed on petri dishes containing 1% soluble cellulose agar and incubated at 30 ± 2°C for 72 hours. Later on, each petri plate was flooded with congo red (0.1% w/v). After sometime the pale clear zone indicated the presence ofCMCase activity.

Characterization of Partially Purified Enzyme

Effect of temperature. For the determination of optimum incubation temperature of enzyme, the enzyme was incubated with substrate at various temperatures from 30 to 90°C in 0.05 M citrate buffer for 15 min at pH 4.0 before assaying the cellulase activity. Then energy of activation (Ea) was calculated by using Arrhenius plot as described by Awan [19].

The effect of temperature on the rate of reaction was expressed in terms oftemperature quotient (Q10), which is the factor by which the rate increases due to a raise in the temperature by 10°C. Q10 was calculated by rearranging the equation given by Dixon and Webb [20]:

Q10 = antilog [E x 10/^T2], where E = Ea = activation energy.

Thermostability of purified enzyme. Thermal inactiva-tion ofnative and modified enzyme was determined by incubating the enzyme solutions in 0.05 M citrate buffer (pH 4) at varying temperatures (40, 45, 50, 55, 60, 65, 70, 75°C) in the absence of substrate. Aliquots were withdrawn at different time intervals, cooled on ice for 3 hours and assayed for cellulase activity as described earlier. This procedure was repeated for all the temperatures and data was analyzed.

Effect of pH. The substrate was prepared in 2 buffer solutions: citrate buffer (0.05 M, pH 3.5 to 7.0) and tris buffer (0.1 M tris HCl, pH 7.5 to 9.0). For the estimation of optimum pH, the enzyme was mixed with substrate at different pH levels (3.5 to 9.0). Reaction mixture was incubated for 30 min and the activity of purified enzyme was measured for the determination ofpKg's of active site residues as described by Dixon and Webb [20].

Effect of Substrate Concentration. The effect of various carboxymethyl cellulose concentrations on fixed amount of partially purified enzyme was also studied by using 0.005 to 0.075% substrate solution suspended in buffer. The purified enzyme was assayed at standard assay conditions and Lineweaver-Burk plots were applied as described by Siddiqui et al. [21].

RESULTS

Partial Purification of Enzyme. Active cellulase enzyme produced by parent strain (T. viride FCBP-142) and its mutants (Tv-UV-5.6 and Tv-Ch-4.3) was isolated as cell free supernatant from fermentation medium by using various ammonium sulfate percentage saturations ranging from 20 to 100%. Cell free supernatant subjected to purification exhibited maximum activity of partially purified protein fraction (enzyme) at 70% saturation (Fig. 1). The precipitation of enzyme from parental as well as mutant derivatives revealed almost similar trend. The onset of precipitation was materialized at about 25% saturation while almost complete precipitation occurred at 70% saturation of ammonium sulfate at 0°C. Data acquired on evaluation of purification of cellulase enzyme revealed low activity ofpellets recovered below 30% ofammonium sulfate saturation while active pellet/protein concentration was recovered upon stepwise increase from 30 to 70% saturation of ammonium sulfate. The results clearly indicate that the protein concentration was markedly greater in crude samples than in purified protein samples. The overall purification was about 4-fold in parental, as well as UV and chemical mutant strains and the specific activity was found to be substantially increased by purification, in all the test strains (table).

Evaluation of Molecular Weight by SDS-PAGE. The

electrophoretic mobility profiles of known molecular weight proteins were compared with that of partially purified cellulase enzyme of T. viride FCBP-142, Tv-UV-5.6 and Tv-Ch-4.3 in 10% SDS-PAGE that exhibited three bands (Fig. 2). The molecular weight assays of these polypeptide protein bands revealed around 30, 45 and 55 kDa values, respectively. These bands (proteins) possibly represent iso-enzymes or different s

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.

Показать целиком