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Being as the second most common polysaccharide in nature, hemicelluloses exhibit 20-30% of lignocellulosic biomass. Xylan is the primary and major constituent of hemicelluloses and is acknowledged as the important means of renewable biomass . Xylan is effectively hydrolyzed by the unified actions of β-1, 4-endoxylanases (EC 18.104.22.168), β-xylosidase (EC 22.214.171.124) [2, 3]. Therefore, lignocellulosic biomas has greater prospect of substantial production of xylooligosaccharides. Due to their efficacious application in paper and pulp industries, food and feed, microorganisms capable of producing xylanolytic enzymes (xylanases) have received utmost consideration in the past few years . But various applications require cellulose free and thermostable xylanases .
Microorganisms including fungi, yeasts and bacteria are used as the precursor of xylanase, and those xylanase are prominent in waste treatment, animal feed, paper and pulp, biofuel industries, baking and brewing in recent years. Xylanase of low molecular mass are tremendous for the pretreatment of kraft cooked pulp because for better hydrolysis xylanase have to enter to the interior part by forming pore and only low molecular weight xylanase can perform this . Furthermore, xylanase exerting low molecular weight and stable over wide range of pH and temperature will nake the process even more economical. These xylooligosaccharide are linked via β-1,4 glycosidic bond and are non-digestable which are known to decrease risk of colon cancer, increase bioavailability of calcium and reduce cholesterol level . Xylanase also has been used to enhance the nutritional property of agricultural silage, grain feed and clarifying wine and fruit juices and reduce the pollution created by chloro-organo compounds in paper-pulp industries .
Till date, different Bacillus species has been reported as their capability of producing xylanase, some of them are as B. cereus BSA1 [M], Bacillus sp. GRE7 [N], B. stearothermophilus T6 [J], Bacillus licheniformis A99 [R], Geobacillus thermodenitrificans TSAA1[S], Bacillus halodurans S7 [K], Bacillus amyloquefaciens [I] etc. This current work report a xylanase produced from Bacillus sp. isolated from Korean soil sample, designated as CSB40. Beside the production and purification of xylanase, we show the biochemical and thermodynamic characterization of this enzyme and its appropriateness in producing xylooligosaccharide which has great potential of being rationally promoted in feed formulations and nutraceuticals [15,16].
2. Materials and methods
Beechwwod xylan was purchased from Sigma-Aldrich (St Louis, USA). Silica gel plates used for Thin Layer Chromatography were from Merck (Darmstadt, Germany). Xylose, xylobiose, xylotriose and xylotetraose which were used as standard xyloolisaccharides were purchased from Megazyme (Ireland). DEAE Sepharose fast flow was purchased from Amersham Bioscience (Uppsala, Sweden). All the reagents used were of the highest analytical grade available.
2.2. Enzyme production and purification
A bacterial strain CSB40 was isolated from the provinces of Korea were cultivated in Beechwood xylan (1.25%), peptone (0.5%), yeast extract (0.25%), K2HPO4 (0.3%), KH2PO4 (0.3%), CaCl2 (0.02%), MgSO4.7H2O (0.03%) and NaCl (0.01%). CSB40 was cultivated in 1000 mL Erlenmeyer flask containing 300 mL medium at 37 °C and 120 rpm for 48 hours. The cultural broths were centrifuged at 10,000xg for 30 minutes and clear supernatant was subjected to purification via ammonium sulphate precipitation method. Purification procedures were performed out at 0 °C unless stated otherwise. Ammonium sulfate was added to supernatant at 30-80% saturation, and the mixture was kept overnight. The precipitate was centrifuged at 10000xg for 45 min at 4°C. The pellets were resuspended in 10 mM Potassium Phosphate buffer (pH 9), and dialyzed against the same buffer overnight and concentrated with an ultra filtration membrane of 50 kDa (Millipore Corp.). The enzyme solution was loaded to DEAE Sepharose Fast Flow (12cm X 2.3 cm) pre-equilibrated with 10mM Phosphate buffer, pH 9. Gradient elution of bound proteins was done using same buffer containing 0 to 1M KCl at the flow rate of at 33 mL/h. Active fractions of xylanase were pooled, concentrated, and analyzed for the purity. Further tests were carried out using the pure enzyme.
2.3. Enzyme assay and protein estimation
Bradford method was used to determine the protein concentration where bovine serum albumin was deployed as standard . The xylanase activity of enzyme was determined by measuring the release of reducing sugar using 3, 5-dinitrosalicyclic (DNS) method described by Miller using xylose as standard . 100µl of 1% (w/v) beechwood xylan prepared in 10mM phosphate buffer (pH 9) and 100µl of appropriately diluted enzyme was used as standard assay mixture. The enzyme and substrate mixture was then subjected to incubation at 50 °C for 45 minutes in water bath. After the incubation, 100µl of DNS reagent was added in the mixture and boiled for 10 minutes, and cooled. The amount of reduced sugar liberated was measured by absorbance at 540nm. The amount of enzyme that discharges 1µm of xylose equivalent reducing sugar per minute is considered as one unit of xylanase activity.
2.4. Polyacrylamide gel electrophoresis
The molecular weight of the purified enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% (w/v) polyacrylamide gel as described by Laemmli . Afterward electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 and then destaining with a solution containing methanol, glacial acetic acid and distilled water. Molecular weight was estimated by comparison with the reference protein. For the reference proteins, protein size marker (Thermo Scientific, USA) was used.
Xylan activity (zymography) was performed as described in . In brief, zymography (activity staining) were obtained by co-polymerizing 0.2% (w/v) Beechwood Xylan with 12% (w/v) polyacrylamide. After electrophoresis, the gel was washed two times by distilled water for 10 minutes and followed by soaking in 25% (v/v) isopropanol with light shaking to remove the SDS which also renature the proteins in the gel. The gel was then incubated for 60 min at 50°C, the zymogram was stained for residual carbohydrates with Congo red solution (0.1%, w/v), destained with 1 M NaCI and fixed with 0.5% (v/v) acetic acid. The activity bands observed and were photographed.
2.5. Effect of temperature and pH
Temperature was optimized at pH 12.5 in the range 40-80°C. To determine temperature stability, enzyme samples were pre-incubated at various temperatures (up to 80 °C) for 75 minutes, and residual activities were measured under standard assay conditions. The pH optimum was determined by carrying out enzyme assay at 50 °C using various pH buffer (pH value 2.5-13.5 and 200 mM concentration). The buffers used were; citric acid/sodium citrate (2.5-4.5), potassium phosphate (6-7), Tris/HCl (8-9), sodium carbonate/sodium bicarbonate(10-11) and KCl/NaOH (12.5-13.5). pH stability was determined after incubating the sample at 10mM with various pH buffers at 0°C for 24 and residual activity was measured at 20mM under standard conditions.
2.6. Effects of metal ions and chemicals
The effects of additives such as detergents, reducing, oxidizing agents, metal ions and chemicals on the activities of xylanase were studied.
For the metal ions, the pure enzyme was incubated with monovalent (K+ and Na+) and divalent (Ca2+, Mg2+, Cu2+, Co2+, Zn2+, Ni2+, Ba2+, Mn2+, and Fe2+) metal ions at final concentration of 5mM. Effect of xylanase activity was seen upon addition of 10% concentrated different chemicals. The relative activities were determined under standard protocol comparing with control without additives respectively (100%).
2.7. Effects of detergents and modulators
The effects of detergents on xylanase activities were calculated by adding Triton X-100, Tween-20, Tween-80, CHAPS, and sodium dodecyl sulfate (SDS) at a concentration of 0.25 %. The impact of oxidizing and reducing agents on xylanase activities were also studied by incubating with hydrogen peroxide, sodium perborate and β-mercaptoethanol whereas the effects of chelating agents like ethylene diamine tetra-acetic acid (EDTA) and ethylene glycol tetra-acetic acid (EGTA) on xylanase activity were studied at a concentration of 5mM. In every case, the relative enzyme activities were measured under standard conditions with the activities without any additives taken as control (100 %) as described above.
2.8. Enzyme production using agro-industrial waste
Different agro wastes such as corncob, wheat bran and malt sprout were utilized as the carbon source for the production of xylanase and were compared to that of beechwood xylan.
2.8. N- terminal amino acids
The N-terminal amino acid sequence of CSB40 was determined by Edman degradation using a Procise Model 492 protein sequencer (Applied Biosystems, CA, USA).
2.9. Substrate specificity and Kinetic parameters
The purified xylanase was assayed using different commercial substrates and polysaccharides as carbon source. CSB40 exhibited high activity in the presence of beechwood xylan compared to other. For kinetic parameters, four different concentrations of beechwood xylan [0.125%-1%(w/v)] and with constant enzyme concentrationwere prepared. Assays were performed under standard assay conditions at optimal conditions. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) were determined from a Lineweaver- Burk plot and Eadie-Hofstee Plot.
2.10 Thermodynamics characterization of xylan hydrolysis
The thermodynamic parameters for beechwood xylan hydrolysis were calculated by Eyring’s absolute rate equation derived from the transition state theory :
where kb is the Boltzmann’s constant (R/N) = 1.38 × 10-23 J K-1, T is the absolute temperature (K), h the Planck’s constant = 6.626 ×10-34 Js, N is the Avogadro’s number = 6.02 ×1023 mol-1, R is the gas constant = 8.314 J K-1 mol-1.
entropy of activation (4)
The free energy of substrate binding (and free energy of transition state formation () was determined using the following equations:
The effect of temperature on the rate of reaction was demonstrated in terms of temperature 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 of Dixon and Webb :
2.10. Enzymatic hydrolysis
Enzymatic hydrolysis of xylanase CSB40 was performed via Thin Layer Chromatography (TLC). Purified xylanase from CSB40 (0.35 mg/mL) was incubated with Beechwood xylan (0.2%), phosphate buffer (pH 6) at 37 °C. After boiling for 10 minutes and samples were then spotted on the silica gel plates (stationary phase) 60F 254 (E. Merck, Germany). The plates were developed with mobile phase containing chloroform—acetic acid—water (6:8:2, v/v/v) followed by spraying the plates with a methanol—sulfuric acid mixture (95:5, v/v) and heated for few minutes at 160 °C. A mixture of xylose, xylobiose, xylotriose and xylotetrose(10 mg/ml) were used as the standard.
3. Results and discussion
3.1. Enzyme production and purification
Xylanase production was highest in 48 hours utilizing beechwood xylan as substrate which is very short time compared to that of xylanase from other Bacillus and steptromyces species [8-14].
A summary of xylanase purification is illustrated in Table 1. Xylanase was purified to 8.12 fold with a recovery yield of 33.71%. CSB40 was purified by ammonium sulphate precipitation method followed by a single-step DEAE Sepharose Fast Flow column chromatography. Active fractions of peaks were pooled, concentrated, and analyzed for purity. The active fraction were found pure and appeared as single band as analyzed by SDS-PAGE.
3.2. Polyacrylamide Gel electrophoresis
Fig. 1 a and l b show the SDS-PAGE and zymography of the purified enzyme were performed to analyze the enzyme purity and estimate the molecular mass of the enzyme, respectively. The enzyme settled as a single band with a molecular mass of approximately 27 kDa confirming its homogeneity in electrophoresis. The xylanase showed a clear band on zymogram gel, detected by congo red staining, indicating that it was active.
3.3. Effect of temperature and pH on the activity and stability of the purified xylanase
pH and temperature had greater influence in the xylanase activity and stability. The effect of pH and temperature on the activity and stability of CSB40 are shown on Fig 2 and 3. Xylanase production was maximum with potassium phosphate buffer of pH 6 and was stable over pH 4.5-12.5 which is the wider stability range so far reported to our best knowledge. pH optimum is comparable with many other Bacillus xylanase B. licheniformis , B.stearothermophilus , and B. thermoalkalophilus ,. The effect of pH in activity is due to the reason that substrate binding and catalysis are dependent on charge distribution of substrate and enzyme. Catalytic reactions carried out from 30-80°C, determined the optimum temperature for the maximum enzymayic activity was found to be 50°C. The enzyme was fully stable after incubation at 50°C for 1 hr. Its tolerability to broad range of pH values, considerable thermostability and high optimum temperature makes CSB40 suitable in different arena of biotechnology such as xylan hydrolysis, biobleaching, bioethanol production etc.
3.4. Effects of metal ions and chemicals
The influences of metal ions on the activities of xylanase CSB40 were studied and illustrated in Table 2. Usually enzyme requires some activators for expressing their catalytic power. Cofactor mediated activation is usually for industrial benefits to increase catalytic efficiency. A siginificant decrese in activities was observed in presence of Cu++ which is similar to the xylanase from Bacillus cereus BSA1 [M], Bacillus halodurans S7 [K], Bacillus amyloquefaciens [I] and Bacillus sp. GRE7 [N]. Cu ions have the ability to catalyze auto-oxidation of cysteines to form intra molecular disulfide bridges or the formation of sulphenic acid may be the reason behind it [W]. Nevertheless the enzyme activity was partially inhibited by Co, Fe, Mn, K and Ba, while it was strongly stimulated by Zn. The enzyme activity remains unaffected in presence of metal ions like Mg, comparable to the xylanase from Bacillus halodurans S7 [K]. Acetone, methanol, ethanol and other chemicals produced discrete inhibition in the activity of enzyme (Table 3). This result precludes the use of this enzyme in industrial processes that present these metal ions and chemicals in relevant concentrations.
3.5. Effects of detergents and modulators
The activation or inhibition of xylanse by detergent and modulators is also important for the study of the structure of the active site and mechanism of action. Non-ionic detergent like Triton-X 100 had been found to have stimulatory effect which is comparable to the xylanase from Steptromyces althioticus LMZM . On the other hand, the enzyme activity was greatly suppressed by SDS (10%), and similar results were seen in xylanase from Bacillus halodurans TSEV1 [T]. Xylanase activity was remarkably stimulated in the presence of β-mercaptoethhanol which suggests the presence of a reduced thiol group of cysteine in the enzyme. In contrast, xylanase activity was highly affected by chelating agents like EDTA and EGTA, suggesting its metallo type and is comparable to the xylanase from Bacillus coagulans BL69 [U].
2.8. Enzyme production using agro-industrial waste
A remarkable production of xylanase was observed by the utilization of agricultural and industrial waste like corncob, wheat bran and malt sprout. The production was highest in hours with U/mL which is highly encouraging because commercially available xylan can be altered as substrate. These findings also recommend that CSB40 is a compelling xylanase producer by utilizing agro-industrial wastes as a substrate and is a prime candidate in various bio-industries.
3.6. N-terminal amino acids
The amino acid sequence of the last 15 N-terminal amino acids of MnSt was found to be GWSVDAPYIAXQPFS. This sequence was compared with available sequences in the National Centre for Biotechnology Information (NCBI) protein database using BLAST (basic local alignment search tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi). The BLAST search did not suggest homology with other mannanase sequences, but showed a high degree of homology with sequences of the 13- mannanase from Streptomyces sp. Tu6071.
3.7. Substrate specificity and Kinetic parameters
Purified CSB40 enzyme was analysed using different substrates that includes beechwood xylan, birchwood xylan and other carbon sources such as agarose, glucose, sucrose, fructose, mannose, potato starch etc. CSB40 showed its highest activity with beechwood xylan (100%). Birchwood xylan has nearly 50% of relative activity whereas other artificial substrates which were used as carbon sources did not showed any activity. These results signify that CSB40 is cellulose-free xylanase which provide a strong recommendation as a biobleaching agent for paper and pulp industries.
The kinetic parameters (Km) and (Vmax)of CSB40 was assessed by using a Lineweaver-Burk Plot to be 0.080 ± 0.00398 mg/mL and 794.63 ± 7.55 mmol/min mg using different concentrations of beechwood xylan (1-0.125%). On the other hand, kinetic parameters (Km) and (Vmax) were found to be 0.0819±0.0029 mg/mL and 797.03±5.22 mmol/min mg respectively on the basis of Eadie-Hofstee Plot. Km signifies the maximum catalytic efficiency at low substrate concentration where Vmax represent theturnover numberof xylanase, which isthe number of substrate molecules hydrolysed by xylanase molecule in a unit time when the xylanase is fully saturated with xylan. The Km value was lower and Vmax value was higher than other xylanase from B. cereus BSA1 [M], Bacillus sp. GRE7 [N], B. polymyxa CECT153, B. stearothermophilus T6 [J], Bacillus licheniformis A99 [R], Geobacillus thermodenitrificans TSAA1[S]. These findings suggest that CSB40 is more efficient and its affinity for the substrate is exceptional compared to that of other Bacillus organisms.
3.8. Enzymatic hydrolysis and oligosaccharide production
The time course enzymatic hydrolyzed products of beechwood xylan were analyzed by TLC shown in Fig. 3. The enzyme degraded xylan at random, and the end products released were xylobiose(X2), xylotriose (X3) and xylotetrose (X4) without a significant accumulation of xylose where xylobiose is the main oligosaccharide. The hydrolysis property of present xylanase is similar to the xylanase from Bacillus sp. Strain 41M-1 [V], New Species of Bacillus [F]. However, xylose was observed in the hydrolysis of xylan by the xylanase from Bacillus sp. strain SPS-0 [L] Thus, CSB40 was suggested to be an endoxylanase that randomly cleaves xylan as a substrate. It is also reported that during xylooligosaccharide production, high xylobiose and low xylose are desirable because for the industrial purpose, it is found that hydrolysate containing more than 12 % of xylose is of low efficacy [Z]. Because of the fact that they positively modulate the intestinal microflora and suppress the growth of pathogenic microbiota, xylobiose and xylotriose can be directly used in food [Y].