Microbial strains and xylanase production
The newly isolated Bacillus licheniformis strain was routinely maintained in a nutrient agar medium at pH 7.0 and temperature 35 °C. The glycerol stock cultures and working stock cultures were revived regularly. The production medium had been optimized to maximize the yield of xylanase (Malhotra and Chapadgaonkar 2020). The optimized conditions used for the production process were as follows: (i) pH 6, (ii) culture temperature 35 °C, (iii) concentration of xylan 2% w/v, and (iv) concentration of wheat bran 2.5% w/v. The broth was centrifuged and the supernatant was used for further purification of xylanase.
Production of xylanase in a 5-L stirred-tank reactor
Bioage 5-L stirred-tank bioreactor was used for xylanase production with previously optimized process conditions. The optimized xylanase production medium was used as described before. The reactor was sterilized by autoclaving for 1 h. After sterilization, the reactor vessel was connected to the control unit and recirculated the cooling water bath and allowed to cool to the process temperature. It was inoculated with 10% v/v of actively growing culture of the strain of Bacillus licheniformis. The reactor was operated at the optimized process parameters viz. pH 6.0, temperature 35 °C, RPM 125, and dissolved oxygen concentration (DO) of 60%. The reactor was harvested at peak xylanase activity achieved after 48h of the culture. The broth was centrifuged at 10,000 RPM for 10 min to remove the cells and stored appropriately for further studies.
Assay of xylanase and protein concentration
Xylanase activity was determined by the 3,5-dinitrosalicylic acid (DNSA) method given by Miller 1959 [9]. In brief, the culture broth was first centrifuged at 10,000 rpm for 15 min at 10 °C to separate the cells. 1% w/v beechwood xylan (Himedia MB141-10G) prepared in 0.05M sodium citrate buffer was used as substrate. One milliliter of the substrate was incubated with 500μl of the enzyme (supernatant obtained from centrifuged culture broth) at 50 °C for 15 min. The reaction was terminated by the addition of 3 ml of DNSA, and the mixture was boiled for 10 min in a water bath. The absorbance was measured at 540 nm after cooling the mixture. Xylose was used as standard. One unit of xylanase activity (U) is defined as the amount of enzyme that liberates 1 μmol of reducing sugar-xylose per min under the standard assay conditions. Protein concentration was measured by lowry's method [10].
$$Xylanase\ activity\ U/ ml=\frac{\upmu moles\ of\ xylose\ liberated\times Dilution\ factor}{volume\ of\ enzyme\ used\times time\ of\ reaction}$$
Lyophilization
The culture supernatant was concentrated by lyophilization in a lyophilizer (Allied Frost) for 6 h. The lyophilized sample was resuspended in phosphate-buffered saline (PBS 0.5M), pH 7.2, and then used for further studies.
Gel filtration
Sephadex G-25 (0.2 × 5) column was used for size-exclusion or gel-permeation chromatography. The column was pre-equilibrated with PBS (pH 7.0), and lyophilized culture broth (200 μl) was loaded carefully on the column. The elution was performed using PBS as given before. Five hundred microliters of fractions were collected and subjected to spectrophotometric protein and xylanase determination.
Molecular mass determination by SDS PAGE and zymogram analysis
The molecular weight of xylanase was estimated using 12% polyacrylamide gels by SDS PAGE [11]. The gel was visualized by Coomassie Brilliant Blue R-250 staining. Broad range protein molecular weight marker Bangalore GeNei, India, was used as standard.
Zymogram analysis was carried out using 0.1% beechwood xylan (w/v) incorporated into the polyacrylamide gel. The native PAGE was run without using SDS in the gel as well as in the running buffer. After the run, the gel was washed 4–5 times with distilled water for 30 min at 4°C and incubated for 24 h at room temperature in PBS. After incubation, the gel was immersed in 0.1% Congo red solution. The stained gel was then washed with an excess of 1M NaCl. The position of xylanase could be obtained as a clear band in the gel. After destaining the gel, it was being treated with 0.5% v/v acetic acid to increase the contrast between the bands and background.
Thermo and pH optima
Temperature optima of the partially purified xylanase were determined by changing the incubation temperature while performing the enzyme assay (Fig. 4). Similarly, pH optima of the partially purified xylanase were obtained by incubating the partially purified enzyme in 0.05M buffer solutions in pH range 6.5–9.5 (citrate in the range of 4.5–6.5, phosphate in the range 7.5–8.5, and tris buffer in the 9.5 range) and determination of activity at that pH at 50 °C.
Study of kinetics of xylanase activity
Initial reaction rates using beechwood xylan as a substrate were determined at a concentration of 0.5–10 mg/ml in 50 mM phosphate buffer (pH 7.0) at 50 °C. The assay method has been detailed in the previous section. The kinetic constants, Km and Vmax were estimated using the linear regression method of the Lineweaver-Burk plot.