A simple and efficient diffusion technique for assay of endo β-1,4-xylanase activity

Abstract

Endo-β-1, 4-xylanases is thought to be of great significance for several industries namely paper, pharmaceuticals, food, feed etc. in addition to better utilization of lignocellulosic biomass. The present investigation was aimed to develop an easy, simple and efficient assay technique for endo-β-1, 4-xylanases secreted by the aerobic fungi. Under the proposed protocol, 9 g/L xylan containing agar was prepared in 100 mM phosphate buffer at different pH (4.5, 5.5 and 6.5). The sterilized xylan agar was dispensed in 90 mm petri dishes. 100 µl of culture supernatant of 12 fungal isolates was added to the wells and left overnight at 31±1 0 C. The petri dishes were observed for zone of clearance by naked eye and diameter was measured.Congo red solution (1 g/L) was applied over the petri dishes as per the established protocol and thereafter plates were flooded with 1M Sodium chloride solution for the appearance of zone of clearance. The diameter for zone of clearance by the proposed method and the established protocol was almost identical and ranged from 21 to 42 mm at different pH depending upon the activity of endo-β-1, 4-xylanases. Change of pH towards alkaline side enabled similar or marginal decrease of diameter for the zone of clearance in most of the fungal isolates. The specific activities of these fungal isolates varied from 1.85 to 11.47 IU/mg protein.The present investigation revealed that the proposed simple diffusion technique gave similar results as compared to the established Congo red assay for endo-β-1, 4-xylanases. Moreover, the present technique avoided the cumbersome steps of staining by Congo red and de-staining by sodium chloride.

Screening of yeasts capable of producing cellulase-free xylanase

“…Similarly, Lopes et al (2011) evaluated 349 yeasts in Petri plates, and found that 9 (2.6 %) showed an enzymatic index above 2.5. The enzymatic index, a semi-quantitative parameter applied to evaluate the ability of yeast strains to produce enzymes in solid medium, has been used by several authors (Ribeiro et al, 2014;Adesina, 2013;Florencio, 2012;Nagar, 2012;Samanta et al, 2011;Tallapragada, 2011), who have considered it an efficient method for screening for microorganisms. The next selection step was performed according to the capacity of yeast strains to produce the xylanolytic enzyme in liquid medium with xylan.…”

Optimization of xylanase from Pseudomonas mohnii isolated from Simlipal Biosphere Reserve, Odisha, using response surface methodology

“…For this purpose, pure cultures of selected colonies were prepared through repeated streak plating, and a loopful of culture from the pure cultures was inoculated on nutrient broth xylan medium and broth cultures were incubated for 18 h. Ten microliters of each bacterial culture (intact bacterial cell in broth) was inoculated inside a 4-mm pore made on the xylan agar plates. Xylan agar plates containing bacterial inoculums were then incubated at 37°C for 72 h. Plates were stained by 0.1% Congo red solution and kept for 1 h followed by destaining using 1 M NaCl for 15 min for determination of xylan hydrolyzing zone surrounding the bacterial growth on xylan agar plates [25]. After destaining, xylan hydrolyzing halo zone (H) and colony diameter (C) were measured for the individual bacterial strain to calculate hydrolysis index (H:C).…”

Mangrove soil as a source for novel xylanase and amylase as determined by cultivation-dependent and cultivation-independent methods

“…The isolates and negative control (E. coli) were cultured in triplicate for 4 days under agitation at 28°C in 15 mL of liquid medium with xylan. To recover crude extracellular enzymes, 2 mL of each sample was centrifuged at 14,000g for 20 min at 4°C to collect the supernatant that contained the extracellular enzymes [31]. The enzymatic assay was performed according to Samanta et al [31] and was read at 540-nm length [32] using a standard curve with known xylose concentration.…”

“…To recover crude extracellular enzymes, 2 mL of each sample was centrifuged at 14,000g for 20 min at 4°C to collect the supernatant that contained the extracellular enzymes [31]. The enzymatic assay was performed according to Samanta et al [31] and was read at 540-nm length [32] using a standard curve with known xylose concentration. Enzymatic activity was expressed as a unit of enzymatic activity (U) and was defined as the enzyme amount required to release 1 μmol of reducing sugar per minute of reaction in 1 min (1 U/mL) [33].…”