Sample collection
20 samples were collected from diverse geographical locations for isolation of xylanase producing bacteria. Samples from soil were collected by a disinfected spatula from 10 cm below soil surface in disinfected plastic bags. Three sequential dilutions were prepared for each sample (10^-1, 10^-2, 10^-3) in normal sterile saline. Selective medium for xylanase (xylanase screening medium) was prepared. 100 µl of each dilution was spread on selective medium and the plates were incubated at optimal temperature 45 °C for 24-72 h. Plates were detected for colonies with different characteristics like size, shape and presence of spores and dissimilar colonies were sub cultured onto freshly prepared nutrient-rich agar medium.⁶ All the isolates were preserved as glycerol stocks at -20 °C for further use.

Isolation of xylanase-secreting bacteria
The xylanase producing bacterial isolates were spot inoculated onto xylanase screening medium (Birch wood xylan 2 g, Yeast extract 3 g, Ammonium sulphate, 0.2 g Sodium bicarbonate 20 g, Agar 15 g, pH 11, Distilled water 1 L) and incubated for 24-48 h. Plates were swamped with Congo red and 0.1 M NaCl, respectively. The bacterial colonies that have xylanase activities will show a region of substrate hydrolysis on solid medium. The bacterial isolates showing clear hydrolysis zones were chosen for further investigation.

Xylanase enzymatic assay
50 mL xylanase production broth was taken, and potential bacterial isolates were inoculated in it. Broth was incubated in a rotatory shaker (250 rpm) at 45 °C for 72 h. 3 mL cultures were withdrawn from flasks on regular intervals and centrifuged. Centrifugation was done at 10,000 rpm for 10 min. The supernatant free of cells thus obtained after centrifugation was selected as the source of xylanase enzyme. Xylanase assay was conducted by measuring the quantity of xylan as a substrate. The reaction mixture contained approximately 0.5 mL diluted xylan. The reaction was performed at 45 °C for a period of 10 min and ceased with the addition of 1 mL of DNSA (di nitro salicylic acid reagent) and followed by 0.4 mL of 33% sodium potassium tartrate. The xylanase released was determined spectrophotometrically at 540 nm. A standard curve was plotted according to Miller.⁷

pH and temperature optimization
The enzyme was incubated on different pH and temperatures to identify the optimum range at which enzyme activity is maximum. The enzyme’s optimal pH range was determined. It is done by incubating reaction mixture on varied pH. The temperature at which the enzyme performs best was also determined. This is done by incubating the reaction mixture at varied temperature. After incubation xylanase activity was assayed.

Identification of xylanase producing bacterial isolates
The bacterial isolates producing xylanase were observed for morphology of colony, microscopic examination (gram staining) and biochemical test were performed such as Catalase, Oxidase, Gelatin liquefaction and endospore staining to identify the gram reaction and physiology of isolates.

Optimisation of submerged fermentation parameters for xylanase production by statistical approaches
A two factorial Plackett-Burman Design was applied for optimizing the parameters for improved fermentation for production of xylanase by selected isolate. This design is used to recognize important parameters including physical and chemical that are necessary for enhanced enzyme production. This is accomplished by performing N+1 experiments in screening N numbers of variables.⁸ In these two minima (-) and two maximum (+) values were chosen for each variable (Table 1). The highest enzyme production is indicated by the peak area. Better results may be obtained if the variation between the two values (minimum and maximum) is large. The experimental design includes the evaluated experimental design. Eleven variables were analysed that includes three dummies (for evaluation of error), seven nutritional and one physical variable. Experimental error was computed using the following equation⁹:

V_eff = ∑(Ed)² / N

Where,
V_eff = the variance of the effect of level
E_d = the effect of level for the dummy variables
N = the number of dummy variables used in the experiments.

As per Plackett-Burman design, three critical variables were identified. Additional, optimization was carried out using Central Composite Design.¹⁰

Table (1):
Showing code values variable, maximum and minimum used in the Plakett-Burman design

Table (2):
Code values of variables in CCD

The statistical tool used for experimental design analysis was ‘Design Expert 6.0’ Stat-Ease, Inc. Minneapolis, USA. The effect substrate concentration, ammonium sulphate and pH on xylanase secretion was observed across five distinct concentrations (Table 2).

Twenty experimental runs were carried out and xylanase activity was measured. Experiments were done in triplicates. The data was analysed by multiple regression procedure and polynomial equation (second order)⁷.
The equation for analysis is as follows:

Y = β₀ + ∑β_ixi + ∑β_iixi² + ∑β_ij + xixj

Where,
y = predicted response
β₀ = offset term
β_i = linear effect
β_ii = squared effect
β_ij = interaction effect
xi = independent variables
xj = independent variables

Using design expert software, contour plots were generated.