Highlights

Highlights

acidophilic xylanase from Bacillus aerophilus KGJ2 medium and process variable were optimized by Response Surface Methodology biochemical characterization of xylanase enzyme xylooigosaccharide production

1

Xylanase production from Bacillus aerophilus KGJ2 and its application 1

in xylooligosaccharides preparation 2

D. Gowdhaman, V S. Manaswini, V. Jayanthi, M. Dhanasri, G. Jeyalakshmi, V. Gunasekar, KR. Sugumaran, V. 3

Ponnusami* 4

School of Chemical & Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, India. 5

ABSTRACT 6

Xylanolytic enzyme was produced using a newly isolated Bacillus aerophilus KGJ2 and 7

low cost lignocellulosic sources in solid state fermentation. Seven different agricultural 8

residues (wheat bran, tea dust, saw dust, paper waste, cassava bagasse, rice straw and rice 9

husk) and six nitrogen source namely yeast extract, beef extract, peptone, ammonium 10

nitrate, ammonium sulphate, and ammonium chloride were examined for xylanase 11

production. Upon initial screening, wheat bran and ammonium chloride were chosen as 12

suitable carbon source and nitrogen source respectively. Plackett–Burman fractional 13

factorial design was employed to screen the important process variables affecting enzyme 14

production. Substrate concentration, nitrogen source, moisture content and MgSO4.7H2O 15

were identified as statistically significant variables. Subsequently Box–Behnken method 16

was used to optimize the process conditions to achieve maximum xylanase yield. Under 17

optimized conditions xylanase yield was 45.9 U/gds. Best xylanase activity was obtained 18

at 70 C and pH 4.0. It retained more than 90% activity after incubation at 80 – 90 C for 19

60 min. The hydrolytic efficiency of xylanase on xylan was examined and xylobiose, 20

xylotriose and xylotetrose were obtained as hydrolytic products. 21

Keywords: Lignocellulosic waste; solid state fermentation; xylooligosaccharides22

* Corresponding author. Tel.: +91 4362 264101; fax: +91 4362 264120. E-mail addresses: [email protected], [email protected] (V. Ponnusami).

2

1. INTRODUCTION 23

Xylooligosaccharides (XOs) are sugar oligomers generated through hydrolysis of 24

xylan. They find applications in a variety of industries including pharmaceutical, 25

agriculture and feed industry. If orally consumed, XOs have prebiotic effects [1]. 26

Xylooligosaccharides can be used to decrease cholesterol, sustain the gastrointestinal 27

health, and enhance the biological availability of calcium etc [2]. Xylooligosaccharides 28

enhance the nutritional and sensory characteristics of food as they are modestly sweet, 29

stable over a wide pH and temperature range and inhibit the starch retrogradation [3]. 30

Enzymatic hydrolysis of xylan proved to be an efficient method in production of 31

XOs as the enzymes are highly selective and do not produce by products. Though, 32

enzymes of fungi, actinomycetes and bacterial origins are available, bacterial xylanases 33

are preferred over other xylanases as they possesses better temperature and pH tolerance 34

[4][5]. 35

Recently solid state fermentation is widely adopted to reduce the cost of 36

production of various biological molecules [6]. In solid state fermentation, various 37

renewable xylan sources can be used for the production of xylanase as a substitute for 38

hardwood xylan. Agricultural residues like wheat bran, paper waste, rice straw, cassava 39

bagasse, tea dust, saw dust and rice husk have been investigated as possible low cost 40

substrates. These are generated in large quantities through industrial processes [7]. Proper 41

utilization of the agricultural substrates can be achieved through solid state fermentation 42

(SSF) technique. The advantages of SSF in enzyme production are: (i) easy recovery of 43

products, (ii) simple setup, (iii) high volumetric productivity, (iv) low energy demand and 44

(v) direct utilization of low cost substrate [8] [9]. 45

3

Thus in the present work, XO producing acidophilic xylanase was produced in 46

solid state fermentation using a newly isolated Bacillus aerophilus strain KGJ2. Wheat 47

bran was used as low cost carbon source in solid state fermentation. Statistical 48

experimental design is widely adopted in bioprocess engineering for identification of 49

influential process variables and for optimisation of process variables with comparatively 50

less number of variables than in conventional experimental design [10–14]. In the present 51

work, this was performed in two stages. First, process variables influencing production of 52

xylanase from wheat bran were first screened by Plackett–Burman fractional factorial 53

design of experiments. After identifying the important variables optimisation was carried 54

out using Box–Behnken method in the second stage. 55

Properties of xylanase were also evaluated and their potential for XOs production 56

was examined. This is the first report on production of xylanase from the new isolate B. 57

aerophilus KGJ2 and its application for XO production. 58

2. Materials and Methods 59

2.1 Microorganism 60

The strain isolated from paper mill effluent collected from a local pulp and paper 61

industry, identified as Bacillus aerophilus KGJ2 (NCBI Gene Bank Accession No. 62

JX027507), was used in this work. The organism was preserved on xylan agar plates that 63

contain (in g/L): birch wood xylan, 5.0 (Sigma Aldrich MA, USA); peptone, 5.0; NaCl, 64

1.0; K2HPO4, 2.0; CaCl2.2H2O, 0.1; MgSO4.7H2O, 0.1; yeast extract, 1.0; bacteriological 65

agar, 15.0. Plates were maintained at 4°C. 66

2.2 Production and extraction of xylanase in solid state fermentation 67

4

Xylanase production was carried out in solid state fermentation in 250 ml 68

Erlenmeyer flasks using 10 g of finely powdered agro residue supplemented with basal 69

salt solution. The composition of the basal salt solution was (g/L): NaCl, 1.0; KH2PO4, 70

2.0; NH4Cl, 2.0; MgSO4.7H2O, 0.5; FeSO4.7H2O, 0.25; MnSO4, 0.25 and CaCl2.2H2O, 71

0.1. This was added to the substrate, with a moisture ratio of 1:1. The flasks were kept for 72

sterilization at 121 C for 15 min. Ten percent (w/v) (1.52 x 107 cfu/ml) of 18 h seed 73

culture was added to each flask and was incubated at 30 C for 24 h. Samples were taken 74

from the flasks periodically and the enzyme was extracted using 0.1 M phosphate buffer, 75

pH 7.0 (1:30 w/v) by mixing the content using an orbital shaker at 150 rpm for 30 min 76

and then filtered through cheese cloth. The filtrate was subsequently centrifuged at 10000 77

g for 20 min at 4 C. The supernatant, crude enzyme, was used for further studies [15]. 78

2.3 Enzyme assay 79

Xylanase activity was measured DNS method [16]. 1 ml of 1% birch wood xylan 80

solution was added with 0.5 ml enzyme solution in a test tube and incubated at 70 °C for 81

5 min in water bath. After 5 min 1.5mL DNS reagent was added and the mixture was 82

incubated for another 10 min in boiling water bath [16]. The blank (1.5 ml 20 mM 83

sodium citrate buffer with 1.5 ml of DNS) and control (0.5 ml of enzyme,1 ml of 20 mM 84

sodium citrate buffer and 1.5 ml of DNS) were also run along with sample. The 85

absorbance was measured at 540 nm. One unit of xylanase activity was defined as the 86

amount of enzyme that liberates 1 mol of reducing sugars equivalent to xylose per 87

minute under the assay conditions described [1]. Total soluble protein concentration was 88

5

determined using bovine serum albumin (BSA) as a standard according to Lowry et al 89

[17]. 90

2.4 Screening of carbon source 91

Seven different agricultural residues (wheat bran, tea dust, saw dust, paper waste, 92

cassava bagasse, rice straw and rice husk) were first chosen as possible low cost carbon 93

source. To this, basal salt solution was added in 1:1 ratio and the mixture was autoclaved. 94

Then 10% (w/v) inoculum was added to the flask and incubated at 30 C. Crude enzyme 95

samples were taken at regular time interval and the xylanase activities were analysed. 96

Effect of carbon sources was statistically examined using Tukey test and the one that 97

showed maximum enzyme activity was chosen as best carbon source and the same was 98

used in further studies. 99

2.5 Screening of nitrogen source 100

The nitrogen sources examined include yeast extract, beef extract, peptone, 101

ammonium sulphate, ammonium nitrate, and ammonium chloride. The basal salt solution 102

was mixed with 0.1% of the above nitrogen source and used for evaluation of xylanase 103

activity. A control devoid of nitrogen source was used for comparison. Once again, using 104

Tukey test results the best nitrogen source was identified. 105

2.6 Plackett Burman design 106

The carbon and nitrogen source screened in previous steps were added to the 107

production medium. Nine variables namely substrate concentration, moisture ratio, 108

inoculum size, concentration of nitrogen source, pH, fermentation time, MgSO4.7H2O, 109

6

CaCl2.2H2O, and NaCl were considered as influential variables and included in the 110

screening experiment. Nine factors were investigated in 14 experimental runs which 111

included 3 replicates at central point as shown in Table 1. The first order model given in 112

eq. (1) was used here. 113

Y= βo + ∑βi xi (1) 114

where, 115

Y - response variable (xylanase yield in U/gds) 116

xi - level of independent variable 117

βo, βi - model intercept and linear coefficients respectively. 118

2.7 Box–Behnken design 119

Box-Behnken design of experiment was performed, with the important variables 120

screened in previous step, to optimize the conditions favourable for xylanase production. 121

Response surface method with the four factors, each varied at three levels was employed 122

to optimize the response variable. The regression analysis was performed using Minitab 123

15 and experimental data were fitted to the following quadratic equation: 124

n

i

n

jjiij

n

iiii

n

iii xxaxaxaaY

1 111

2

10 (2) 125

Where, 126

ao - model constant term 127

ai, aii, aij - regression coefficients of linear, square and interation terms [17]. 128

7

2.8 Partial purification of enzyme 129

Crude enzyme produced under optimized conditions was partially purified by 130

ammonium sulphate precipitation followed by dialysis [9]. The molecular weight of the 131

protein was determined using SDS–PAGE electrophoresis. After electrophoresis, the gel 132

was stained with coomassie brilliant blue R-250. A low molecular weight marker protein 133

purchased from Bangalore Genei Pvt. Ltd. was used as the molecular weight standard. 134

Zymogram analysis was done using native PAGE (7%) with 1% xylan mixed with 135

separating gel. After electrophoresis the gel was stained using 2% congo red for 20 min 136

and further washed with 1 M NaCl solution for 10 to 20 min to visualize the band [1]. 137

2.9 Effect of temperature and pH on xylanase activity and stability 138

Effect of temperature on xylanase activity was investigated by determining 139

enzyme assay at different temperatures (30 C – 90 C) optimum pH 4 by using 20 mM 140

sodium citrate buffer. 141

Thermostable nature of the xylanase was investigated by pre-incubation of the 142

enzyme in 20 mM sodium citrate buffer (pH 4) at different temperatures (30 C – 90 C) 143

by varying time period, and then residual activity was determined as described in section 144

2.3. Residual activity of enzyme before incubation was taken 100%. 145

The effect of initial pH on xylanase activity was studied using various buffers, 146

such as sodium citrate (pH 2.0–6.0), sodium phosphate (pH 7.0), Tris hydrochloride (pH 147

8.0) and glycine – sodium hydroxide (pH 9.0–10.0) each at 20 mM concentration at 70 148

°C. 149

8

To determine the xylanase stability at different pH values, the xylanase was pre-150

incubated at different pH values (4.0 - 10.0) using appropriate buffers for 12 h. At regular 151

time interval, the residual activity of xylanase enzyme was determined as described in 152

section 2.3. Residual activity of enzyme before incubation was taken 100%. 153

2.10. Production of xylooligosaccharides (XOs). 154

To investigate efficiency of the xylanase enzyme to produce XOs hydrolysis of 155

xylan was performed. 10 ml of crude xylanase (0.29 mg/ml) was added to 90 ml of 1% 156

birch wood xylan in 100 mM sodium citrate buffer (pH 4.0) and incubated at 70 C. 157

Samples were withdrawn periodically for every 15 min up to 60 min and were examined 158

for reducing sugar [1]. Products of hydrolysis in the course of time were qualitatively 159

analyzed by thin layer chromatography (TLC) as described elsewhere [1]. 160

3. RESULTS AND DISCUSSION 161

3.1 Screening of carbon source on enzyme production 162

Carbon source is very important media constituent for the growth of the 163

microorganisms, and also it alter overall cellular and metabolic process. Different agro 164

residues were tried as alternate carbon source for xylanase production by Bacillus 165

aerophilus KGJ2. The comparison of different carbon sources is shown in Fig. 1 and 166

Tukey’s test has confirmed that the difference in xylanase yield was statistically 167

significant at 99% confidence level (p < 0.01). Wheat bran supported highest xylanase 168

production (18.6 U/gds) followed by rice straw (16.3 U/gds) and rice husk (12.2 U/gds). 169

Preferred position for Figure 1 170

9

Chapla et al. [18] had earlier reported that wheat bran contained necessary ingredients 171

required to support xylanase production. Large surface area provided by the loose 172

particles used in SSF under moist conditions favoured xylanase production. Previous 173

literature supports the suitability of wheat bran for xylanase production in solid state 174

fermentation. Wheat bran has been reported to serve as reasonably good substrate for 175

xylanase production by different Bacillus sp. [18–22] and even proved to be a more 176

effective carbon source than xylan for xylanase production by Bacillus sp. SPS- 0 [22]. 177

Other bacterial species viz. Pseudomonas and Streptomyces have also been reported to 178

utilize wheat bran efficiently and produce high xylanase titer [23]. 179

3.2 Effect of nitrogen sources on enzyme production 180

The nitrogen source significantly influences the pH of the medium during 181

fermentation and in turn enzyme production. Different nitrogen sources were tested, with 182

wheat bran as the substrate, and the results were compared using Tukey’s test. The results 183

confirmed that difference in xylanase yield was significant at 99% confidence level (P < 184

0.01). It was observed that ammonium chloride gave the best result of 18.93 U/gds 185

xylanase activity (Fig. 2). 186


3.3 Plackett-Burman Design 188

Plackett-Burman Design was used to screen essential variables for xylanase yield 189

from wheat bran in solid state fermentation. Xylanase yields (U/gds) were determined 190

under various experimental conditions as shown in Table 1. Regression results including 191

estimated coefficients, t – values and p – values are listed in Table 2. From the table it 192

10

can be seen that p-value for the variables such as substrate wheat bran (WB), Nitrogen 193

source (AmC), Moisture content (M), and MgSO4.7H2O (MS) were lesser than 0.05 and 194

these variables were thereafter considered for further optimisation. On the other hand, p-195

values for pH, Fermentation time (t), Inoculum size (In), CaCl2 (CC), and NaCl (SC) 196

were found to be greater than 0.05. These factors were, therefore, considered to be not 197

significant and not included in optimisation studies. 198

3.4 Box-Behnken design 199

The four variables chosen from the previous screening step, namely, WB, AmC, 200

M, and MS were further optimized by Box-Behnken statistical design. The experimental 201

design and corresponding xylanase yield (U/gds) are shown in Table 3. Table 4, shows 202

that the p - value for interaction of WB with MS was 0.568. Therefore, corresponding 203

regression coefficient of this interaction term was removed from the regression model 204

and the regression was performed again. The regression coefficients for reduced model 205

along with their statistical parameters are listed in Table 5. As illustrated in the table, 206

from the p-values of the terms, it was evident that all the terms incorporated in the 207

reduced model had statistically significant effect on the response variable. 208

High R2 and R2adj values (99.72% and 99.44% respectively), suggested that the 209

model fitted the experimental data very well. To ensure the adequacy of the model, 210

ANOVA was performed and results are presented in Table 6. The results confirmed that 211

all linear, quadratic and interaction terms in the model were statistically significant with 212

corresponding low p-values (< 0.01). Meanwhile, it could be seen from the ANOVA 213

table that the p- value for the lack of fit was 0.185. It only illustrate the fact the model 214

11

adequately explained the variation in xylanase yield as a function of medium variables. 215

Nevertheless, to further test the adequacy of the model, residual analysis was carried out 216

using normal plot shown in Fig. 3 (a). It was observed that the points corresponding to 217

run numbers 1 and 19 could be possible outliers in the experiments. 218


Therefore, regression was repeated after removing these points. Model coefficients were 220

once again determined after eliminating the possible outliers. Table 5 and 6 list the 221

regression coefficients and ANOVA respectively, for the reduced model without outlier. 222

Normal probability plot, after elimination of residual, shown in Fig. 3 (b) was also 223

examined and it was found that the standardized residuals for all the points were normally 224

distributed and fall in the range of + 2 to – 2. Moreover, p – value for the lack of fit was 225

found to be 0.519 which was approximately three times greater than that for the reduced 226

model with outlier. These observations confirmed that the run numbers 1 and 19 were 227

real outliers. Substituting the regression coefficients shown in Table 5 in eq. (2), the 228

following regression model was obtained in terms of coded units: 229

y = 43.8 + 6.76 WB + 2.15AmC + 0.59MS – 3.20 M – 7.25WB2 – 1.35AmC2 – 5.00MS2 230

– 3.77M2 – 4.54WB×AmC – 1.78WB×M + 4.55AmC×MS + 0.73AmC×M + 4.30MS×M231

… (3) 232

R2 of the model was determined to be 99.9%. The regression model was considered to be 233

a good correlation [24]. 234


12

The contour plot (Fig. 4) elucidates the interactions among the variables. The 236

shape of the curves (non-linear, elliptical) clearly demonstrated the interaction between 237

the variables. Maximum yield of xylanase with corresponding medium variables of 238

substrate WB, AmC, MS, and M was obtained by solving the second order polynomial 239

model given by eq. (3). The model was analytically solved using inverse matrix method. 240

Optimum medium variables were estimated to be: substrate (WB) – 6.0 g, AmC – 3.2 241

g/L, MS – 1 g/L) and M – 54.2%. Corresponding xylanase yield was found to be 45.9 242

U/gds. The predicted xylanase yield was confirmed and validated by performing an 243

experiment with statistically optimised variables in the production medium, and the 244

recovery of xylanase was 43.4 U/g ds, which was close to the predicted one. 245

3.5 SDS-PAGE analysis 246

The partially purified xylanase enzymes showed a single denatured protein band on SDS-247

PAGE. A 12% separating gel with standard molecular weight marker protein shows 248

approximately around 29 kDa (Fig. 5(a)). Zymogram analysis is shown in Fig. 5(b) along 249

with native PAGE of the protein. The figure clearly demonstrate the presence of 250

xylanase. 251


3.6 Effect of temperature and pH on enzyme activity and stability 253

It is essential to study the effect of pH and temperature on enzyme activity as 254

these two variables profoundly influence the enzyme activity. Similarly stability of the 255

enzymes in wide pH and temperature range determines the choice and suitability of the 256

enzymes for various industrial applications. Effect of solution pH and temperature on 257

13

enzyme activity and stability of the crude xylanase are shown in Fig. 6 and 7. The 258

enzyme activity was observed in the pH range 2.0 to 10 and the maximal relative activity 259

was found at pH 4. The enzyme retained more than 70-80 % activity for the first 2 h from 260

pH 2 to 10. The enzyme shows stability at pH 4 (74.56%) for first 4 h and 56.47% after 6 261

h. The stability of the enzyme slowly declined to 33%, 22% and 12% after 8, 10, 12 h of 262

incubation respectively. B. licheniformis P11 (C) xylanase exhibited 77 to 78% stability 263

over a wide pH range of 5 – 6 when pre-incubated at 60 C for 1 h [25]. Most of the 264

bacterial xylanase showed optimum pH in the acidic (or) slightly alkaline range [26]. 265

Bacillus pumilus SV-85S showed activity and stability over broad range of pH 5-11 [27]. 266



Activity of partially purified xylanase was maximal at about 70 C. In order to 269

check the thermo stability, the residual enzyme activity was measured after incubating 270

enzyme at 80 and 90 C. More than 90% activity was retained after incubation at 80 – 90 271

C for 30 min. The thermal stability of the enzyme was comparable with those of 272

previously reported xylanases. Xylanase with similar temperature optima had been 273

reported from Bacillus licheniformis in the broad range of 40 C to 100 C [25]. In 274

Bacillus sp., 11-1S, the temperature optimum was 80 C [28]. Similarly, Bacillus 275

steareothermophilus showed highest reaction rate at 75 C [29]. Previous reports on 276

xylanase showed wide range of optimum temperature for various xylanase. Bajaj and 277

Singh (2010) [26] reported that most of the bacterial xylanases showed optimum activity 278

at 50-60 C. B. halodurans xylanase exhibited optimum activity at 80 C [30]. But, B. 279

14

halodurans PPKS-2 xylanase was active at a temperature 70 C. Previous literature also 280

suggested the thermo stability of xylanase from Bacillus sp. varied between 55 and 80 C 281

[31]. A comparison of acido-thermal stability of xylanase from various other Bacillus sp. 282

with that of xylanase from Bacillus aerophilus KGJ2 is shown in Table 7 [5], [28], [32–283

36]. Use of enzymes with thermostability, in industrial processes has been expected to 284

remarkably reduce the need for pH and temperature adjustments before the enzyme were 285

added [37] [38]. 286

3.7 Hydrolysis of xylan and production of XOs 287

The xylanase from Bacillus aerophilus KGJ2 was examined for its potential to 288

produce XOs from birch wood xylan (using 1% birch wood xylan at pH 4.0, and 289

temperature 70 C). Thin layer chromatography (TLC) of hydrolyzed products of xylan is 290

shown in Fig. 8. The hydrolysis products released by xylanase from birch wood xylan 291

consist of XOs ranging from xylose to xylotetrose (X1–X4) at different time intervals. 292


The present results suggested that xylanase cleaved the substrate, xylan, to liberate 294

mainly XOs suggesting that it is an endoxylanase. Similarly xylanase, obtained from 295

Thermoasus auranticus fungus, was employed to hydrolyze xylan through endo-acting 296

mechanicsm [39]. Bacillus stearothermophilus xylanase reacted with oat spelt xylan 297

liberated xylobiose and xylotriose [40]. Similarly, Bacillus sp. NO.C-125, xylanase gave 298

end products like xylobiose, xylotriose and xylotetrose, when it hydrolyzed xylan [41]. 299

Xylooligosaccharides produced by hydrolysis of xylan are industrially important sugar 300

oligomers. Applications of these XOs are numerous in both food and non-food 301

15

applications. The XOs are well known for their ability to inhibit intestinal pathogens. 302

There is an excellent market demand for XOs as ingredients for functional food. To cite 303

few examples XOs are used in soft drinks, yogurt, cakes, pastries etc [42]. The XOs are 304

used as a carbon source for probiotic microorganism like Bifidobacterium sp [43]. It 305

possesses anti-inflammatory activity [44], immunostimulating potential, anti-cancerous 306

activities against sarcoma and other tumors [45], antioxidant [46] anti-allergy, anti-307

infection and anti-inflammatory properties [47]. Application of agricultural waste as a 308

raw material for the production such a valuable industrial product is beneficial to the 309

society as it simultaneously helps to reduce solid waste on one hand and to produce value 310

added product on the other hand. 311

Conclusion 312

Bacterial xylanase was produced in solid state fermentation using new isolate 313

Bacillus aerophilus KGJ2 and wheat bran as carbon source. Influential process variables 314

were screened by Plackett – Burman fractional factorial design. Four variables namely 315

concentration of carbon source, nitrogen source, moisture content, and magnesium 316

sulphate were identified as statistically important variables among seven variables chosen 317

for the study. Further optimization of these variables was carried out using Box-Behnken 318

design of experiment. Optimum values were found to be: substrate (WB) – 6.0 g, AmC – 319

3.2 g/L, MS – 1 g/L) and M – 54.2%. Under optimum conditions xylanase activity was 320

45.9 U/gds. Optimum pH and temperature for xylanase activity were found to be 4 and 321

70 C respectively. The enzyme retained more than 90 % of its activity after incubation at 322

80 – 90 C for 60 min. The enzyme was examined for its potential to produce 323

xylooligosaccharide using brich wood xylan as carbon source. The results confirmed the 324

16

isolated strain can be used for the production of valuable xylanase enzyme with potential 325

application in xylooligosaccharide production. 326

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481

24

List of figures 482

Fig. 1. Effects of different carbon sources on xylanase production. [RS- Rice Straw; 483

PW- Paper Waste; RH- Rice Husk; CB-Cassava Baggasse; WB-Wheat Bran; TD- Tea 484

Dust; SD- Saw Dust] 485

Fig. 2. Effect of different nitrogen sources on xylanase production by Bacillus 486

aerophilus KGJ2. [A.N-Ammonium nitrate; B.E-Beef extract;A.S-Ammonium sulphate; 487

A.C-Ammonium chloride; P- Peptone; Y-Yeast extract] 488

Fig. 3. (a) Residual plot for reduced model (b) Residual plot for reduced model without 489

outlier 490

Fig. 4. Contour plots showing response and interactive effects between variables 491

Fig. 5 (a) SDS PAGE analysis of xylanases from Bacillus aerophilus. Lane 1: Molecular 492

marker, Lane 2: crude xylanase; Lane 3: Partially purified xylanase (ammonium sulphate 493

precipitation); Lane 4: Partially purified xylanase (after dialysis) (b) N - native PAGE of 494

the crude enzyme from Bacillus aerophilus; Z - Zymogram of the crude enzyme from 495

Bacillus aerophilus detected with 1% xylan. 496

Fig. 6. Effect of pH on activity and pH stability of Bacillus aerophilus KGJ2 xylanase. 497

The activity assay was conducted using following buffers: sodium citrate (pH 2–6), 498

sodium phosphate (pH 7), Tris-HCl (pH 8) and glycine – NaOH (pH 9–10). The pH 499

stability of xylanase was determined by incubating the enzyme in different pH buffers as 500

mentioned above for 12 h at 70 °C. The residual activity was also measured at 70 °C. 501

Fig. 7. (a) Effect of temperature on xylanase activity. (b) Thermostability of Bacillus 502

aerophilus KGJ2 xylanase. The enzyme was pre-incubated at different temperatures (30-503

90 °C) and residual activity was assayed after 1 h. 504

25

Fig. 8. Thin layer chromatography (TLC) of hydrolyzed products of xylan using xylanse 505

from Bacillus aerophilus KGJ2. (M is the standard mixture of sugars with X1- xylose, 506

X2-xylobiose, X3- xylotriose and X4- xylotetrose, S1, S2 and S3 are samples hydrolysed 507

at 60 min, 45 min and 30 min time course respectively.) 508

509

List of Tables 510

Table 1. Plackett - Burman factorial design for yield of xylanase in solid state 511

Fermentation. 512

Table 2. Statistical parameters from Plackett - Burman factorial design 513

Table 3. Experimental design for xylanase production in Box Benhen Design. 514

Table 4. Estimation of regression coefficients for full model 515

Table 5. Estimation of regression coefficients for reduced model with and without outlier 516

Table 6. ANOVA for reduced model with and without outlier 517

Table 7. Comparison of optimum pH, temperature and stability of xylanase produced by 518

various Bacillus species. 519

Fig. 1. Effects of different carbon sources on xylanase production. [RS – Rice Straw; PW – Paper Waste; RH – Rice Husk; CB – Cassava Baggasse; WB – Wheat Bran; TD – Tea Dust; SD – Saw Dust]

0

5

10

15

20

25

RS PW RH CB WB TD SD

Spec

ific

enzy

me

activ

ity (U

/g d

s)

Fig. 2. Effect of different nitrogen sources on xylanase production by Bacillus aerophilus KGJ2. [AN – Ammonium nitrate; BE – Beef extract; AS – Ammonium sulphate; AC – Ammonium chloride; P – Peptone; Y – Yeast extract]

0

5

10

15

20

25

AN BE AS AC P Y

Spec

ific

enzy

me

activ

ity (U

/g d

s)

(a)

(b)

Fig. 3. (a) Residual plot for reduced model (b) Residual plot for reduced model without outlier

Fig. 4. Contour plots showing response and interactive effects between variables

(a) (b)

Fig. 5 (a) SDS PAGE analysis of xylanases from Bacillus aerophilus. Lane 1: Molecular marker, Lane 2: crude xylanase; Lane 3: Partially purified xylanase (ammonium sulphate precipitation); Lane 4: Partially purified xylanase (after dialysis) (b) N - native PAGE of the crude enzyme from Bacillus aerophilus; Z - Zymogram of the crude enzyme from Bacillus aerophilus detected with 1% xylan.

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11pH

% R

esid

ual a

ctiv

ity

Effect of pH on activityStability (2 h)Stability (4 h)Stability (8 h)Stability (12 h)

Fig. 6. Effect of pH on activity and pH stability of Bacillus aerophilus KGJ2 xylanase. The activity assay was conducted using following buffers: sodium citrate (pH 2–6), sodium phosphate (pH 7), Tris-HCl (pH 8) and glycine – NaOH (pH 9–10). The pH stability of xylanase was determined by incubating the enzyme in different pH buffers as mentioned above for 12 h at 70 °C. The residual activity was also measured at 70 °C.

(a)

(b)

Fig. 7. (a) Effect of temperature on xylanase activity. (b) Thermostability of Bacillus aerophilus KGJ2 xylanase. The enzyme was pre-incubated at different temperatures (30-90 °C) and residual activity was assayed after 1 h.

Fig. 8. Thin layer chromatography (TLC) of hydrolyzed products of xylan using xylanse from Bacillus aerophilus KGJ2. (M is the standard mixture of sugars with X1- xylose, X2-xylobiose, X3- xylotriose and X4- xylotetrose, S1, S2 and S3 are samples hydrolysed at 60 min, 45 min and 30 min time course respectively.)

Table 1 Plackett - Burman factorial design for yield of xylanase in solid state Fermentation

Run order WB AmC pH t I M CC SC MS

Observed xylanase activity (U/gds)

Predicted xylanase activity (U/g ds)

1 10 0 8 1 3 30 0.4 2 1 20.4 23.3 2 10 4 3 5 3 30 0 2 1 13.8 10.9 3 2 4 8 1 11 30 0 0 1 23.8 23.4 4 10 0 8 5 3 70 0 0 0 24.1 23.7 5 10 4 3 5 11 30 0.4 0 0 13.1 15.9 6 10 4 8 1 11 70 0 2 0 15.8 16.1 7 2 4 8 5 3 70 0.4 0 1 33.8 34.1 8 2 0 8 5 11 30 0.4 2 0 34.9 32 9 2 0 3 5 11 70 0 2 1 33.6 36.4

10 10 0 3 1 11 70 0.4 0 1 35.6 32.7 11 2 4 3 1 3 70 0.4 2 0 17.9 17.5 12 2 0 3 1 3 30 0 0 0 13.9 14.2 13 6 2 5.5 3 7 50 0.2 1 0.5 40.6 41.7 14 6 2 5.5 3 7 50 0.2 1 0.5 41.9 41.7 15 6 2 5.5 3 7 50 0.2 1 0.5 42.7 41.7

Table 2. Statistical parameters from Plackett - Burman factorial design

Term Effect Coeff. SE

Coeff. t p Constant 23.392 1.045 22.38 0 WB -5.85 -2.925 1.045 -2.8 0.049 AmC -7.383 -3.692 1.045 -3.53 0.024 pH 4.15 2.075 1.045 1.99 0.118 t 4.317 2.158 1.045 2.07 0.108 In 5.483 2.742 1.045 2.62 0.059 M 6.817 3.408 1.045 3.26 0.031 CC 5.117 2.558 1.045 2.45 0.071 SC -1.317 -0.658 1.045 -0.63 0.563 MS 6.883 3.442 1.045 3.29 0.030 Ct Pt 18.342 2.337 7.85 0.001

Table 3. Experimental design for xylanase production in Box-Behnken Design.

Run WB AmC MC M

Observed xylanase activity (U/g ds)

Predicted xylanase activity (U/g ds)

1 2 0 0.5 50 23.4 22.5 2 10 0 0.5 50 44.2 44.2 3 2 2 0.5 50 35.4 34.8 4 10 2 0.5 50 39.7 40.0 5 6 1 0 30 42.0 41.8 6 6 1 1 30 34.7 34.2 7 6 1 0 70 26.9 26.9 8 6 1 1 70 36.8 36.4 9 2 1 0.5 30 27.5 27.5

10 10 1 0.5 30 44.8 44.4 11 2 1 0.5 70 24.5 24.6 12 10 1 0.5 70 34.7 34.5 13 6 0 0 50 39.5 39.5 14 6 2 0 50 34.5 34.4 15 6 0 1 50 31.5 31.4 16 6 2 1 50 44.7 44.5 17 2 1 0 50 24.0 24.3 18 10 1 0 50 37.7 37.5 19 2 1 1 50 24.1 25.0 20 10 1 1 50 38.5 38.8 21 6 0 0.5 30 40.2 40.7 22 6 2 0.5 30 43.0 43.3 23 6 0 0.5 70 32.6 32.9 24 6 2 0.5 70 38.3 38.4 25 6 1 0.5 50 43.5 43.8 26 6 1 0.5 50 43.9 43.8 27 6 1 0.5 50 44.0 43.8

Table 4 Statistical parameters for full regression modelsa

Term Coeff SE

Coeff t P Constant 43.86 0.314 139.51 0 WB 6.73 0.157 42.82 0 AmC 2.02 0.157 12.82 0 MS 0.48 0.157 3.06 0.01 M -3.19 0.157 -20.30 0 WB2 -7.24 0.236 -30.72 0 AmC2 -1.19 0.236 -5.05 0 MS2 -5.18 0.236 -21.96 0 M2 -3.80 0.236 -16.12 0 WB×AmC -4.13 0.272 -15.15 0 WB×MS 0.16 0.272 0.59 0.568 WB×M -1.76 0.272 -6.47 0 AmC×MS 4.54 0.272 16.66 0 AmC×M 0.74 0.272 2.72 0.019 MS×M 4.31 0.272 15.81 0 aR-Sq = 99.7%, R-Sq(adj) = 99.4%

Table 5 Statistical parameters for reduced regression models with and without outlier

With outliera Without outlierb

Term Coeff SE

Coeff t p Coeff SE

Coeff T p Constant 43.86 0.31 143.16 0.00 43.80 0.16 267.4 0.00 WB 6.73 0.15 43.94 0.00 6.76 0.10 69.1 0.00 AmC 2.02 0.15 13.16 0.00 2.15 0.09 23.8 0.00 MS 0.48 0.15 3.15 0.01 0.59 0.09 6.8 0.00 M -3.20 0.15 -20.83 0.00 -3.20 0.08 -38.9 0.00 WB2 -7.22 0.23 -31.53 0.00 -7.25 0.13 -54.2 0.00 AmC2 -1.16 0.23 -5.18 0.00 -1.35 0.13 -10.7 0.00 MS2 -5.17 0.23 -22.53 0.00 -5.00 0.13 -39.4 0.00 M2 -3.78 0.23 -16.54 0.00 -3.77 0.13 -30.1 0.00 WB×AmC -4.13 0.27 -15.55 0.00 -4.54 0.18 -25.0 0.00 WB×M -1.78 0.27 -6.64 0.00 -1.78 0.14 -12.4 0.00 AmC×MS 4.55 0.27 17.09 0.00 4.55 0.14 31.9 0.00 AmC×M 0.73 0.27 2.79 0.02 0.73 0.14 5.2 0.00 MS×M 4.30 0.27 16.23 0.00 4.30 0.14 30.3 0.00 aR-Sq = 99.7%, R-Sq (adj) = 99.5% bR-Sq = 99.9% , R-Sq(adj) = 99.8%

Table 6 ANOVA for reduced model with and without outlier With outlier Without outlier Source DF Seq SS Adj SS Adj MS F p DF Seq SS Adj SS Adj MS F p Regression 13 1313.2 1313.2 101.0 358.8 0 13 985.9 985.9 75.8 939.8 0 A 1 543.6 543.6 543.6 1930.9 0 1 297.2 385.2 385.2 4773.2 0 B 1 48.7 48.7 48.7 173.1 0 1 24.8 45.8 45.9 568.3 0 J 1 2.8 2.8 2.8 9.9 0.008 1 14.6 3.7 3.7 45.6 0 F 1 122.2 122.2 122.2 433.9 0 1 122.2 122.2 122.2 1514.1 0 A*A 1 180.9 279.8 279.8 993.8 0 1 124.3 237.3 237.3 2941.7 0 B*B 1 7.1 7.5 7.5 26.8 0 1 8.3 9.3 9.3 115.4 0 J*J 1 91.7 142.9 142.9 507.7 0 1 84.9 125.3 125.3 1552.4 0 F*F 1 77.0 77.0 77.0 273.6 0 1 88.2 73.0 73.0 904.8 0 A*B 1 68.1 68.1 68.1 241.7 0 1 50.3 50.3 50.3 623.6 0 A*F 1 12.4 12.4 12.4 44.1 0 1 12.4 12.4 12.4 153.9 0 B*J 1 82.3 82.3 82.3 292.2 0 1 82.3 82.3 82.3 1019.5 0 B*F 1 2.2 2.2 2.2 7.8 0.015 1 2.2 2.2 2.2 27.1 0 J*F 1 74.1 74.1 74.1 263.3 0 1 74.1 74.1 74.1 918.7 0 Residual error 13 3.7 3.7 0.3 11 0.9 0.9 0.9 Lack of fit 11 3.5 3.5 0.3 4.8 0.185 9 0.7 0.7 0.1 1.3 0.519 Pure error 2 0.1 0.1 0.1 2 0.1 0.1 0.1 Total 26 1316.9 24 986.8

Table 7. Comparison of optimum pH, temperature and stability of xylanase produced by various Bacillus species. Xylanase producing Bacillus sp.

Optimum pH

Optimum temperature

Temperature and pH stability

Xylanase yield

Reference

Bacillus sp. 1 1-1S 3.5-4.5 65 0C 2.5, 45-70 0C

ND [28]

Bacillus licheniformis A99 7.0 50 0C 16.3U/g DBB

[5]

Bacillus arseniciselenatis DSM 15340

8.0 50 0C 10.0, 30-40 0C

ND [9]

Bacillus licheniformis SVD1

6.0-7.0 55 0C 5.0-7.0, 55 0C

ND [36]

Bacillus mojavensis A21 7.0 – 9.0 60 0C 7.0-9.0, 30-60 0C

7.54 U/ml

[1]

Bacillus pumilus SV- 205 6.0 60 0C 6.0-11.0, 60 0C

7382.7± 1200 IU/ml

[27]

Bacillus sp TAR-1 5.0-9.5 50 0C 9.0, 75 0C 10 U/ml [32] B. pumilus ASH 8.0 37 0C, 7.0, 70 0C 5407

IU/ml

B. subtilis ASH

7.0 37 0C 37-42 0C, 7.0

8964 U/g ds

[35]

Bacillus sp.GRE7

7.0 70 0C 60-80 0C, 7.0

3950 IU/g

[33]

Bacillus stearothermophilus SDX

6.0 37 0C 6.0-12.5, 37-850C

3446U/g ds

[31]

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