Original Research Paper

Enhanced production of cellulase-free, thermo-alkali-solvent-stablexylanase from Bacillus altitudinis DHN8, its characterization andapplication in sorghum straw saccharification

Dharmesh N. Adhyaru a, Nikhil S. Bhatt a,n, H.A. Modi b

a P.G. Department of Microbiology, Gujarat Vidyapeeth, Sadra-382 320, Gujarat, Indiab Department of Life Sciences, School of Sciences, Gujarat University, Ahmadabad, Gujarat, India

a r t i c l e i n f o

Article history:Received 16 September 2013Received in revised form7 October 2013Accepted 11 October 2013Available online 23 October 2013

Keywords:Bacillus altitudinis DHN8Cellulase-free xylanaseSubmerged fermentationPartial purificationChemical pretreatmentsSorghum straw saccharification

a b s t r a c t

A newly isolated Bacillus altitudinis DHN8 was assessed for xylanase production by utilizing sorghumstraw. The highest xylanase production was recorded at sorghum straw 3% w/v, inoculum size 1% v/v,inoculum age 18 h, incubation time 42 h, pH 7.0, temperature 35 1C and agitation speed 250 rpm.Moreover, xylose 0.5%, gelatine 0.5% and KNO3 0.3% (w/v) further enhanced the production. The detailedoptimization study resulted in a 3.74-fold increase in xylanase production as compared to that ofthe initial conditions. The partially purified xylanase showed �70% pH stability after 18 h at pH range of6–10. Thermostability study revealed more than 60% xylanase activity at temperature range 45–65 1Cafter 60 min. The presence of metal ions (10 mM CaCl2, MnCl2 and FeCl3) and solvents (10% v/visopropanol, methanol, ethanol and acetone) were increased xylanase activities remarkably. Duringsaccharification study, 3% alkaline hydrogen peroxide treatment was found to be beneficial for themaximum enzymatic hydrolysis of sorghum straw (34.94 mg/g reducing sugar) after 36 h. As such, thisxylanase could be considered as a cellulase-free, thermo-alkali-solvent stable biocatalyst being importanttool for many biotechnological industries.

To the best of our knowledge, this is the first report on the production of xylanase by this Bacillusspecies.

& 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Xylan is the principal hemicellulose which is the major compo-nent of plant cell wall. It is the second plentiful polysaccharide aftercellulose, in both hard woods and annual plants (Qie et al., 2010;Polizeli et al., 2005) and accounts for 20–35% of the total dry weightin tropical plant biomass. Endo-xylanases (EC 3.2.1.8) are an impor-tant group of industrial enzymes responsible for complete hydrolysisof xylan in to short xylooligosaccharides and xylose in synergismwith other accessory enzymes such as β-xylosidase, α-L-arabinofur-anosidase and α-glucuronidase (Chavez et al., 2006; Collins et al.,2005). Xylanases have been reported from bacteria, fungi, actino-mycetes and yeast (Qie et al., 2010; Sunna and Antranikian, 1997)but bacteria due to their high metabolic diversity are widely used forxylanase production. Several Bacillus sp. are known to secrete highlevels of extracellular xylanases which are either poor or free incellulase activity. In recent years, xylanases have attracted a great

deal of attention because of their biotechnological potential in thevarious industrial processes such as, in food industry in order toenhance the digestibility of animal feed, in textile industry and inthe paper and pulp industry for bleaching purpose resulting in adecrease of chlorine utilization and consequently lowering environ-mental impact (Chivero et al., 2001). Xylanases in synergism withseveral enzymes can be used for the generation of biological fuels,such as ethanol and xylitol from lignocellulosic biomass (Cormanaet al., 2005; Beg et al., 2001).

The major obstacle for wide range application of the xylanasesin industries is high cost of the production. Pure substrates beinghighly expensive and thus it cannot be afforded at the industrial levelbulk production of enzymes. Therefore, it is necessary to explorecheap substrates for cost-effective enzyme production. Agriculturalresidues represent one such cheap raw material for industrialproduction of enzymes. Furthermore, each bacterium has its ownspecial conditions for maximum enzyme production. Thus, the hyperproduction of industrial enzymes by optimizing various physiologicaland nutritional parameters is of prime importance because animproper optimization of these growth parameters leads to a lowerenzyme production. In addition of the above problems, majorityof the reported xylanases do not withstand the robust industrial

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1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bcab.2013.10.003

n Corresponding author. Tel.: þ91 98794 83847/079 23274274;fax: þ079 232 74 272.

E-mail addresses: bhattnikhil2114@gmail.com, bhattnikhil_brc@yahoo.in(N.S. Bhatt).

Biocatalysis and Agricultural Biotechnology 3 (2014) 182–190

process conditions of high alkaline pH and high temperatures(Sharma and Bajaj, 2005). Therefore, search for still better enzymeswhich comply well with the industrial processes is in progress(Kumar and Satyanarayana, 2011).

In view of the demand for novel xylanases with industriallyimportant properties, an attempt has been made to assess thepotentiality of newly isolated Bacillus species for the xylanaseproduction using low cost agricultural residue under submergedfermentation. The partially purified enzyme was characterizedfor few properties and further examined for its application inthe saccharification of sorghum straw.

2. Material and methods

2.1. Isolation, screening and identification of xylanase producingstrain

2.1.1. Primary screeningDifferent soil samples were collected from the active compost

pit (�10 cm depth) during mid of the April 2011. Each sample wasmixed thoroughly and 1 g of that was suspended in 50 ml steriledistilled water. Mixtures were allowed to settle down and then serialdilutions were prepared. From each dilution, 0.1 ml was spread onoat spelt xylan (1% w/v) agar plates and incubated at 37 1C for 48 h.Those colonies showed a clear zone of xylan hydrolysis were isolatedand retained for further secondary screening.

2.1.2. Secondary screeningThe isolates from the primary screening were cultured in basal

liquid media containing (g/l): Oat spelt xylan, 10.0; beef extract, 3.0;NaCl, 5.0; KNO3, 2.0; K2HPO4, 1.0 and MgSO4 �7H2O, 0.5 at pH 7.0 in100 ml Erlenmeyer flasks and incubated at 37 1C under shaking(150 rpm). After 48 h of incubation the fermented broth wascentrifuged at 10,000g for 10 min and the supernatant was usedfor enzyme assay.

2.1.3. Bacterial identificationA total of 15 bacterial cultures were selected for the xylanase

production based on the secondary screening. The most promisingisolate DHN8 was identified on the basis of its morphological andbiochemical characterization. Further confirmation was done by16S rRNA gene sequencing at Gujarat State Biotechnology Mission(GSBTM), Gandhinagar, India. The gene sequence has been sub-mitted to Gene Bank at NCBI (GenBank accession no. KF258832).The sequence was compared with sequences available in NCBIdatabase and dendrogram was generated using Mega 5.2 software.

2.2. Selection of agro-residue for enhanced cellulase free xylanaseproduction

For enhanced xylanase production various agro-residues suchas caster shell, sugarcane bagasse, saw dust, rice straw, barleystraw, sorghum straw, wheat straw, groundnut shell and maizestraw were evaluated. Xylanase production was carried out using2% (w/v) agro-residue in the 100 ml basal liquid medium (pH 7.0)containing same medium components but without oat speltxylan. The flasks were sterilized at 121 1C for 15 min, cooled andinoculated with 1% (v/v) of bacterial culture. The contents wereincubated at 37 1C under shaking condition (150 rpm) for 48 h.The fermented broth was centrifuged at 10,000g for 10 min at 4 1Cand the clear supernatant was analysed for enzyme assay.

2.3. Analytical methods

All the experiments were done in triplicates and the valuespresented are mean values7SD.

Chemicals such as oat spelt xylan, carboxy methyl cellulose(CMC), bovine serum albumin (BSA) and dinitrosalicylic acid(DNSA) were purchased from Hi Media Laboratories Ltd. All theother chemicals, media, salts and reagents used were of analyticalgrade (Sigma- Aldrich, Hi Media, Qualigens and Merck).

The xylanase activity was measured according to Bailey et al.(1992). The reaction mixture consisting 450 ml of 1% oat spelt xylanin 50 mM sodium phosphate buffer (pH 7.0) and 50 ml of enzymewas incubated for 10 min at 50 1C. The amount of reducing sugarreleased was quantified using 3,5-dinitrosalicylic acid (DNS) methodas described by Miller (1959). The CMCase and FPase activities weredetermined according to IUPAC recommendation (Ghose, 1987). Oneunit of xylanase or cellulase activity was defined as the amount ofenzyme required to liberate 1 mmol of xylose or glucose equivalentper min under the specified conditions.

The protein content was measured according to Lowry et al.(1951) with BSA as the standard.

2.4. Assessment of process parameters for cellulase free xylanaseproduction

2.4.1. Optimization of physiological parameters2.4.1.1. Inoculum size. To study the effect of inoculum size, produc-tion media were inoculated at a level of 0.5, 1.0, 2.0, 3.0, 4.0 and5.0% (v/v) from the 12 h old bacterial culture broth.

2.4.1.2. Inoculum age. To study the effect of inoculum age onxylanase production, inoculum was prepared by inoculating 50 mlof basal liquid medium with bacterium DHN8 and incubated at37 1C under shaking condition (150 rpm). At regular interval of 6 hincubation, 1% (v/v) of inoculum was transferred to 100 ml freshfermentation medium followed by incubation at 37 1C for 48 hunder shaking at 150 rpm. The culture filtrate was centrifuged andfinally used for enzyme assay.

2.4.1.3. Incubation time. In order to achieve maximum xylanaseproduction, incubation time was varied from 12 to 60 h. The crudeenzyme was extracted and assayed at regular interval of 6 h.

2.4.1.4. pH. Initial pH of the fermentation mediumwas varied from3 to 10. The pH was adjusted by using 1 M—HCl or NaOH prior toautoclaving.

2.4.1.5. Temperature. Xylanase production was studied at differenttemperatures ranging from 25 to 50 1C.

2.4.1.6. Agitation speed. The rate of agitation was studied at 0 to300 rpm with a difference of 50 rpm.

2.4.2. Optimization of nutritional parameters2.4.2.1. Influence of carbon sources. Influence of various carbon sourceson the xylanase production were assessed at a concentration of0.5% (w/v).

2.4.2.2. Influence of organic and inorganic nitrogen sources. Variousorganic and inorganic nitrogen sources were evaluated for enhan-ced xylanase production at concentration of 0.5 and 0.3% (w/v),respectively.

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2.5. Partial purification and characterization of xylanase

Solid ammonium sulphate was slowly added to the culturesupernatant in an ice bath under constant stirring to achieve 70%saturation. After centrifugation at 10,000g for 10 min at 4 1C,the supernatant was removed and the precipitate was resus-pended in 10 ml of 50 mM sodium phosphate buffer (pH 7.0).The final solution was dialysed against the same buffer overnightat 4 1C with three intermittent changes of the buffer.

2.5.1. Effect of temperature on xylanase activity and stabilityThe optimum temperature was determined by incubating the

enzyme extract in 1% oat spelt xylan solution at various tempera-tures (35–75 1C). For thermostability assay, enzyme was incubatedat different temperatures in the absence of substrate for 120 min.Aliquots were withdrawn at regular time intervals to test theresidual xylanase activity.

2.5.2. Effect of pH on xylanase activity and stabilityThe pH optima was determined by measuring relative activity at

various pH values using sodium citrate (pH 3–6), sodium phosphate(pH 6–8) and glycine-NaOH (pH 8–10) buffers (50 mM). The stabilityof xylanase was assessed by incubating enzyme samples for 24 h indifferent buffers. At regular interval of 3 h samples were withdrawnand residual enzyme activity was determined.

2.5.3. Effect of metal ions on xylanase activityThe effect of metal ions on enzyme activity was determined by

incubating the xylanase preparations in 10 mM metal solution for30 min. Residual activity was measured by standard enzyme assay.

2.5.4. Effect of various solvents on xylanase activityThe enzyme was incubated with various alcoholic and non

alcoholic solvents at 10% (v/v) concentration for 30 min and residualactivity was measured by the standard assay procedure.

2.6. Chemical pretreatment of sorghum straw

Prior to the enzymatic hydrolysis, the sorghum straw wasgiven three separate chemical pretreatments. Ten grams of driedand powdered sorghum straw was taken for each pretreatment

experiment. The biomass was mixed with 200 ml of 1 M NaOH (for24 h), 1 M HCl (for 12 h) and 3% alkaline hydrogen peroxide, pH11.0 (for 12 h) separately to obtain a solid:liquid ratio of 1:20. Thecontents were kept under shaking (100 rpm) at 50 1C. The pre-treated substrates were washed until neutrality and dried in ovenat 50 1C till constant weight was achieved.

2.7. Enzymatic hydrolysis

Enzymatic hydrolysis of sorghum straw was carried out in50 ml Erlenmeyer flask with 2.5% (untreated and pre-treated)substrate and 20 ml of appropriately diluted crude enzyme at50 1C, 100 rpm for 48 h. Controls were kept in which activeenzyme was replaced with heat inactivated enzyme. The reactionsystem was supplemented with 0.005% sodium azide to preventmicrobial growth. Aliquots were taken at regular intervals of 12 h,centrifuged and the supernatant was assayed for total reducingsugar by 3,5-dinitrosalysilic acid method (Miller, 1959).

2.8. Analysis of hydrolysed products by TLC

The products of enzymatic hydrolysis of sorghum straw wereexamined by ascending thin-layer chromatography (TLC) on pre-coated silica gel plates (60 F254, Merck) using acetone/ isopropylalcohol/water (6:3:1.5, v/v/v) as mobile phase. At defined times,reaction mixtures were sampled, the enzyme activity was stoppedby boiling for 10 min and 3.0 ml samples were applied on TLCplates. The separated products were detected by spraying the platewith α-naphthol (3.5% in ethanol and 10% sulphuric acid) followedby heating at 100 1C.

3. Results and discussion

3.1. Isolation and identification of strain DHN8

The newly isolated DHN8 strain was Gram positive, strictlyaerobic, spore forming, rod shaped and peritrichous. It reactedpositively in the catalase and oxidase tests. According to theseresults, it was clear that the bacterium belonged to the genusBacillus species. Based on the results of 16S rRNA gene sequencingthe bacterium was identified as Bacillus altitudinis (accession no.

Fig. 1. The phylogenetic dendrogram of Bacillus altitudinis DHN8 was labelled with an aster and related species by the neighbour-joining approach. Bootstrap values obtainedwith 1000 resamplings are indicated as percentages at all branches. The scale bars represent 0.05 substitutions per nucleotide position. Numbers following the names of thestrains are accession numbers of published sequences.

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KF258832) and designated as B. altitudinis DHN8. Through thealignment of homologous sequences of known bacteria, phyloge-netic position of the strain is shown in Fig. 1.

3.2. Effect of various agro-residues on xylanase production

Among tested agro residues, B. altitudinis DHN8 exhibited clearpreference towards sorghum straw for highest xylanase produc-tion (65.4972.18 IU/ml) followed by wheat straw, sugarcanebagasse, barley straw and rice straw (62.0071.91, 47.0071.68,42.6871.16 and 34.7371.21 IU/ml, respectively). However, lowerxylanase activities were observed in presence of maize straw,caster shell, groundnut shell and saw dust (Fig. 2). The higherxylanase production in presence of sorghum straw might be dueto the nature of hemicellulose, the presence of some activatorsin the carbon source, surface, pore size and favourable degrad-ability of carbon source (Gomes et al., 1993). However, manyresearch groups used hemicellulosic substrates such as wheatbran, rice straw, wheat straw, soybean flakes, rice bran, sugarcanebagasse and groundnut shells for xylanase production (Battanet al., 2007; Sharma et al., 2007; Taneja et al., 2002; Gawandeand Kamat, 1999). These data suggested that the xylanase induc-tion is a complex phenomenon and the level of response todifferent inducers varies with the strains. This study highlightedthat choosing an appropriate agro-residue can improve enzymeproduction markedly.

Further different concentrations of sorghum straw rangingfrom 1.0 to 6.0% (w/v) were tested under submerged fermentation.The maximum xylanase production (68.4972.37 IU/ml) wasachieved at 3% (w/v) sorghum straw (Fig. 3). The gradual decreasein xylanase production was observed above 3% sorghum strawconcentration. The possible reason behind such xylanase produc-tion behaviour could be formation of thick suspension in presenceof higher substrate concentration resulted in improper mixing ofthe substrate under agitation condition.

3.3. Effect of inoculum size, inoculum age and incubation period

An optimum inoculum level is necessary for maintaining balancebetween the proliferating biomass and available nutrients to producemaximum enzyme level. The highest xylanase production (72.8472.64 IU/ml) was achieved when the production medium was inocu-lated with 1% (w/v) of 12 h old inoculum (Fig. 4). The enzyme titredeclined drastically with increase in inoculum size beyond theoptimum values could be due to faster nutrient consumption. Inearlier studies, Battan et al. (2007) and Nagar et al. (2010) also showed

the use of 1.0–5.0% (v/v) inoculum size for hyper xylanase production.Higher concentration of inoculum is not preferable in industrialfermentations (Lincon, 1960).

The effect of inoculum age was studied from 6 to 48 h oldculture of B. altitudinis DHN8. The maximum xylanase production91.3872.68 IU/ml was obtained with 18 h old inoculum (Fig. 5)but decreased thereafter. These results obtained might be due tothe fact that, the maximum enzyme titre is produced during earlyto late exponential phase of the organism. It also suggests that thepartial association existed between growth of the organism and itsenzyme production pattern. Kumar et al. (2012) and Nagar et al.(2010) also showed the use of 18 h old inoculum for xylanaseproduction.

The time of fermentation for maximum enzyme productionvaries among different bacteria and is dependent upon the organism

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Fig. 2. Effect of various agro residues on xylanase production. CS: caster shell,SB: sugarcane bagasse, SD: saw dust, RS: rice straw, BS: barley straw, SS: sorghumstraw, WS: wheat straw, GS: groundnut shell, MS: maize straw. The fermentationmedium containing 2% w/v agro residue, beef extract, 3.0; NaCl, 5.0; KNO3, 2.0;K2HPO4, 1.0 and MgSO4 � 7H2O, 0.5 at pH 7.0 was inoculated with 12 h old bacterialculture at 1% v/v. The enzyme production was carried out at 37 1C under shaking(150 rpm) for 48 h.

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Fig. 3. Effect of sorghum straw concentration (1–6% w/v) on xylanase production.The fermentation medium (pH 7.0) with varying concentration of sorghum strawwas inoculated with 12 h old bacterial culture at 1% v/v. The enzyme productionwas carried out at 37 1C under shaking (150 rpm) for 48 h.

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Fig. 4. Effect of inoculum size on xylanase production. The 3% w/v sorghum strawcontaining fermentation medium (pH 7.0) was inoculated with 12 h old bacterialculture at 1 to 5% v/v. The enzyme production was carried out at 37 1C undershaking (150 rpm) for 48 h.

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Fig. 5. Effect of inoculum age on xylanase production. The fermentation medium(pH 7.0) was inoculated at 1% v/v level with 12 to 60 h old bacterial culture. Theenzyme production was carried out at 37 1C under shaking (150 rpm) for 48 h.

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type, its enzyme production pattern, cultural conditions and geneticmakeup of the organism. During course of incubation period, high-est xylanase production (81.5672.85 IU/ml) was obtained at 42 hof incubation and thereafter it declined gradually (Fig. 6). Thereduction in xylanase yield could be due to nutrients depletion ordue to proteolysis (Flores et al., 1997). Xylanase produced by Bacillussp. was found to be growth-associated, reaching a maximum after24 h and the enzyme production remained more or less constantup to 48 h (Anuradha et al., 2007). On the other hand, Bacillushalodurans PPKS-2 (Prakash et al., 2011) and Bacillus SSP-34(Subramaniyan and Prema, 2000) produced maximum xylanaseafter 48 and 96 h, respectively.

3.4. Effect of pH, temperature and agitation speed

The B. altitudinis DHN8 was cultivated in the production mediumadjusted at different pH (3.0–10.0). The bacterium did not producesatisfactory xylanase yield at initial medium pH 3.0 to 5.0 but at pH6.0 xylanase titre was increased and reached maximum at pH 7.0(85.7372.23 IU/ml). Moreover, afterwards from pH 8.0 to 10.0substantial xylanase production was observed (Fig. 7). Xylanaseproduction by various bacteria and fungi has been shown to bemarkedly dependent on pH (Wong et al., 1982). Acidic pH (4.0–6.0)generally favours fungal xylanases (Bajpai, 1997) while higher pHfavours bacterial xylanases (Bisaria and Ghose, 1991). In tuning withthis finding, Bacillus pumilus ASH (Battan et al., 2007) and Bacillussubtilis ASH (Sanghi et al., 2008) showed elevated xylanase produc-tion at pH 7.0. However, Bacillus mojavensis AG137 (Sepahy et al.,2011) and Bacillus NT 9 (Han et al., 2004) showed maximumxylanase production at medium pH 8.0 and 10, respectively.

The xylanase production was conducted at different temperatures(25–50 1C). The maximum xylanase production (91.1472.01 IU/ml)

was obtained at 35 1C (Fig. 8) and reduced sharply at highertemperature range of 40 to 50 1C. Although the physiological changesinduced by high temperatures during enzyme production are notcompletely understood, it has been suggested that at higher tem-peratures microorganisms may synthesize only a reduced numberof proteins essential for growth and other physiological processes(Gawande and Kamat, 1999). These results might also be correspondsto the growth profile of the microorganism where as no othertemperature was suitable to such extant for growth and enzymesecretion. Many researchers reported 37 1C temperature for max-imum xylanase production from Bacillus sp. (Nagar et al., 2010;Sanghi et al., 2008; Battan et al., 2007).

During submerged fermentation process, agitation and aerationare two important parameters used for uniform mixing of thenutrients and to meet the oxygen demand. The B. altitudinis DHN8exhibited significant amount of xylanase production in the rangeof 150 to 300 rpm agitation speed with optimum xylanase yield(103.8773.46 IU/ml) at 250 rpm. However, under static and loweragitation (50 rpm) conditions only 10.21 and 45.31% of the maximumxylanase activity was observed (Fig. 9). The lower xylanase produc-tion under static to low agitation conditions may be attributed to thedissolved oxygen (DO) limitation, improper mixing of media compo-nents and cell clumping. Various researchers showed maximumxylanase production at an agitation rate of 200–250 rpm (Kumaret al., 2012; Battan et al., 2007; Taneja et al., 2002; Beg et al., 2001;Gawande and Kamat, 1999).

3.5. Influence of carbon and nitrogen sources

Carbon source is one of the most important factors duringthe growth and metabolic process of microorganisms. The presence

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Fig. 6. Effect of incubation time on xylanase production. The fermentation medium(pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzymeproduction was carried out at 37 1C under shaking (150 rpm) and samples wereanalysed at 6 h intervals.

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Fig. 7. Effect of pH on xylanase production. The fermentation medium containingdifferent pH from 3 to 10 was inoculated at 1% v/v level with 18 h old bacterialculture. The enzyme production was carried out at 37 1C under shaking (150 rpm)for 42 h.

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Fig. 8. Effect of temperature on xylanase production. The fermentation medium(pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzymeproduction was carried out at different temperature range from 25 to 50 1C undershaking (150 rpm) for 42 h.

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Fig. 9. Effect of agitation speed on xylanase production. The fermentation medium(pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzymeproduction was carried out at 35 1C under static and shaking (0 to 300 rpm)conditions for 42 h.

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of carbon sources in the fermentation medium exerted profoundeffect on the enzyme production behaviour of the bacterium. Amongvarious monosaccharide and disaccharides tested only xylose,sucrose and starch (145.1373.85, 115.4173.66 and 111.073.91 IU/ml, respectively) stimulated xylanase production. However, othercarbon sources failed to support high yield of xylanase production ascompared to control (Table 1). The most plausible explanation fordecrease in xylanase yield with other carbon sources is that thesesources exerted catabolite repression. Such carbon source dependentenzyme production regulation has been noticed in different xylanaseproducing microbial strains (Lakshmi et al., 2009; Oliveira et al.,2006; Gassesse and MaMo, 1999).

To check the effect of nitrogen sources on xylanase productiondifferent organic and inorganic nitrogen sources were addedindividually to the fermentation medium (Table 2).

Amongst various organic nitrogen sources, maximum xylanaseproduction was obtained with gelatine (197.9476.00 IU/ml) followedby urea, yeast extract, tryptone and beef extract. However, casein,meat extract, malt extract and peptone did not showed stimulatoryeffect on xylanase production. The enhanced production of xylanasein presence of gelatine as organic nitrogen sources may be attributedto organic nitrogen source mediated regulation of microbial growthand metabolism (Gupta et al., 2000). Moreover, it has been alsoobserved that nitrogen can significantly affect the pH of the mediumduring the course of fermentation (Haapala et al., 1994) therebyinfluences the microbial metabolism. Bajaj and Manhas (2012)reported 24.0% xylanase improvement in the presence of gelatine.The most suitable organic nitrogen source for the xylanase productionby Geobacillus thermoleovorans (Sharma et al., 2007) and Bacilluscirculans AB16 (Dhillon and Khanna, 2000) was tryptone. B. moja-vensis AG147 yielded maximum xylanase when tryptone and yeastextract was used in combination (Sepahy et al., 2011).

During the evaluation of inorganic nitrogen sources KNO3 showedmaximum xylanase production (245.0473.15 IU/ml). All other testedinorganic nitrogen sources also showed increase in xylanase produc-tion except (NH4)2H2PO4 (103.7974.76 IU/ml) as compared to thecontrol set devoid of any nitrogen source. In accordance with theseresults, Nagar et al. (2012) observed stimulatory effect of KNO3 incombination with peptone and yeast extract on xylanase production.

3.6. Partial characterization of xylanase produced by B. altitudinisDHN8

3.6.1. Effect of temperature on activity and stabilityThe partially purified xylanase was active in the broad range

of temperatures 35 to 75 1C, with temperature optima at 50 1C.The enzyme exhibited more than 60% of its activity in the rangefrom 45 to 65 1C. The xylanase activity was dropped at temperature

values above 70 1C and only 20% of relative xylanase activity wasdetected at 75 1C (Fig. 10).

The enzyme thermostability depends upon molecular interac-tions such as hydrogen bonds, electrostatic and hydrophobic inter-actions, disulfide bonds, and metal binding which can promote asuperior conformational structure for the enzyme with a higherpacking efficiency, reduced entropy of unfolding, conformationalstrain release and stability of α-helices (Li et al., 2005). Thermo-stability studies of B. altitudinis DHN8 xylanase showed residualactivity between 70 and 90% at 45 to 60 1C temperature after 60 minof incubation. Even at 70 1C after 30 min of incubation 47% of theresidual activity was noticed. The obtained results clearly indicatethe nature of xylanase as thermostable. The suitable temperaturerange for industrial application of present xylanase lies in 35 to 65 1C(Fig. 11). Most of the bacterial xylanases show optimum activity at 50to 60 1C (Bajaj and Singh, 2010). Identical temperature optimaat 50 1C were reported for the xylanases from Bacillus sp. (Nagaret al., 2010; Polizeli et al., 2005). However, xylanase produced byB. halodurans exhibited activity over 30–100 1C, with an optimum at

Table 1Influence of carbon sources on cellulase-free xylanase production by Bacillusaltitudinis DHN8.

Carbonsources

Xylanase activity(IU/ml)

Protein(mg/ml)

Specific activity(IU/mg)

Controla 103.1872.80 2.5470.09 40.5371.11Glucose 62.0773.04 2.1070.06 29.6072.29Maltose 83.9372.29 2.2870.07 36.7170.99Starch 111.0073.91 2.2870.07 36.7172.30Cellulose 68.3672.86 2.4470.09 27.9370.16Sucrose 115.4173.66 2.9370.15 39.4170.80Fructose 88.2972.66 2.2670.11 39.1171.69Xylose 145.1373.85 3.1370.13 46.3172.02Galactose 68.8773.70 2.3370.16 29.6672.72Lactose 62.1271.71 2.3370.14 26.7272.36Mannitol 85.3374.01 2.9370.06 29.1371.38

a The control medium used was devoid of carbon source.

Table 2Influence of nitrogen sources on cellulase-free xylanase production by B.altitudinis DHN8.

Nitrogensource

Xylanase activity(IU/ml)

Protein(mg/ml)

Specific activity(IU/mg)

OrganicPeptone 133.9673.13 3.0470.13 44.1071.40Beef extract 144.2974.61 3.3370.07 43.3572.04Yeast extract 173.6476.38 3.5470.08 48.9970.75Casein 83.0372.66 2.5070.11 33.1470.54Malt extract 101.0672.52 2.9070.10 34.8572.00Trypton 154.7672.40 3.4970.14 44.3471.67Gelatine 197.9476.00 3.8770.10 51.1772.12Skim milk powder 144.8273.47 3.4270.09 42.2771.13Meat extract 94.7873.94 2.3870.11 39.7971.21Urea 183.8477.93 3.1870.08 57.6971.87

Inorganic(NH4)2HPO4 195.6578.69 3.8670.11 50.6770.79(NH4)2H2PO4 103.7974.76 3.2670.14 31.8470.93KNO3 245.0473.15 4.0070.17 61.3171.95(NH4)2S2O8 163.7372.49 3.6470.14 45.0071.10NH4NO3 213.8874.90 3.9170.16 54.6471.25NH4Cl 177.3176.09 3.1070.09 57.1172.93NaNO3 198.1372.18 3.6670.15 54.0871.70(NH4)2 SO4 207.0673.27 4.0870.19 50.8372.64(NH4)2HPO4 195.6578.69 3.8670.11 50.6770.79Controla 135.3973.87 3.1370.14 43.2371.08

a The control medium used was devoid of nitrogen source.

0

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120

35 40 45 50 55 60 65 70 75

Rel

ativ

e ac

tivity

(%)

Temperature ( C)

Fig. 10. Effect of temperature on the activity of partially purified xylanase. Theenzyme extract was incubated in 1% oat spelt xylan solution at different tempera-tures (35 to 75 1C). The relative activity was measured using standard assay after10 min.

D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182–190 187

80 1C (Kumar and Satyanarayana, 2011). B. halodurans PPKS-2xylanase showed optimum temperature of 70 1C (Prakash et al.,2011). Thermostability of different Bacillus sp. xylanases variesbetween 55 and 80 1C (Kumar and Satyanarayana, 2011).

3.6.2. Effect of pH on activity and stabilityEnzyme activity is markedly affected by pH because substrate

binding and catalysis are often dependent on charge distributionof both substrate and particularly enzyme molecules. The xylanaseof B. altitudinis DHN8 exhibited the highest xylanase activity at pH7.0 and retained more than 60% relative activity over broad rangeof pH 5 to 10 (Fig. 12).

pH stability study revealed that the present xylanase was stablein pH range of 6 to 10 and showed �70% residual activity after18 h of incubation (Fig. 13). The results indicated that the partiallypurified xylanase was alkali stable in nature. In similar study,xylanase from Bacillus sp. GRE7 showed pH stability over the pHrange of 5–11 for 30 min (Kiddinamoorthy et al., 2008). Bacillusstearothermophilus T-6 xylanase had stability at pH 6.5–10.0 for5 min (Khasin et al., 1993). The differences in pH and temperaturestability for extracellular xylanases might be due to the posttranscriptional modifications in xylanase excretion process, suchas glycosylation, that improved stability in more extreme pH andtemperature conditions (Savitha et al., 2007).

Thus, B. altitudinis DHN8 xylanase possessed cellulase-freenature, broad pH stability spectrum and stability at elevatedtemperature, could be important tool for application in manyindustrial processes.

3.6.3. Effect of metal ions and additives on xylanase activityMetal ions can be involved in enzyme catalysis in a variety

of ways: they may accept or donate electrons; they may them-selves act as electrophiles; they may mask nucleophiles to preventunwanted side reactions; they may bring together enzyme andsubstrate by coordinate bonds; they may hold the reacting groupsin the required 3D orientation and they may simply stabilizea catalytically active confirmation of the enzyme (Palmer, 2001).As shown in Table 3, xylanase activity was assayed in the presenceof several metal solutions (10 mM). The xylanase activity wasmarkedly stimulated in the presence of metal ions such as CaCl2(168.00%), MnCl2 (126.95%) and FeCl3 (106.01%). However, signifi-cant inhibition in xylanase activity was found in the presenceof other metal compounds. HgCl2 was acted as strong inhibitorycompound (�64% inhibition), suggesting that there is an impor-tant cystein residue in or close to the active site of the enzyme(Khandeparkar and Bhosle, 2006). Kumar and Satyanarayana(2011) reported that the activity of B. halodurans xylanase wasstrongly inhibited by Snþ2, Hgþ2, Cuþ2 and Cdþ2. More thana quarter of all known enzymes require the presence of metalatoms for fully catalytic activity (Palmer, 2001). These effectsreveal which kind of ions should be precluded or included inindustrial processes.

The reports on the solvent stable xylanases are very rare.The influence of various alcoholic and non alcoholic solvent additiveson the activity of xylanase is shown in Table 3. The xylanase activitywas stimulated in the presence of isopropanol (126.07%), methanol(117.81%), ethanol (108.69%) and acetone (102.96%). However, pre-sence of 1-butanol, 2-butanol, benzene and toluene resulted inreduced xylanase activity. Similar observations for xylanase stimula-tion and suppression in presence of straight chain alcohols werereported by Li et al. (2010). Biocatalysis in organic media offers several

0

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0 30 60 90 120

Res

idua

l act

ivity

(%)

Time (min)

35 C

40 C

45 C

50 C

55 C

60 C

65 C

70 C

75 C

Fig. 11. Effect of temperature on the stability of partially purified xylanase.The enzyme extract was incubated at different temperatures (35 to 75 1C) for120 min. The residual activity was measured after each 30 min intervals.

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100

120

3 4 5 6 7 8 9 10

Rel

ativ

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tivity

(%)

pH

pH 3-6 pH 6-8 pH 8-10

Fig. 12. Effect of pH on the activity of partially purified xylanase. The enzymeextract was incubated in different buffers: sodium citrate (pH 3–6), sodiumphosphate (pH 6–8) and glycine-NaOH (pH 8–10) buffers (50 mM).

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pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

Fig. 13. Effect of pH on the stability of partially purified xylanase. The enzymeextract was incubated for 24 h in different buffers (pH 3 to 10) for 24 h. At regularinterval of 3 h samples were withdrawn and residual enzyme activity wasdetermined.

Table 3Effect of metal compounds and solvents on cellulase-free xylanase activity.

Metal compound(10 mM)

Relative xylanaseactivity (%)

Solvents(10% v/v)

Relative xylanaseactivity (%)

Controla 100.0071.42 Control 100.0071.42CoCl2 72.7072.45 Methanol 117.817 5.04FeCl3 106.017 4.98 Ethanol 108.6972.56MnCl2 126.9573.21 Isopropanol 126.0774.90CaCl2 168.7278.66 1-Butanol 89.3472.47HgCl2 46.8772.03 2- Butanol 92.5673.83ZnCl2 62.3673.00 Acetone 102.9672.81NiCl2 32.3571.21 Toluene 67.2172.99MgCl2 62.9571.27 Benzene 76.1071.60

a The control medium used was devoid of any modulator or solvent.

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advantages viz. higher solubility of hydrophobic substrate enabalingtheir effective reactions, reduced microbial contamination and reusa-bility (Sardessai and Bhosle, 2004). These results provide essentialdata for the application of xylanase in an organic phase industrialreactions.

3.7. Enzymatic hydrolysis of sorghum straw

The utilization of enzymatic hydrolysis to obtain sugars fromagricultural residues is of great interest in modern biotechnologyparticularly for bio-solvent production. Fig. 13 depicts the effect ofenzymatic hydrolysis on reducing sugar production from differ-ently pretreated sorghum straw. The 3% alkaline hydrogen per-oxide pretreated sorghum straw yielded maximum reducing sugar(34.94 mg/g) after 36 h of enzymatic hydrolysis. Whereas, alkalipretreated, acid pretreated and untreated biomass yielded 29.56,23.81 and 2.58 mg/g reducing sugar after 48 h. In 3% alkalinehydrogen peroxide pretreated sorghum straw after an initial phaseof rapid sugar formation during 36 h the rate of hydrolysisdecreased which could be due to enzyme inactivation or depletionof an easily hydrolysable fraction of hemicellulose. As compared toalkali and acid pretreatments, alkaline hydrogen peroxide treat-ment is more effective in lignin solubilisation (Chen et al., 2008)and improving of crop residue digestibility (Talebnia et al., 2010)(Fig. 14).

4. Conclusion

Due to the increasing economic relevance of xylanases presentstudy was designed and attempt was made to optimize differentprocess parameters. The detailed optimization study resulted in a3.74-fold enhancement in xylanase production as compared tothat of the initial conditions. The cellulase-free and thermo-alkali-solvent stable xylanase produced by the newly isolated B. altitu-dinis DHN8 using least reported sorghum straw as substrate is oneof the rare xylanases because of its stability at extreme processconditions prevailing in the paper industry. The xylanase was ableto release reducing sugars mainly xylose from alkaline hydrogenperoxide treated sorghum straw biomass which could be furtherfermented to biofuel.

Acknowledgement

Authors are greatly acknowledges the financial assistance fromDepartment of Science and Technology (DST), Ministry of Science

and Technology, Govt. of India, for giving INSPIRE Fellowship toMr. Dharmesh N. Adhyaru, under INSPIRE Program.

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