Isolation, Purification and Characterization of Xylanase produced by Bacillus sp. NIORKP76 strain under solid state fermentation and its application in saccharification of various agro-residues into fermentable sugars

doi:10.21203/rs.3.rs-3193700/v1

Download PDF

Research Article

Isolation, Purification and Characterization of Xylanase produced by Bacillus sp. NIORKP76 strain under solid state fermentation and its application in saccharification of various agro-residues into fermentable sugars

https://doi.org/10.21203/rs.3.rs-3193700/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Microbial xylanases are xylan hydrolyzing enzymes which has congregated attention due to their immense potential in many industries. Increasing demand for these enzymes versus inadequate supply makes these biomolecules a value-added product. The current study is focused on xylanase from bacterial isolate identified as Bacillus sp. NIORKP76. The bacterial isolate used in this study showed ability to produce xylanase on an inexpensive agro-industrial waste (wheat bran) under solid-state fermentation. The isolate showed maximum xylanase production in growth media supplemented with phosphate, NaCl and NH₄Cl concentration of 64 mM, 15 mg/mL and 0.3 mg/mL respectively. The maximum xylanase titer volume was obtained with 1:3 substrate to moisture ratio (w/v). Using optimized conditions maximum xylanase production in wheat bran was achieved in 72 h at room temperature 28 ±2°C. Xylanase exhibited pH optima of 8.0 and retained 92% of its residual activity after 24h incubation period at pH 8.0, thus proving its high stability at alkaline pH. Xylanase displayed optimum activity at 60°C. Xylanase stability at 30°C and 40 °C remained unhindered even after 12h of incubation period. The xylanase isolated in this study was purified up to homogeneity and its molecular weight was found to be ~28kDa. Xylanase produced by Bacillus sp. NIORKP76 strain was found to have essential qualities required for saccharification of various agro-residues to generate fermentable sugars which can be a raw-material for biofuel production. Wheat bran with heat pre-treatment was found to be par excellence agro-waste as compared to other heat pre-treated and untreated lignocellulosic agro-wastes in production of fermentable reducing sugars. 141mg/g fermentable sugars were whipped up when reaction mixture of pre-treated wheat bran with 5U/g partially purified xylanase incubated at 40°C for 8h, which was found to be best results among all agro-residues studied.

Agro-residues

Marine bacteria

Bacillus

Xylanase

SSF

saccharification

Xylan together with cellulose (1,4-b-glucan) and lignin (a complex polyphenolic compound) make up the major polymeric constituents of the plant cell wall (Kukarni et al., 1999). Xylans are straight homopolymers that contain D-xylose monomers connected through β-1, 4–glycosyl bonds (Kukarni et al., 1999; Beg et al., 2001). Because of its heterogeneity and intricacy, the total hydrolysis of xylan requires a huge assortment of agreeably acting catalysts (Puls et al., 1987; Biely, 1985; Subramanian and Prema, 2002). Xylanolytic enzymes are the bio-catalysts which can break down complex xylan molecule into its oligomers and further monomers. Endo-1,4-b-D-xylanase (EC 3.2.1.8) is a primary xylanolytic enzyme mainly accountable for arbitrarily hydrolysis of the xylan spine. The other accessory xylanolytic enzymes sever auxiliary residues and xylose monomers from the non-reducing end of xylo-oligosaccharides and xylobiose. There are various microbial sources of xylanases are reported which mainly include bacteria, fungi, actinomycetes and yeast (Battan et al., 2006). Xylanases have got immense importance in several sectors of industries worldwide which includes pulp and paper industry, food and beverage, animal fodder, fruit juice, vegetable processing, textile, detergent, leather, bioethanol production, bioconversion of lignocellulosic matter etc. (Khandeparker and Bhosle, 2005b; Helianti et al., 2016). In recent decades xylanases gathered remarkable attention due to their prospective application in biobleaching and biopulping processes as an environment friendly alternative for chlorinated compounds used during conventional bleaching and pulping processes which subsequently lead to environmental pollution. Xylanases can be employed easily in delignification process without major alteration in existing pulping and bleaching processes (Hiremath and Patil, 2011).

The major limitation of industrial usage of xylanases is its production cost. The most prominent reason of high cost of enzymes is the high purity xylan used as substrate during production of these xylanase. If inexpensive alternate source of substrate is used for the production the total enzyme cost can be reduced significantly (Battan et al., 2006; Knob et al., 2014).

An agro industrial waste are nothing but the residues generated from the growing and processing of raw agricultural products such as crops, fruits, vegetables, meat, poultry and dairy products (Obi et al., 2016). The disposal of these invoked by-products is becoming major concern in agricultural countries since it causes environmental pollution (Wang et al., 2016). Agro industrial residues majorly comprised of lignocellulosic biomass containing hemicelluloses and treated as waste in many agricultural countries due to infeasibility to convert them into beneficial products for mankind (Obi et al., 2016). Developments of new and potential bio-catalytical processes which can utilize these agro industrial residues and convert it into valuable products, simultaneously competing with economically well-established chemical processes are required (Knob et al., 2014). Lignocellulosic biomasses are the major stake holders among all the categories of agro-industrial wastes mentioned above, which mainly includes: wheat bran, rice bran, sugarcane bagasse, corn cob, straw, stem, deciduous wood, seaweeds etc. Out of these agro-residues, wheat bran is readily available, cheap lignocellulose biomass which is rich source of xylan but utilized in least possible way and often considered to be waste. Biotechnological utilization of wheat bran for production of xylanase under solid state fermentation (SSF) not only serves as economical substrate but it will also seize environmental advantages. SSF has got a number of unambiguous advantages over submerged fermentation such as economy of space, media simplicity, comprehensible machinery, equipment and control systems, lower water volume due to compact fermentation vessels, greater product yields, reduced energy demand, lower capital and recurring expenditures in industry, easy scale up of fermentation process, simple product recovery with less solvents, superior yields, absence of foam build-up and easy control over contamination in system due to low moisture level (Khandeparker and Bhosle, 2005b; Kamble and Jadhav, 2011).

In recent times these lignocellulosic biomasses have been under prominence due to their ability to be an inexpensive medium for microorganisms in production of fermentable sugars, which are the precursors of variety of biofuels generated (Bhatia et al., 2012) along with other harmless by-products. Although there are several processes to generate fermentable sugars from agro-industrial wastes (Kleawkla and Chuenkruth, 2016; Shet et al., 2018), current study is mainly focused on enzymatic hydrolysis of the same by microorganism. Different microorganisms such as bacteria, fungi, yeast etc. utilizing these agro-residues and generating fermentable sugar has been studied previously (Parab et al., 2017; Baig et al., 2004; Akin-Osanaiye et al., 2005).

By considering all aspects, the prime objective of our current study is to isolate, purify and characterize xylanase produced by Bacillus sp. NIORKP76 strain when grown on SSF under optimized physio-chemical conditions and apply onto different agro-residues for generation of fermentable sugars. This study will give us insights about best possible agro-residue used in this study for generation of reducing sugars by enzymatic hydrolysis process.

Microbial strain and growth conditions

The bacterial strain used in this study was obtained from sediment and water samples collected from mangrove regions of Goa, India and it was identified as Bacillus sp. NIORKP76 strain with GenBank accession number MH767158 (stored at in house culture repository facility). Bacillus sp. NIORKP76 strain inoculum was prepared in Erlenmeyer flasks (250 mL) containing 100 mL sterile Basal salt solution (BSS) with 0.5% xylan as sole carbon source, 0.2% yeast extract and 0.03% peptone. At regular time intervals bacterial growth in inoculated media was measured spectrophotometrically (600 nm) as well as by counting colony-forming units (CFU).

Wheat bran, an inexpensive agro industrial residue was collected from local vendor to use as growth media for xylanase production. Optimization of physio-chemical conditions, required for maximum production xylanase was carried out by one variable at a time (OVAT) approach as follows:

Effect of salts

The concentration of nutrients such as phosphate (0-128 mM), sodium chloride (10–35 mg/mL) and ammonium chloride (0-1.1 mg/mL) were varied sequentially to determine optimum concentration required for maximum xylanase production under SSF. The optimized nutrient concentration was used in preparation of modified basal salt solution (MBSS) which subsequently used in other nutrient optimization experiment. Sterile MBSS was added aseptically to sterile wheat bran containing tubes with substrate to moisture ratio of 1:2 (w/v). 1% of 24h old pre-grown Bacillus sp. NIORKP76 strain was inoculated in all tubes. Blank was maintained for all nutrient concentrations without inoculating bacterial cells. All the tubes were incubated at RT for varying time intervals (0-120 h).

Effect of Moisture content

The effect of substrate to moisture content (w/v) ratio on xylanase production was studied using wheat bran and MBSS (prepared with optimized nutrients concentration). The ratio was varied from 1:1.5 to 1:4 (w/v). The test (with bacterial inoculum) and blank (without bacterial inoculum) tubes with respective substrate : moisture content were incubated at RT for varying time intervals.

Growth curve and xylanase production

The contents of the fermentation tubes were used to determine xylanase production and bacterial growth at respective time interval. After each time interval, phosphate buffer (pH 7.0) was added to fermentation tube and vortex to homogenize entire content. Homogenized mixture was centrifuged at 12,000 rpm for 5 min to obtain clear supernatant (SN) and pellet. SN was used as crude enzyme mixture to determine extracellular xylanase activity. Collected pellet was used for extraction of total protein. Cellular protein content was used as parameter to determine bacterial growth as per method described by Khandeparker et al, (2008). In brief, pellet was treated with 0.1N NaOH and kept in boiling water bath for 30 min, this was done to extract total proteins, further sample was cooled and neutralized by adding equal volume of 0.1N HCl. Sample was vortex and supernatant was collected which used for protein estimation. Protein estimation was carried out using protein assay dye reagent concentrate (BioRad, USA) as per manufacturer’s instructions.

Growth and xylanase production under optimized conditions

Growth and xylanase production by Bacillus sp. NIORKP76 strain was monitored on wheat bran using MBSS. Erlenmeyer flasks (25 mL) containing 1g sterile wheat bran each, were moistened with sterile 3mL MBSS. All the flasks were inoculated with 1% pre-grown bacterial culture and incubated at RT under static condition for varying time intervals. At each time interval flasks were selected in duplicate to extract crude enzyme and total protein as per protocol mentioned above. Production of xylanase (Xyl) was measured by carrying out respective enzyme assay.

Xylanase assay

Xylanase activity was assayed as per the method described by Khandeparkar and Bhosle, (2005b) with minor modifications. 1% beechwood xylan (SRL chemicals) solution was used as substrate for xylanase. Reaction mixture containing crude enzyme and substrate was incubated at RT for 20 min. The reducing sugars liberated were measured by 3, 5-dinitrosalicylic (DNS) method (Miller, 1959).

Extensive production of xylanase under SSF

1% overnight grown bacterial inoculum was inoculated in five 500 mL Erlenmeyer flasks, each containing 5g sterilized fermentation media moistened with sterile 15mL MBSS (1:3). Fermentation flasks were incubated at room temperature (28°C ± 2) for 72 h under static conditions. After incubation period 100 mL sodium phosphate buffer (pH 7.0) was added to each flask containing fermented product. Fermentation medium completely homogenized using vortex and centrifuged at 10,000g for 10 min at 4°C. The supernatant containing extracellular crude xylanase was collected and refrigerated until further use.

Protein precipitation and dialysis

The total protein content from supernatant was precipitated by adding ammonium sulphate up to saturation point (80%). The precipitated was collected after overnight refrigeration at 4°C. The collected precipitate was dissolved in sodium-phosphate buffer (50 mM, pH 7.0) and dialyzed using centrifugal devices (Merck-Millipore, Amicon ultra centrifugal filters) with molecular weight cutoff 13kDa. The dialyzed fractions were concentrated using rotary evaporator (Equitron Roteva, Medica Instrument) and stored at 4°C. The concentrated partially purified xylanase sample was further purified by ion exchange chromatography technique.

Ion exchange chromatography

The anion exchanger resin (DEAE-Sepharose FF, Sigma-Aldrich) was packed in column (10mm diameter and height 150mm) and flow rate was adjusted to 0.5 mL/min. The packed column was equilibrated with 50mM Na-phosphate buffer to remove unbound impurities. 1mL of concentrated partially purified enzyme sample was loaded onto column and linear gradient of NaCl from 0 to 3M applied. 2 mL fractions were collected in fresh Eppendorf tubes and analyzed for xylanase activity and protein content. The protein content in collected fractions was measured spectrophotometrically at 280nm and xylanase activity in same fractions was measured by standard xylanase assay. All active fractions were pooled together, concentrated and further applied onto column packed with cation exchange resins (CM-Sepharose). The volume of resins in column and flow rate was maintained same as anion exchanger column. All the active fractions obtained were concentrated and stored at 4°C for further analysis.

Molecular weight and zymogram analysis

Crude and purified xylanase sample was electrophoresed on SDS-PAGE (stacking gel-6%, resolving gel-12%) along with low range protein marker (BioRad, 14.4kDa to 97.4kDa) to determine molecular weight of xylanase. The electrophoresed gel was stained with silver stain technique to visualize protein bands. Zymogram analysis of purified xylanase carried out on SDS-PAGE (stacking gel-6%, resolving gel-12%) as per method described by Khandeparker et al., (2017). 0.1% xylan was added to resolving gel (12%) as substrate for xylanase. The electrophoresed gel with separated protein bands was washed thoroughly (4 times) in sodium phosphate buffer (50 mM, pH 8.0), with initial two washes contained 25% isopropanol to remove SDS from gel and renature protein followed by two washes with sodium phosphate buffer (50mM, pH 8.0). The washed gel was incubated at optimum temperature of xylanase for 60 min and soaked in 0.1% Congo red stain solution for 2 min. Excess stain was washed with 1% NaCl solution until active xylanase band were visible. The visibility of active xylanase band was improved by adding 0.5% acetic acid solution which changed gel background color to dark blue and clear colorless active xylanase band were visible.

Effect of pH on xylanase activity and stability

The effect of pH on xylanase was measured by determining enzyme activity of crude xylanase mixed in 50mM buffer solutions of pH range 5 to 10. Evaluation of xylanase stability at different pH was carried out by preparing mixture of crude enzyme in respective buffer (4.0–5.0, Citrate buffer; 6.0–8.0, sodium phosphate buffer; 9.0–10.0, Glycine-NaOH buffer) in 1:1 ratio and incubating it at RT (28°C ± 2) for 24h. At different time intervals, aliquots from mixture were withdrawn to determine residual enzyme activity. Xylanase assay was performed as per standard protocol mentioned above.

Effect of Temperature on xylanase activity and stability

The influence of temperature on xylanase was determined by incubating enzyme-substrate reaction mixture at different temperatures (30°C-80°C). The stability of xylanase at different temperatures was determined by incubating enzyme solution prepared in optimized buffer at temperature range of 30°C-80°C for 24h. At different time intervals aliquots from enzyme solution was withdrawn to determine residual enzyme activity. Xylanase assay was performed as per standard protocol mentioned above. Under optimum assay conditions activity of xylanase was considered to be 100%.

Kinetic determinations

Rate of xylan hydrolysis reaction was determined using different concentrations of beechwood xylan (SRL) ranging from 0.25 to 5.0 mg/mL. The varying substrate concentration solutions were prepared in sodium-phosphate buffer (50mM, pH-8.0). Standard xylanase assay method mentioned above was followed. The kinetic constants K_m and V_max were determined as described by Lineweaver and Burk (1934).

Xylanase gene sequence determination

Xylanase gene amplification from Bacillus sp. NIORKP76 strain was carried out by PCR technique. Previously reported xylanase gene primers (xynF and xynR) by Khandeparker et. al., (2017) were engaged in gene amplification procedure. The amplified gene fragment sequence was determined and analyzed as per Khandeparker et al., (2017). Furthermore, gene sequence similarity analysis was executed with corresponding gene sequences using online gene bank databases.

Collection and preparation of agro-residues

Different types of agro-residues such as Wheat bran (WB), Rice bran (RB), Rice husk (RH), Sugarcane bagasse (SB) and Seaweed (SW) were used for xylanase hydrolysis study. WB, RB and RH was collected from local stores, whereas SB was collected from local sugarcane juice vendor. SW sample was collected from the coast of Goa, India (15.5809° N, 73.7448° E). All the agro-residues were washed thoroughly with tap water to eliminate excess amounts of undesired debris and were dried at 50°C temperature. After drying, all the agro-residue samples were powdered using grinder and sieved through 0.25mm pore size strainer to have uniform size particle (Fernandes et al., 2020).

Pre-treatment of Agro-residues

Pre-treatment of agro-residues was performed with 10% biomass at 121°C, for 45 min (Parab et. al. 2017). Following the pre-treatment step, using vacuum filtration technique solids were separated from the solution and washed several times with distilled water and air dried. These air-dried biomass samples were used for further experiment.

Hydrolysis of agro-residues by partially-purified Xylanase

All the untreated and pre-treated agro-residue biomasses were added to phosphate buffer (0.05 M, pH 8.0) separately, and enzymatic hydrolysis was initiated by adding 5U/mg partially purified xylanase to agro-residue biomass. The reaction mixture was incubated at 40°C for various time intervals (0-8h). The product was centrifuged and RS (reducing sugar) from hydrolysate were analyzed by DNS method (Parab et. al., 2017).

Bacterial strain and its characteristics

Bacillus sp. NIORKP76 strain used in this study displayed morphological colony characteristics such as circular in shape with entire margin, raised elevation, moist consistency and white color upon growing on sterile ZMA plates. It showed cell properties such as Gram positive, rod-shape, motile and facultative anaerobic in nature. It showed ability to produce β-galactosidase and catalase. It was able to utilize sugars such as sucrose, mannitol, glucose, xylose, arabinose and trehalose but showed negative results for utilization of malonate and citrate. Acetoin production and nitrate reduction was not observed with this bacterial strain (Table. 1). Based on morphological, bio-chemical and molecular analysis (16s rRNA sequencing) it was identified as Bacillus sp. and designated as Bacillus sp. NIORKP76 strain (GenBank accession number: MH767158).

Optimization of growth conditions and growth media for xylanase production

Bacterial biomass as well as xylanase production was significantly influenced by varying physio-chemical conditions in growth medium. Utmost xylanase titer was produced in growth media containing BSS with 64mM phosphate ion (Fig. 1a). When NaCl concentration varied from 1–4% in BSS, maximum xylanase production was seen in growth media containing 1.5% NaCl (Fig. 1b). Similarly, maximum xylanase production was observed in growth media containing 0.03% NH₄Cl concentration (Fig. 1c).

The substrate to moisture ratio also played a very crucial role in xylanase production. It was observed that low moisture containing growth media was more favorable for bacterial growth as well as xylanase production as compared to high content of free moisture. The culture showed maximum xylanase production in growth media supplemented with modified BSS containing 64mM phosphate ion, 1.5% NaCl, 0.03% NH₄Cl and with substrate to moisture ratio of 1:3 (w/v) (Fig. 1d).

Growth and xylanase production by Bacillus sp. NIORKP76 strain under SSF

Xylanase being primary xylanolytic enzyme, optimized physio-chemical parameters were used for scaling up xylanase production by SSF. While growing in fermentation media using inexpensive agro-industrial waste that is wheat bran as carbon source Bacillus sp. NIORKP76 strain showed maximum growth at 84h. Observed growth was of diauxic (Schmiedel and Hillen, 1996) pattern. Such a pattern observed when bacterial isolate undergoes stress conditions in growing media. Utilization of simpler sugars leads to first log phase which is followed by lag phase due to exhaustion of simple sugars and switching over of cellular mechanism to utilize more complex nutrients available in fermentation media. First log phase was observed for 12h (24h to 36h) followed by lag phase of 12h (36h-48h) and further second log phase of 36h (48h-84h). Maximum xylanase production was observed at 72h, irrespective of maximum growth which occurred at following time interval (Fig. 2).

Purification of xylanase

The crude xylanase produced by Bacillus sp. NIORKP76 strain under SSF was precipitated and collected for further purification. Partially purified xylanase was obtained after dialysis carried out using centrifugal devices of molecular weight cut off 13kDa. The dialyzed fractions were further applied to column chromatography for further purification of xylanase till homogeneity. The purification steps are summarized in Table.2.

When xylanase was loaded on to anion exchanger column (DEAE-Sepharose) three protein peaks were observed, one of which was single isolated peak whereas other two were in close proximity with each other. Single protein peak showed maximum xylanase activity (Fig. 3). The xylanase containing fractions were pooled together concentrated and further purified on cation exchanger column (CM-Sepaharose). Approximately 47% xylanase recovery with 99-fold purification was achieved with specific activity of 2650 Umg^-1.

Molecular weight determination and zymogram analysis

As purification of xylanase preceded the decrease in number of protein bands observed thus confirming xylanase purification up to homogeneity. Crude xylanase showed multiple protein bands (Lane:2) as compared to very few protein bands to single band (~ 28kDa) after passing through anion (Lane:3) and cation exchanger columns (Lane:4) respectively. Zymogram analysis revealed active xylanase bands corresponding to molecular weight of ~ 28kDa in protein samples obtained after ion exchange chromatography (Fig. 4). From SDS-PAGE and zymogram results it was confirmed that the xylanase produced by Bacillus sp. NIORKP76 strain was having molecular weight of ~ 28kDa.

Kinetic parameters of xylanase

The K_m value of xylanase for beechwood xylan was found to be 4mg/mL and the V_max was found to be 100µmol/mL (Figs. 5 and 6). The K_m and V_max values were obtained from Lineweaver-Burk plot of xylanase activity at pH 8.0 and 60°C using various concentrations of beechwood xylan as substrate.

Effect of pH and temperature on the activity and stability of xylanase

The optimum pH of the xylanase produced by Bacillus sp. NIORKP76 strain was 8.0 (sodium phosphate buffer) (Fig. 7). Xylanase retained 92% of its activity at pH 8.0 for 24h (Fig. 8). The optimum temperature of xylanase was found to be 60°C at its respective optimum pH value (Fig. 7). Standard assay of xylanase diluted in suitable buffer incubated at various temperatures unveiled the influence of temperature on xylanase. Over the incubation period, xylanase from enzyme aliquot incubated at 40°C displayed increase in activity and retained its 100% activity for 24h (Fig. 9).

Nucleotide sequence analysis of Xylanase gene

Amplified PCR product was acquired from Bacillus sp. NIORKP76 strain using xynF and xynR primers. When obtained gene sequence subjected to similarity analysis with GenBank database it exhibited 99% similarity with partial sequence of endo-1,4-beta-xylanase reported from Bacillus velezensis (GenBank accession number: QCC30342.1). GenBank accession number: ON861829 was allotted to xylanase gene sequence isolated from Bacillus sp. NIORKP76 strain. The complete gene sequence comprised of 579-bp open reading frame encoding a protein of 20950.71Da, comprised of 193 amino acid residues with theoretical pI = 9.05. The amino acid sequence unveiled conserved catalytic domain belonging to glycosyl hydrolases family 11 observed from 17–189.

Enzymatic hydrolysis of agro-residues

Succeeding preliminary processing of all agro-residues, uniform particle size of 0.25mm was obtained. In current study effect of heat pre-treatment prior to xylanase hydrolysis (5U/g) on yield of reducing sugars from various agro-residues can be clearly noted. With increase in incubation period from 0 to 8h, there was ascending trend in release of reducing sugar observed in all five agro-residues (untreated and pre-treated). At all studied time intervals pre-treated biomass samples liberated additional reducing sugars as compared to untreated ones, thus proving hot water pre-treatment is simple yet effective for superior enzymatic hydrolysis of lignocellulosic biomass.

While, untreated and pre-treated wheat bran exhibited noteworthy results with maximum release of reducing sugars 137.67mg/g ± 2.3 and 141.07mg/g ± 1.8 respectively, untreated and pre-treated rice-husk seems to be least favorable for reducing sugars production with merely 29.47mg/g ± 3.8 and 52.67mg/g ± 5.2 respectively when incubated with 5U/g xylanase for 8h at 40°C. Other untreated agro-residues such as rice bran, sugarcane bagasse and seaweed generated reducing sugar concentrations of 65.87, 35.00 and 28.73mg/g respectively, which was escalated in pre-treated rice bran, sugarcane bagasse and seaweed samples leading to sugar concentrations of 81.07, 54.13 and 57.33mg/g respectively at the end of incubation period of 8h at 40°C with 5U/g xylanase enzyme (Figs. 10 and 11).

Xylanases have been classified as essential industrial enzymes due to their immense potential in multiple biotechnological applications in various industries (Battan et al., 2006). These enzymes with desirable pH tolerant and thermotolerant ability can be used in various processes in different industries. One of the major applications of this enzyme is found in paper and pulp industry for bleaching of kraft pulp. In order to obtain industrially compliant xylanase either natural microbial diversity is exploited or genetic engineering techniques being employed for enhanced xylanase production in host cells (Khandeparker et al., 2017; Yin et al., 2010). Various microbial sources of xylanase such as bacteria, fungi, actinomycetes and yeast have been reported (Battan et al., 2006). There are several reports found in literature describing production of xylanase using microbial systems under SmF (Hiremath and Patil, 2011; Gissesse and Gasha, 1997) but SSF technique is preferred over SmF due to its numerous advantages in production of xylanase, mainly use of inexpensive substrate (Kamble and Jadhav, 2011; Khandeparker and Bhosle, 2005a).

Bacillus sp. NIORKP76 strain used in this study was found to be an excellent producer of moderate thermotolerant and alkalitolerant xylanase under SSF. Bacillus sp. has been previously reported for the production of xylanase, which is the key hemicellulolytic enzyme among all others (Helianti et al., 2016; Kamble and Jadhav, 2011; Irfan et al., 2016; Gowdhaman et al., 2014; Kumar et al., 2013; Ho and Heng, 2014; Boucherba et al., 2017; Virupakshi et al., 2004). Apart from Bacillus sp. there are other bacterial strains such as Arthrobacter sp., Lactobacillus sp., Caldicellulosiruptor sp., Glaciecolamesophila and Paenibacillus sp. which are also reported for production of xylanase (Khandeparker and Jalal, 2014; Mi et al., 2014; Guo et al., 2013; Teeravivattanakit et al., 2016).

There are various substrates used for production of xylanase which include wheat bran, tea dust, saw dust, paper waste, cassava bagasse, rice straw, rice husk, palm kernel cake, barley husk, corn cob, sugar cane bagasse, oat bran, oat spelt, beet pulp and pineapple peel, among which wheat bran reported to be the best carbon source for xylanase production (Khandeparker et al., 2008; Gowdhaman et al., 2014; Ho and Heng, 2014).

As per report of Raimbault, (1998) the free moisture content in SSF is mainly influenced by type of substrate used and its water binding ability, which subsequently has pronounced effect on growth kinetics. In current study optimum substrate to moisture ratio was found to be 1:3 (w/v) which was similar to the reports described by Kumar et al., (2017), Khandeparker et al., (2008), Khandeparkar and Bhosle, (2005a). In other reports such as Sindhu et al., (2006); Kamble and Jadhav, (2011) reported 1:1.5 and 1:1.8 (w/v) ratio for production of xylanase under SSF (Wheat bran) respectively, whereas Sanghi et al., (2007) (Wheat bran) and Virupakshi et al., (2004) (Rice bran) used 1:2 (w/v) ratio for the same. This clearly indicates that substrate to moisture ratio may vary with microbial strain used in the study.

Bacillus sp. NIORKP76 isolate produced optimum xylanase at the end of 72h incubation period using wheat bran as substrate which was also reported by Kamble and Jadhav, (2011), Khandeparkar and Bhosle, (2005b) and Virupakshi et al., (2004) in their studies. The moistening agent used in this study was a modified basal salt solution (MBSS) containing optimized salt concentration (buffer ion: 64mM; NH₄Cl: 0.03%; NaCl: 1.5%). Battan et al., (2006) and Sanghi et al., (2007) described effect of moistening agent on xylanase titer under SSF. To enhance xylanase production under SSF by Bacillus sp. NIORKP76 modification in moistening agent was carried out.

SDS-PAGE analysis displayed single protein band with purified enzyme sample. Based on relative mobility of protein samples in SDS-PAGE molecular weight of xylanase was found to be ~ 28kDa. Zymogram analysis of purified xylanase sample confirmed the molecular weight with presence of active xylanase band. Xylanases of different molecular weight have been reported from Bacillus sp. Khandeparker et al., (2017) reported 23.3kDa xylanase, Kamble and Jadhav, (2011) reported 29.8kDa and Tseng et al., (2001) reported 45kDa xylanase from Bacillus sp. Xylanase isolated from Bacillus sp. by Sa-Pereira et al., (2001) reported to have high molecular weight of 340kDa.

The xylanase enzymes produced by Bacillus sp. NIORKP76 isolate exhibited optimum pH and temperature of 8.0 and 60°C respectively. Xylanases reported by Khusro et al., (2016) showed optimum pH 7.0 and temperature optima at 35°C. Chaturvedi et al., (2015) reported xylanase having pH and temperature optima of 6.5 and 45°C respectively. Teeravivattanakit et al., (2016) also reported xylanase with pH and temperature optima of 7.0 and 50°C respectively. Similarly, Boucherba et al., (2017) reported xylanase with pH and temperature optima of 7.0 and 55°C. Adigüzel and Tunçer, (2016) reported xylanase having temperature optima of 60°C which was similar to our reported xylanase but pH optima were only 6.0.

The xylanase enzyme was found to be very stable, retaining 92% of residual activity when incubated in buffer pH 8.0 and 137% residual activity was recorded when incubated at 40°C for 24h, unlike xylanase reported by Adigüzel and Tunçer, (2016) which was having half-life of 10h when incubated at pH 7.0 and temperature 40°C. Xylanase reported by Khusro et al., (2016) was able to retain approximately 80% activity, when incubated for 4h in pH 8.0 and at 40°C.

The overall cost of any product determined by the complexities of production procedure and materials used. The current study follows very simple materials and methodologies which gives cost effective products. Hydrolysis of lignocellulosic biomass is predominantly carried out by two approaches, acid or alkali hydrolysis and enzymatic hydrolysis. These are either executed discretely or sequentially provided type of research objective. Unlike, acidic and alkali hydrolysis processes the enzymatic hydrolysis is regulated at mild conditions, eliminating corrosion drawbacks which enhances overall process feasibility (Soccol et al., 2011). As per report by Bansal et al., (2021) the saccharification efficiency of enzymatic hydrolysis process does not affect significantly in terms of fermentable sugar production as compared to acidic hydrolysis process. Enzymatic hydrolysis in certain processes added as pre-treatment phase to truncate chemical wastage and energy requirements which subsequently minimizes cost and environmental problems associated with chemical hydrolysis process (Sreeraj et al., 2022). Fermentable sugar production from various lignocellulosic material by use of xylanase has been previously reported. The efficacy of xylanase in terms of dose (5U/mg) used in this study is much more superior than those which used by Parab et al., (2017): 50U/mg and Kaur et al., (2023): 10U. The xylanase dose of 5U/g generated 137.67mg/g and 141.07mg/g fermentable sugar with untreated and pre-treated wheat bran biomass, which is superior to results obtained by Mazlan et al., (2019) in which 50U/mL enzymatic dose liberated merely 174.74mg/g reducing sugars from pre-treated biomass.

Increasing demand of xylanase versus inadequate supply is the major reason of its high cost. Xylanase production using inexpensive agro-industrial residues and potential bacterial isolate under SSF can be ideal way of fulfilling demand of xylanase at lower cost. The Bacillus sp. NIORKP76 strain used in this study produced elevated titre of xylanase while growing on wheat bran under optimized conditions. The xylanase produced was of better-quality in terms of pH and temperature stability. Thus, our obtained results suggest that Bacillus sp. NIORKP76 isolate can be used to scale up the xylanase production under SSF and enzymes produced can be used in various industrial applications due to their extraordinary stability at different pH. The efficacy of currently studied xylanase concerning fermentable sugar production from untreated and pre-treated agro-residues is better than previously studied reports, which makes it advantageous for industrial processes engaged in similar work.

Acknowledgements:

The authors are thankful to Director, CSIR-National Institute of Oceanography, Goa (India), and Head, BOD, for giving all necessary services and encouragement. The authors thank the Council of Scientific and Industrial Research (CSIR, India) for providing financial support through the Senior Research Fellowship (SRF) to the first author. This article is part of doctoral work being submitted by the first author at Goa University in the Department of Microbiology under the corresponding author’s guidance.

Compliance with ethical standards:

Conflict of interest: The authors declare that they have no conflict of interest.

Data Availability:

The authors confirm that the data supporting the findings of this study are available within the article. Raw data that support the findings of this study are available from the first and corresponding author, upon reasonable request.

Adigüzel AO, and Tunçer M (2016) Production, Characterization and Application of a Xylanase from Streptomyces sp. AOA40 in Fruit Juice and Bakery Industries. Food Biotechnol 30:3, 189-218. DOI:10.1080/08905436.2016.1199383
Akin-Osanaiye BC, Nzelibe HC, and Agbaji AS (2005) Production of ethanol from Carica papaya (pawpaw) agro waste: effect of saccharification and different treatments on ethanol yield. African J Biotechnol 4(7):657-659. DOI:10.5897/AJB2005.000-3118
Baig MM, Baig ML, Baig MI, Yasmeen M (2004) Saccharification of banana agro-waste by cellulolytic enzymes. African J Biotechnol 3(9):447-50. DOI:10.5897/AJB2004.000-2088
Battan B, Sharma J, Kuhad RC (2006) High-level of xylanase production by alkaliphilic Bacillus pumilus ASH under solid-state fermentation. World J Microbiol Biotechnol 22:1281-1287. https://doi.org/10.1007/s11274-006-9173-x
Beg QK, Kapoor M, Mahajan L, Hoondal GS (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56(3-4):326-38. https://doi.org/10.1007/s002530100704
Bhatia L, Johri S and Ahmad R (2012) An economic and ecological perspective of ethanol production from renewable agro waste: a review. AMB Expr 2, 65 (2012). https://doi.org/10.1186/2191-0855-2-65
Biely P (1985) Microbial xylanolytic systems. Trends in Biotechnol 3(11):286-290. https://doi.org/10.1016/0167-7799(85)90004-6
Boucherba N, Gagaoua M, Bouanane‑Darenfed A, Bouiche C, Bouacem K, Kerbous MY, Maafa Y, Benallaoua S (2017) Biochemical properties of a new thermo‑ and solvent‑stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3. Bioresour Bioprocess 4, 29 (2017). https://doi.org/10.1186/s40643-017-0161-9
Chaturvedi S, Singh R, Khurana SPM (2015) Production of Bacterial - Xylanolytic Enzyme using Agricultural Waste by Solid State Fermentation. Int J Curr Microbiol App Sci 4(4): 9-16.
Fernandes ÉS, Bueno D, Pagnocca FC, Brienzo M. Minor biomass particle size for an efficient cellulose accessibility and enzymatic hydrolysis. ChemistrySelect. 5(25):7627-31. https://doi.org/10.1002/slct.202001008
Gessesse A, Gashe BA (1997) Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkaline soda lake. J Applied Microbiol 83:402-406. https://doi.org/10.1046/j.1365-2672.1997.00242.x
Gowdhaman D, Manaswini VS, Jayanthi V, Dhanasri M, Jeyalakshmi G, Gunasekar V, Sugumaran KR, Ponnusami V (2014) Xylanase production from Bacillus aerophilus KGJ2 and its application in xylooligosaccharides preparation. Int J Biol Macromol 64:90-8. https://doi.org/10.1016/j.ijbiomac.2013.11.012
Guo B, Li PY, Yue YS, Zhao HL, Dong S, Song XY, Sun CY, Zhang WX, Chen XL, Zhang XY, Zhou BC, Zhang YZ (2013) Gene Cloning, Expression and Characterization of a Novel Xylanase from the Marine Bacterium, Glaciecola mesophila KMM241. Mar Drugs 11:1173-1187. https://doi.org/10.3390/md11041173
Helianti I, Ulfah M, Nurhayati N, Suhendar D, Finalissari AK, Wardani AK (2016) Production of Xylanase by Recombinant Bacillus subtilis DB104 Cultivated in Agro-industrial Waste Medium. HAYATI J Biosci 23:125-131. https://doi.org/10.1016/j.hjb.2016.07.002
Hiremath KS, Patil CS (2011) Isolation, production and characterization of alkalo thermostable xylanase from newly isolated Bacillus sp. Int J Bioechnol applications 3(1):48-51. 10.9735/0975-2943.3.1.48-51
Ho HL, Heng KL (2014) Xylanase Production by Bacillus subtilis in Cost-Effective Medium Using Soybean Hull as Part of Medium Compostion under Submerged Fermentation (Smf) and Solid-State Fermentation (SsF). J Biodivers Biopros Dev 2(1):143. 10.4172/2376-0214.1000143
Irfan M, Asghar U, Nadeemb M, Nelofer R, Syed Q (2016) Optimization of process parameters for xylanase production by Bacillus sp. in submerged fermentation. J Rad Res Appl Sci 9(2):139-147. https://doi.org/10.1016/j.jrras.2015.10.008
Kamble RD, Jadhav AR (2011) Isolation, Purification, and Characterization of Xylanase Produced by a New Species of Bacillus in Solid State Fermentation. Int J Microbiol 2012:683193. https://doi.org/10.1155/2012/683193
Khandeparkar R, Bhosle NB (2005a) Purification and characterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112. Res Microbiol 157:315–325. https://doi.org/10.1016/j.resmic.2005.12.001
Khandeparkar RDS, Bhosle NB (2005b) Isolation, purification and characterization of the xylanase produced by Arthrobacter sp. MTCC 5214 when grown in solid-state fermentation. Enz Micro Technol 39:732–742. https://doi.org/10.1016/j.enzmictec.2005.12.008
Khandeparker R, Jalal T (2014) Xylanolytic enzyme systems in Arthrobacter sp. MTCC 5214 and Lactobacillus sp. Biotechnol Appl Biochem. 62(2):245-254. https://doi.org/10.1002/bab.1253
Khandeparker R, Numan MTH, Mukherjee B, Satwekar A, Bhosle NB (2008) Purification and characterization of a-L-arabinofuranosidase from Arthrobacter sp. MTCC 5214 in solid-state fermentation. Process Biochem 43(7):707-712. https://doi.org/10.1016/j.procbio.2008.02.014
Khandeparker R, Parab P, Amberkar U (2017) Recombinant Xylanase from Bacillus tequilensis BT21: Biochemical Characterisation and Its Application in the Production of Xylobiose from Agricultural Residues. Food Technol Biotechnol 55(2):164-172. https://doi.org/10.17113/ftb.55.02.17.4896
Khusro A, Kaliyan BK, Al-Dhabi NA, Arasu MV, Agastian P (2016) Statistical optimization of thermo-alkali stable xylanase production from Bacillus tequilensis strain ARMATI. Elect J Biotechnol 22:16–25. https://doi.org/10.1016/j.ejbt.2016.04.002
Kleawkla A, Chuenkruth P (2016) Reducing Sugar Production from Agricultural Wastes by Acid Hydrolysis. KEM 675–676:31–4. https://doi.org/10.4028/www.scientific.net/kem.675-676.31
Knob A, Fortkamp D, Prolo T, Izidoro SC, Almeida JM (2014) Agro-residues as Alternative for Xylanase Production by Filamentous Fungi. BioRes 9(3):5738-5773. http://ncsu.edu/bioresources
Kulkarni N, Shendye A, Rao M (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23(4):411-456. https://doi.org/10.1111/j.1574-6976.1999.tb00407.x
Kumar L, Kumar D, Nagar S, Gupta R, Garg N, Kuhad RC, Gupta VK (2013) Modulation of xylanase production from alkaliphilic Bacillus pumilus VLK-1 through process optimization and temperature shift operation. 3 Biotech 4:345-356. https://doi.org/10.1007/s13205-013-0160-2
Kumar VS, Karthi V, Ramkumar A, Ramesh R, Stephen A, Kumaresan S (2017) Isolation, Screening, Identification, and Optimization of Xylanase Producing Bacteria from Forests of Western Ghats, India. Int J Agri Env Biotechnol 11(1): 173-179.
Mazlan NA, Samad KA, Yussof HW, Saufi SM, Jahim J (2019) Xylooligosaccharides from potential agricultural waste: Characterization and screening on the enzymatic hydrolysis factors. Industrial Crops Products. 129, 575-584. https://doi.org/10.1016/j.indcrop.2018.12.042.
Mi S, Jia X, Wang J, Qiao W, Peng X, Han Y (2014) Biochemical Characterization of Two Thermostable Xylanolytic Enzymes Encoded by a Gene Cluster of Caldicellulosiruptor owensensis. PLoS One 9(8): e105264. https://doi.org/10.1371/journal.pone.0105264
Miller GL (1959) Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem 31(3):426-428. https://doi.org/10.1021/ac60147a030
Obi FO, Ugwuishiwu BO, Nwakaire JN (2016) Agricultural waste concept, generation, utilization and management. Nigerian J Technol 35(4). DOI:10.4314/njt.v35i4.34
Parab P, Khandeparker R, Amberkar U, Khodse V (2017) Enzymatic saccharification of seaweeds into fermentable sugars by xylanase from marine Bacillus sp. strain BT21. 3 Biotech. 7:1-7. https://doi.org/10.1007/s13205-017-0921-4
Puls J, Schmidt O, Granzow C (1987) α-Glucuronidase in two microbial xylanolytic systems. Enz Microbial Technol 9(2):83-88. https://doi.org/10.1016/0141-0229(87)90147-5
Raimbault M (1998) General and microbiological aspects of solid substrate fermentation. Electron. J. Biotechnol 1(3). http://dx.doi.org/10.4067/S0717-34581998000300007
Sanghi A, Garg N, Sharma J, Kuhar K, Kuhad RC, Gupta VK (2007) Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation. World J Microbiol Biotechnol 24:633–640. https://doi.org/10.1007/s11274-007-9521-5
Sa-Pereira P, Costa-Ferreira M, Aires-Barros MR (2001) Enzymatic properties of a neutral endo-1,3(4)-_-xylanase Xyl II from Bacillus subtilis. J Biotechnol 94(2002):265-275. https://doi.org/10.1016/S0168-1656(01)00436-9
Schmiedel D, Hillen W (1996) Contributions of XylR, CcpA and cre to diauxic growth of Bacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose. Mol Gen Genet 250(3):259-266. https://doi.org/10.1007/BF02174383
Shet VB, Varun C, Aishwarya U, Palan AM, Shama, Rao U, Goveas LC, Raja S, Rao CV, Puttur U (2018) Optimization of reducing sugars production from agro-residue coconut leaflets using autoclave-assisted HCl hydrolysis with response surface methodology. Agri Nat Res 52(3), 280-284. https://doi.org/10.1016/j.anres.2018.09.015
Sindhu I, Chhibber S, Capalash N, Sharma P (2006) Production of Cellulase-Free Xylanase from Bacillus megaterium by Solid State Fermentation for Biobleaching of Pulp. Curr Microbiol 53:167–172. https://doi.org/10.1007/s00284-006-0051-4
Soccol CR, Faraco V, Karp S, Vandenberghe LPS, Thomaz-Soccol V, Woiciechowski A, Pandey A, Lignocellulosic Bioethanol: Current Status and Future Perspectives. Biofuels 101-122. https://doi.org/10.1016/B978-0-12-385099-7.00005-X
Sreeraj PR, Mishra SK, Singh PK (2022) Characteristic features and functions of nanocellulose for its feasible application in textile industry. Nanocellulose Materials (pp. 105-122). Elsevier https://doi.org/10.1016/B978-0-12-823963-6.00013-2
Subramaniyan S, Prema P (2002) Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 22(1):33-64. https://doi.org/10.1080/07388550290789450
Teeravivattanakit T, Baramee S, Phitsuwan P, Waeonukul R, Pason P, Tachaapaikoon C, Sakka K, Ratanakhanokchaia K (2016) Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β-D-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities. Appl Environ Microbiol 82:6942–6951. https://doi.org/10.1128/AEM.02256-16
Tseng MJ, Yap NM, Ratanakhanokchai K, Kyu KL, Chen ST (2001) Purification and characterization of two cellulase free xylanases from an alkaliphilic Bacillus firmus. Enz Microbiol Technol 30(2002):590-595. https://doi.org/10.1016/S0141-0229(02)00018-2
Virupakshi S, Babu KG, Gaikwad SR, Naik GR (2004) Production of a xylanolytic enzyme by a thermoalkaliphilic Bacillus sp. JB-99 in solid state fermentation. Process Biochem 40:431–435. https://doi.org/10.1016/j.procbio.2004.01.027
Wang B, Dong F, Chen M, Zhu J, Tan J, Fu X, Wang Y, Chen S (2016) Advances in recycling and utilization of agricultural wastes in China: Based on environmental risk, crucial pathways, influencing factors, policy mechanism. Procedia Env Sci 31:12 – 17. https://doi.org/10.1016/j.proenv.2016.02.002
Yin T., Miao L, Guan F, Wang G, Peng Q, Li B, Guann G, Li Y (2010) Optimized Medium Improves Expression and Secretion of Extremely Thermostable Bacterial Xylanase, XynB, in Kluyveromyces lactis. J Microbiol Biotechnol 10(11):1471-1480. doi: 10.4014/jmb.1005.05041

Table. 1 Morphological, physiological and biochemical characteristics of the isolate

Tests	Results	Tests	Results
*Colony Morphology*		*Carbohydrate Utilization*
Shape	Circular	Xylose	+
Margin	Entire	Glucose	+
Elevation	Raised	Sucrose	+
Consistency	Moist	Mannitol	+
Color	White	Arabinose	+
Opacity	Opaque	Trehalose	+
Gram Nature	Gram Positive	Lactose (ONPG)	+
Shape of the Cell	Rods	Malonate	-
*Growth Conditions*		Arginine	-
Temperature	30°C-40°C	VP	-
pH	7.0-8.0	Citrate	-
Aerobe/Anaerobe	Facultative anaerobe	Nitrate Reduction	-
Motility	Motile	Catalase	+

+: Positive, -: Negative

Table.2 Purification steps of xylanase isolated from Bacillus sp. X4PI8 strain

Purification steps	Xylanase activity (U/ml)	Proteins (mg)	Specific activity (Umg^-1)	Purification fold	Yield (%)
Filtrate	15	0.557	27	1.00	100.00
(NH₄)₂SO₄ precipitation	14	0.237	59	2.24	95.24
DEAE-Sepharose FF	8	0.006	1345	50.62	52.54
CM-Sepharose FF	7	0.003	2650	99.71	47.77

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Isolation, Purification and Characterization of Xylanase produced by Bacillus sp. NIORKP76 strain under solid state fermentation and its application in saccharification of various agro-residues into fermentable sugars

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Microbial strain and growth conditions

Effect of salts

Effect of Moisture content

Growth curve and xylanase production

Growth and xylanase production under optimized conditions

Xylanase assay

Extensive production of xylanase under SSF

Protein precipitation and dialysis

Ion exchange chromatography

Molecular weight and zymogram analysis

Effect of pH on xylanase activity and stability

Effect of Temperature on xylanase activity and stability

Kinetic determinations

Xylanase gene sequence determination

Collection and preparation of agro-residues

Pre-treatment of Agro-residues

Hydrolysis of agro-residues by partially-purified Xylanase

Results

Bacterial strain and its characteristics

Optimization of growth conditions and growth media for xylanase production

Purification of xylanase

Molecular weight determination and zymogram analysis

Kinetic parameters of xylanase

Effect of pH and temperature on the activity and stability of xylanase

Nucleotide sequence analysis of Xylanase gene

Enzymatic hydrolysis of agro-residues

Discussion

Conclusions

Declarations

References

Tables

Additional Declarations

Status:

Version 1