Biomass hydrolyzing enzymes from plant pathogen Xanthomonas axonopodis pv. punicae: optimizing production and characterization

Abstract

Xanthomonas axonopodis pv. punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28 °C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55 °C while xylanase had highest activity at 45 °C. Heat treatment of enzyme extract at 75 °C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.

Introduction

Ever increasing energy demand, depletion of fossil fuels and increased environmental concern has shifted the focus of energy generation towards renewable alternatives. Bioenergy from biomass is promising and is available in the form of solid fuel, liquid biofuel and biogas. Bioenergy from biomass, especially second generation biofuels, has significant advantages over other types of renewable energies because biomass is the most abundant, renewable material on our planet and helps in C sequestration. Lignocellulose, consisting of cellulose, hemicelluloses and lignin, is the major structural component of all plant biomass. Cellulose, the most abundant organic molecule on Earth, is a glucose homopolymer while hemicelluloses, the second most abundant component of lignocellulosic biomass, are heterogeneous polymers of pentoses (including xylose and arabinose), hexoses (mainly mannose, less glucose and galactose) and sugar acids. The sugars in these two polysaccharides are fermented into fuel molecules; however, prior to fermentation, the polysaccharides have to be hydrolyzed into an assimilable and fermentable form.

Enzymatic hydrolysis of cellulose and hemicellulose to fermentable sugars is achieved using a complex set of enzymes called cellulases and hemicellulases. Bacteria and fungi are good sources of cellulases and hemicellulases. The classical cellulase system includes endoglucanase, exoglucanase, and cellobiase. Hemicellulases include enzymes that break down β-1,4 xylan and various other side chains. Currently, enzymatic saccharification of cellulose is achieved using preparations from saprophytic organisms Trichoderma and Aspergillus. However, there are several limitations, such as the crystalline recalcitrance of cellulose, product inhibition, enzyme deactivation, etc., which hinder high saccharification yields and make enzymatic hydrolysis the most costly step in biomass to ethanol process. It has been estimated that the greatest returns in cost savings will be realized by improving conversion of biomass to sugars, increasing hydrolysis yields, reducing enzyme loadings, and eliminating or reducing pretreatment (Lynd et al. 2008). A broader suite of enzymes is required for hydrolysis of cellulose and hemicelluloses to fermentable sugars (McMillan et al. 2011). Enzymatic hydrolysis can be carried out effectively if a mixture of different cellulolytic and accessory enzymes is used. Development of low cost, commercial enzyme mixtures with hemicellulases that work synergistically with cellulases is one of the goals of current research activities in this field. Having a diverse library of cellulases and other complementary enzymes will enable tailoring of such cocktails. Many bacterial and plant pathogens are known to produce an arsenal of plant-cell-wall-degrading enzymes, which can be more potent as the pathogens have to overcome plant defenses (King et al. 2011). A number of plant pathogenic fungi (Fusarium oxysporum, Phoma betae, Collectotrichum gloeosporioides, Myrothecium verrucaria, etc.) have been reported to elaborate high levels of cellulases (Ortega 1994; Moreira et al. 2005). The pathogenicity of a large number of Gram negative bacteria (Xanthomonas, Erwinia and Pseudomonas) relies on their ability to secrete hydrolytic enzymes into external medium (Kamoun and Kado 1990; Kazemi-Pour et al. 2004). Many plant pathogens, including those belonging to genus Xanthomonas, secrete extracellular depolymerizing enzymes, such as cellulases, xylanases, proteases and pectinases (Singh and Verma 1975; Chapon et al. 2001). Although the role of depolymerizing enzymes in necrosis and pathogenicity is well documented, studies on the commercial production of cellulases and xylanases from other plant pathogens, including Xanthomonas, are almost lacking. A literature survey revealed scant information regarding purification and characterization of cellulase enzyme from Xanthomonas. Thus, the present study aims at production, optimization and characterization of cellulolytic and xylanolytic enzymes produced by X. axonopodis pv. punicae, a potent plant pathogen of a number of crops like rice, soybean, cotton, pomegranate, etc., for use in saccharification of lignocellulosic biomass.

Material and methods

Organism and strain maintenance

Xanthomonas axonopodis pv. punicae strain was procured from the germplasm collection at the Division of Microbiology, IARI, New Delhi. It was grown and maintained on nutrient agar at 28  °C for 3 days and slants were stored at 6–8 °C in a refrigerator.

Determining cellulolytic and xylanolytic abilities

The cellulolytic and xylanolytic potential of X. axonopodis was judged qualitatively on the basis of its ability to grow on mineral medium [2.0 g KH₂PO₄; 1.4 g (NH₄)₂SO₄, 1.4 g KNO₃; 0.3 g MgSO₄⋅7 H₂O; 0.3 g CaCl₂; 5.0 mg FeSO₄⋅7 H₂O; 1.6 mg MnSO₄⋅H₂O; 2.0 mg CoCl₂; 1.4 mg ZnSO₄⋅7 H₂O in 1 l at pH 7] with 1 % acid swollen cellulose or 0.1 % xylan as sole carbon source (Rautela and Cowling 1966). The ability of X. axonopodis pv. punicae strain to produce endoglucanases was also assessed by its ability to produce zones of hydrolysis on carboxy methyl cellulose (CMC) agar as proposed by Teather and Wood (1982). The cultures were point inoculated on basal agar medium containing 1 % CMC and incubated at 28 °C for 5 days. Plates were flooded with aqueous congo red solution (1 mg ml⁻¹). After 15 min, dye was drained and plates were washed with 1 M NaCl and observed for production of yellow zones around growth.

For quantitative assay, X. axonopodis strain was grown in 25 ml basal mineral medium dispensed in 100 ml Erlenmeyer flasks with 1 % CMC as C source supplemented with 0.1 % peptone and yeast extract under submerged culture at 28 °C. A bacterial overnight culture grown in nutrient broth was used as inoculum at a rate of 10 %. After 7 days growth on a shaker at 150 rpm and 28 °C, the contents of flask were centrifuged at 10,000 rpm for 10 min and the supernatant was used for estimation of different enzyme activities.

Assay of cellulase and xylanase activity

Activity of cellulase complex was assayed in terms of filter paper activity (FPA /exo-β-1,4- glucanase) and CMCase (endo-β-1,4-glucanase) using Whatman no. 1 and CMC as substrate and incubation at 50 °C as described by Ghose (1987). Xylanase activity was assayed by the method described by Ghose and Bisaria (1987) using xylan. Reducing sugars released were measured by the DNSA method (Miller 1959). One unit of FPA or CMCase corresponded to 1 μmol glucose formed per minute during hydrolysis and one enzyme unit of xylanase was expressed as 1 μmol xylose formed per minute during hydrolysis. β- glucosidase assay was performed using p-nitrophenyl-β-d-glucopyranoside as substrate (Wood and Bhat 1988) and activity was calculated in terms of micromoles of p-nitrophenol produced per milliliter of culture filtrate per minute.

Optimization of cellulases and xylanase production

The one-factor-at-a-time methodology was employed to determine the best C and N source. The effect of different C sources on the production of cellulases and xylanases was studied by supplementing basal medium with 1 % of one of the following C substrates: CMC, cellulose powder, starch or xylan. Cultures were incubated at 28 °C and, after 7 days of incubation, enzyme activities were assayed as described above. Different organic N sources were added to basal medium at 0.5 % concentration to test their effect on enzyme production. Incubation and enzyme assays were performed as mentioned above. The effect on extracellular levels of enzymes produced by X. axonopodis pv. punicae of addition of surfactants like Tween-20, Tween-80, Triton X-100 was also studied. Basal medium was amended with surfactant at a concentration of 0.1 % (w/v or v/v and, after appropriate incubation, enzyme activities were assayed as mentioned above.

Effect of temperature and pH on cellulolytic and xylanolytic enzyme production

The optimum temperature for production of extracellular enzymes was determined by incubating the cultures at 28 °C or 37 °C. To determine the optimum pH leading to maximum enzyme production, the final pH value of the culture medium was adjusted in the range 4–8 by adding 1 N HCl or 1 N NaOH. Incubation and enzyme assays were carried out as described above.

Physico-chemical characterization and thermal stability of enzyme activities

To determine the optimum pH for enzyme activity, the enzyme reactions were performed in 0.05 M citrate phosphate buffer with pH ranging from 4 to 8 in which the substrate was dissolved and incubated at 50 °C. The optimum temperature for enzyme activity was determined by incubating the standard reaction mixtures at temperatures between 45 °C and 75 °C.

To test the thermal stability of the enzymes, the supernatant was kept at 75 °C for 1 h in a water bath. It was then cooled and residual activities determined by performing enzyme assays at 50 °C using the standard protocols described above.

Growth of X. axonopodis under optimized conditions and levels of enzymes produced

The organism was grown in 1 l Erlenmeyer flasks containing 250 ml medium under optimum conditions using the best C and N source, and enzyme activities were assayed after 7 days.

Saccharification of biologically pretreated paddy straw using enzyme extract produced by Xanthomonas axonopodis pv. punicae

Enzymatic saccharification of lignocellulosic substrate, i.e., biologically pretreated paddy straw, was carried out following the procedure described by Saritha (2011). Biologically pretreated paddy straw was obtained from the laboratory (i.e., prepared by solid state fermentation of paddy straw with the isolated strain of Aspergillus niger for 10 days at 30 °C). Compositional analysis of raw and biologically pretreated paddy straw was performed following the procedure of Goering and Van Soest (1970). Biologically pretreated paddy straw had 68 % holocellulose content, 5.21, 7.94, and 18.85 % protein, lignin and ash content, respectively, whereas raw paddy straw had 39.4 % cellulose, 16.2 % lignin, 21.6 % hemicellulose, 3.42 % protein and 19 % ash content.

Saccharification or hydrolysis of the biologically pretreated paddy straw samples was carried out according to the NREL LAP-009 protocol (Brown and Torget 1996), with some modifications. Pretreated samples were used in wet form for the enzymatic digestibility test; 1 g biologically pretreated paddy straw was placed in a 50-ml plastic bottle to which 1 ml enzyme extract was added and the volume made up to 10 ml by adding 0.05 M citrate buffer (pH 4.8) and incubated in shaker water bath at 50 °C. Periodically, 0.5 ml aliquots were withdrawn, heated in a boiling water bath for 2 min to stop the reaction and total reducing sugars estimated by the DNSA method as described above. In another set, biologically pretreated paddy straw samples were incubated with a standard cellulase and β-glucosidase mixture from Megazyme International (10 U E-CELAN, an endo-1,4-β-glucanase from A. niger supplemented with 5 U EBGLUC, a β-glucosidase from A. niger. This set served as a positive control for studying enzymatic saccharification.

Results and discussion

Xanthomonas axonopodis pv. punicae, an original isolate from infected pomegranate fruit during an epidemic in Sholapur, Maharashtra, India, was selected for this study (Supplementary Fig. 1). It showed good growth on basal medium with acid-swollen cellulose and xylan as sole C source, and produced a zone of hydrolysis on CMC agar, indicating its cellulolytic and xylanolytic potential (Supplementary Fig. 2). Quantitative assays to measure extracellular levels of cellulolytic and xylanolytic enzymes revealed that this strain was a good producer of xylanase and FPA, showing high specific activities of 0.743 and 0.168, respectively. Many plant pathogens, including those belonging to the genus Xanthomonas, have been reported to secrete extracellular depolymerizing enzymes such as cellulases, xylanases, proteases and pectinases (Singh and Verma 1975; Chapon et al. 2001). These enzymes serve as virulence factors for invading pathogens and contribute to their pathogenicity. The enzymes elaborated by these organisms promise to be more efficient because plants produce proteins as counter defense to inhibit cell wall degrading enzymes (CWDE), and this interaction drives plant pathogens to evolve unique and ever more potent hydrolytic enzymes (Juge and Svensson 2006; Gibson et al 2011). Different authors have advocated that total cellulolytic activities shown on insoluble substrates like filter paper and cellulose powder are more relevant for biomass conversion as compared to activity on soluble and synthetic substrates (Maki et al. 2009; Banerjee et al. 2010).

The effect of different carbon sources on the production of different cellulolytic and xylanolytic enzyme activities was studied (Table 1). Maximum specific activity (IU mg⁻¹ protein) of cellulolytic enzymes, i.e., CMCase (0.071), FPA (0.059) and xylanolytic activity, i.e., xylanase (0.702) were observed when 1 % CMC (w/v) was used as a C source, except for β-glucosidase, which was maximum when 1 % starch was used as sole C source. Thus, CMC was a better inducer for xylanases and FPA while starch was found to be a better inducer of β-glucosidase. However, starch and xylan being soluble and are thus an easier C source than CMC, and cellulose powder, supported better growth in terms of higher extracellular proteins. While studying production of hydrolytic enzymes from the plant pathogenic fungus Myrothecium verrucaria, Moreira et al. (2005) found that, amongst the various C sources tested, wheat bran produced the highest levels of enzyme activities as it contains insoluble carbohydrates. Ortega (1994) reported that, in the plant pathogen Collectotrichum gloeosporioides, the highest endoglucanase levels were induced by xylan, whereas in Fusarium oxysporum the highest levels were induced on CMC. In the case of Streptomyces sp., glucose gave highest levels of endoglucanases indicating its constitutive nature (Jaradat et al. 2008). While producing thermostable cellulases from a Streptomyces transformant, Jang and Chen (2003) found CMC to be the best C source to stimulate cellulase production. Wheat bran was found to be the best C source for β-glucosidase production by Humicola brevis followed by soyabean protein (Masui et al. 2012). Bakshi (1988) also observed higher levels of β-glucosidase from Streptomyces griseus when starch was provided as a C source. Studies have shown that carbon source is a main factor in inducing high levels of lignocellulolytic enzymes and their cost (Gao et al. 2008). Regarding N sources, the data presented in Table 2 showed that soya meal and yeast extract were good nitrogen sources for cellulolytic and xylanolytic enzyme activities, whereas peptone was found to be a good nitrogen source for production of cellobiase and xylanase enzymes. But when urea was used as a nitrogen source, enzyme activities diminished considerably. Jang and Chen (2003) also found that, in the case of Streptomyces transformants, nitrogen sources like urea and peptone made no difference to enzyme production. On the contrary, Jaradat et al. (2008) found that, in the case of Streptomyces sp., the inorganic nitrogen source ammonium chloride yielded a higher level of cellulases than yeast extract and asparagines.

Table 1 Effect of different carbon sources on the production of enzyme activities by Xanthomonas axonopodis pv. punicae strain. CMC Carboxymethylcellulose, CMCase carboxymethylcellulase, FPA filter paper activity, SEM standard error of the mean, CD critical difference

Table 2 Effect of nitrogen source on the production of enzymes (IU ml⁻¹) by X. axonopodis pv. punicae strain

As shown in Table 3, there was no significant effect of surfactants on the production of extracellular cellulolytic and xylanolytic enzymes. Controls showed highest production of extracellular enzyme activities. Extracellular proteins increased with all the surfactants tested compared to the control (Fig. 1). Although surfactants did cause leaching of proteins, the levels of these enzymes did not increase as they are extracellular by nature. The substrates for endoglucanases (CMCase activity), cellobiohydrolases (FPA) and xylanases are large polysaccharide molecules that cannot enter the cell. Therefore, the organisms excrete most of these activities into extracellular milieu so that they can act on polysaccharide substrates and break them into much smaller units, i.e., oligo-, di- and mono-saccharides. Hulme and Stranks (1970) and Yazdi et al. (1990) also reported no significant effect of surfactants on cellulase production by fungi because of inhibition of oxygen supply. However, Tribak et al. (2002) found a stimulatory effect of surfactants on the production of endo-xyloglucanases and their release by cellulolytic fungi.

Table 3 Effect of surfactants on the production of enzyme specific activities by X. axonopodis pv. punicae strain

Fig. 1

Effect of different surfactants on the release of extracellular proteins by Xanthomonas axonopodis pv. punicae strain

Maximum enzyme production was observed between pH 6 and 8, with highest at 7 and optimum temperature for growth and extracellular enzyme production at 28 °C, which coincides with its optimum physiological temperature and the pH of the organism (Fig. 2). Enzymes were produced under optimized conditions and the results are presented in Table 4. There was an increase in all enzyme activities. CMCase and FPA increased around 4- to 5-fold, while a 1.72-fold increase was observed in the case of xylanase and cellobiase activities.

Fig. 2

Optimum pH for cellulolytic and xylanolytic enzymes production by X. axonopodis pv. punicae strain

Table 4 Production of different enzymes under optimized conditions by X. axonopodis pv. punicae

The activity profiles of different enzymes as affected by temperature and pH are presented in Figs. 3 and 4. All the enzymes showed activity over a broad range of pH (4–8) with an optimum at 8, except FPA, which showed optimum activity at pH 4 and 6 showing that several isoforms of the enzyme are involved. Xylanase also showed more than one pH optimum (at 5 and 8). Gibson et al. (2011) stated that selected phytopathogenic bacterial genomes revealed diversity among glycosyl hydrolases families, and that Xanthomonas campestris was rich in both cellulose- and xylan-degrading enzymes, which appeared to be important virulence factors. Cellulolytic enzymes showed a temperature optimum at around 55 °C, while xylanase activity was highest at 45 °C and declined as the temperature increased. Heat treatment of the supernatant showed xylanase and cellobiase activities to be more thermostable than FPA and CMCase, which indicates that these enzymes are better suited to biomass conversion. Residual activities of 56 % and 80 % were found in xylanase and cellobiase, respectively, after treatment of enzyme extract at 75 °C for 1 h (Table 5), indicating that they are more thermostable than endoglucanase and cellobiohydrolase activities. Song and Wei (2010) reported that cellulases and xylanases produced by Cellulomicrobium cellulans grown on pretreated and extracted bagasse can be applied to the saccharification of steam- and alkali-extracted lignocellulosic substrates. Cellulase and xylanase produced by Cellulomicrobium cellulans retained 64 % and 87 % of their original activity after 1 h of incubation at 45 °C, and are thus classified as moderately thermostable (Yano and Poulos 2003). Alves-Prado et al. (2010) characterized crude xylanases from both bacterial and fungal sources and found that bacterial xylanase showed an optimum pH for enzyme activity at 6.0, whereas a fungal xylanase had optimum pH at 5.0–5.5. Bacterial and fungal xylanases were stable in the pH range 5.0–10.0 and 5.5–8.5, respectively. The optimum temperatures for bacterial and fungal xylanase were 55 and 60 °C, respectively, and they were thermally stable up to 50 °C. Bernier et al. (1983) purified multiple forms of xylanases from Aeromonas sp. It was found that these xylanases were most active at 50–60 °C. Kang et al. (1996) purified two xylanases, which gave the highest activity at 50 °C and showed relatively high stabilities at this temperature. Kamble and Jadhav (2012) reported that 50 °C is the most favorable temperature for xylanase activity from Bacillus arseniciselenatis DSM 15340. Stability of the enzyme was found to be the most important factor when studying the characteristics of this enzyme; it was stable at temperatures of 30 °C and 40 °C for 4 h of incubation, retaining almost 93 % activity. At higher temperatures, xylanase stability gradually declined. Grigorevski de Lima et al. (2005) reported that CMCase from Streptomyces drozdowiczii shows optimum activity at temperatures of 50–60 °C. Jang and Chen (2003) also found in the case of a Streptomyces transformant that the optimum temperature for CMCase was 50 °C, with optimum activity at pH 7. Moreira et al. (2005) reported that xylanase produced by the plant pathogenic fungus Myrothecium verrucaria in submerged cultures has maximum activity from pH 4.5 to pH 5.5. Masui et al. (2012) suggested that thermal stability of xylanase in crude extracts of Humicola brevis var. thermoidea was a notable feature that can be useful when preparing efficient enzymatic cocktails for lignocellulosic treatment.

Fig. 3

Optimum pH of cellulolytic and xylanolytic enzymes produced by X. axonopodis pv. punicae strain

Fig. 4

Optimum temperature of cellulolytic and xylanolytic enzymes produced by X. axonopodis pv. punicae strain

Table 5 Residual enzyme activities after heat treatment of enzyme extract of X. axonopodis pv. punicae strain at 75 °C for 1 h

Saccharification of biologically pretreated paddy straw with enzyme extracts from Xanthomonas resulted in release of sugars that increased with time of incubation; after 96 h the levels of sugars released from Xanthomonas extracts were higher than those obtained with the standard enzyme mixture (Fig. 5). Vlasenko et al. (1997) observed that generally, the optimum conditions for hydrolysis depend on the properties of enzyme and substrate. Enzyme dose, inactivation and inhibition by-products are the main factors affecting carbohydrate conversion. Teymouri et al. (2005) observed that enzyme loading in the saccharification step determines the rate and extent of polysaccharide hydrolysis. In the present study, since crude preparation was used to include all the enzymes, the units of enzymes loaded vs substrate were low therefore the sugar yields were not very high, although they were comparable to yields from the standard enzyme mixture.

Fig. 5

Saccharification of biologically pretreated paddy straw using Xanthomonas enzyme extracts and standard cellulase and β-glucosidase mixture (E-CELAN endoglucanase 10 U + E-BGLUC β-glucosidase 5 U)

This X. axonopodis strain produced endoglucanases, cellobiohydrolases, β-glucosidase and xylanases. Berlin et al. (2005) reiterated that endogenous levels of β-glucosidase and xylanase appear to be important for increasing the ability of cellulase preparations to hydrolyze lignocellulosic biomass. Thus, the X. axonopodis pv. punicae strain tested here has the potential to be exploited for production of biomass-hydrolyzing enzymes.

Conclusion

The X. axonopodis strain used in the present study produced high levels of CMCase, cellobiase and xylanase. The enzymes were versatile with respect to their pH and temperature optima and were thermostable. They provide an indication of the biomass-hydrolyzing ability of the enzyme preparation. The enzymes could be overproduced after further optimization and could find application in different industries including the food, pulp and paper industries and in biorefineries. Accessory hemicellulase enzymes such as xylanases can have a significant effect on the performance of cellulases, presumably by increasing cellulose accessibility. Supplementation of cellulase preparations with xylanases is a useful strategy for cellulase improvement.