Cellulase-free xylanases from Bacillus and other microorganisms

Abstract

Xylanases are used mainly in the pulp and paper industries for the pretreatment of Kraft pulp prior to bleaching to minimize use of chlorine, the conventional bleaching agent. This application has great potential as an environmentally safe method. Hydrolysis by xylanases of relocated and reprecipitated xylan on the surface of cellulose fibres formed during Kraft cooking facilitates the removal of lignin by increasing permeability to oxidising agents. Most of the xylanases reported in the literature contained significant cellulolytic activity, which make them less suitable for pulp and paper industries. The need for large quantities of xylanases which would be stable at higher temperatures and pH values and free of cellulase activity has necessitated a search for novel enzymes. We have isolated and characterised several xylanase-producing cultures, one of which (an alkalophilic Bacillus SSP-34) produced more than 100 IU ml⁻¹ of xylanase activity. The SSP-34 xylanases have optimum activity at 50°C in a pH range 6–8, with only small amounts of cellulolytic activity (CMCase (0.4 IU ml⁻¹, pH 7), FPase (0.2 IU ml⁻¹, pH 7) and no activity at pH 9).

Xylanase, Cellulase, Cellulose binding domain, Paper, Pulp industry

1 Introduction

The growing public concern regarding the environmental impact of pollutants from paper and pulp industry is the driving force behind the search for novel bleaching techniques. Chlorinated phenolic compounds and polychlorinated biphenyls produced during conventional pulp bleaching arise from residual lignin present in the pulp. Residual lignin is very dark in colour because it has been extensively oxidised and modified, and its covalent attachment to hemicelluloses and cellulose fibres makes it very difficult to remove [1]. Several studies have been conducted to assess the deleterious effects of effluents from paper and pulp industries. Most of the chloroaromatic compounds released during the pulp bleaching process like chlorophenols, chlorobiphenyls and other chlorolignin derivatives are toxic and accumulate in the biotic and abiotic components of the ecosystem [2–6]. Larsson et al. [7] found a negative impact of Kraft mill effluents on fish populations, even 10 km away from the plant. The use of chemical pulp paper for the manufacturer of baby diapers and food packaging is also of concern since it is sometimes associated with chlorinated compounds including the animal carcinogen, dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) [1]. The three major constituents of wood are cellulose (35–50%) hemicellulose (20–30%) and lignin (20–30%) [8]. Xylans, with a linear backbone of β-1,4-linked xylose residues, form the major group of hemicelluloses. Endoxylanases (1,4-β-D-xylan xylano hydrolases, EC 3.2.1.8) hydrolyse xylan to xylooligosaccharides and xylose residues, while β-xylosidases (1,4-β-D-xylan xylo hydrolases, EC 3.2.1.37) catalyse the release of xylosyl residues by the terminal attack of xylooligosaccharides [9–11]. Xylanases are of great importance to pulp and paper industries since the hydrolysis of xylan facilitates the release of lignin from paper pulp and reduces the use of chlorine as the bleaching agent [1]. The Kraft pulping process at higher temperatures and pH ranges necessitates the search for alkaline thermophilic xylanases useful in the pretreatment of cooked pulp. Jurasek et al. [12] and Viikari et al. [13] were the first to demonstrate that xylanases could be useful in the paper and pulp industry. Studies with fungal xylanases have resulted in the reduction of chlorine consumption; however, there was also an unacceptable drop in viscosity. This drop was presumably due to cellulase contamination of the xylanase preparations. Use of cellulase-free xylanases in pulp and paper industries would permit the production of rayon grade or superior quality dissolving pulp, as cellulase-free xylanases selectively remove hemicellulose components with minimal damage to cellulose [12,14]. The alkaline cooking results in the dissolution of xylan present in plant cell walls, which later reprecipitates on the surface of pulp fibers. According to Kantelinen et al. [15] this layer of xylan over pulp fibers prevents easy removal of lignin by providing a physical barrier to the oxidizing chemicals. Cellulase-free xylanase hydrolyzes this xylan, leaving the cellulose less disrupted. The enzymatic hydrolysis results in pores in the xylan coat and the oxidizing agents can now easily degrade the residual lignin in the paper pulp. Using this technology, superior quality bleached pulp could be produced with minimal consumption of chlorine. Another, less important, effect of enzymatic pretreatment is hydrolysis of the residual, non-dissolved hemicellulose, which acts as a chromophoric xylan because the Kraft or alkaline cooking of monosaccharides leads to the production of chromophores and aromatic compounds [16,17]. The search for microorganisms producing xylanases with the desired characteristics of higher pH and temperature stabilities with minimal cellulase production were initiated elsewhere. Although Bacillus spp. are industrially important sources of hemicellulases, little is known about the relationship between cellulolytic and xylanolytic activities produced by these organisms.

2 General problems associated with fungal xylanases

The 15 leading companies manufacturing xylanase preparations invariably use fungal xylanases, all of which have an optimum pH<5.5 [18]. The optimum pH for xylan hydrolysis is about 5 for most of the fungal xylanases, which are normally stable at pH 2–9. Application of fungi in paper and pulp industries is further impaired by the acidic pH growth requirement of the fungi themselves. However, this is not the case with bacteria (Table 1).

1

Xylanase and cellulase production from microorganisms

Microorganism	Initial pH of the culture medium	Xylanase (IU ml−1)	Cellulase		Reference
			FPase	CMCase
Arthrographis sp. strain F4	5.5	8.95	2.54	13.37	[19]
Aspergillus awamori VTT-D-75028	5.5	12.0	0.1	3.2	[20]
Aspergillus niger KKS	7.0	138	3.9	1.2	[21]
Chaetomium globosum 11-Ch.g./5	4.8	65.3	1.5	2.2	[22]
Fusarium oxysporum VTT-D-80134	5.5	3.7	0.1	0.7	[20]
Irpex lacteus KY 2902	4.5	353	9.8	252	[23]
Pencillium pinophilum NTG III/6	–	27.3	8.14	165	[24]
Phanerochaete chrysosporium	4.5	15–20	–	1.8–2.4	[25]
Piromyces sp. strain E 2	–	7.96	0.009	0.77	[26]
Schizophyllum commune	5–5.5	1244	65.3	5.0	[27]
Schizophyllum radiatum	6	5.7	2.3	2.4	[28]
Sclerotium rolfsii	5	267	1235	10	[29]
Sporotrichium pulverulentum	4.5–5.0	20.4	0.13	9.48	[30]
Talaromyces emersonii CBS 814.70	4.5	56	26.7	2.41	[31]
Thermomyces lanuginosusa	6.0	650–780	0.01	0.01	[32]
Thielavia terrestris ATCC 26917	–	25.5	0.11	8.7	[33]
Tiarosporella phaseolina	5.5	5.09	0.04	1.49	[34]
Trichoderma harzianum	–	450	2.8	66.0	[35]
Trichoderma reesei RUT C-30 ATCC 56765a	5.0	400	–	6.0	[36]
Trichoderma reeseib	4.4	960	0.7	9.6	[37]
Trichoderma reesei PC-3-7	4.0	1.67	–	1.76	[38]
Trichoderma viride	5.5	188.1	0.55	–	[39]
Bacillus circulans	–	400	0.05	1.38	[40]
Bacillus stearothermophilus Strain T-6 a	7.0	2.33	–	0.021	[1,41,42]
Bacillus sp.	9.0	120	–	0.05	[43]
Bacillus sp.	7.2	11.5	–	1.2±0.13	[44]
Cellulomonas sp. ATCC 21399	–	0.301–0.888	–	0.013–0.117	[45]
Cellulomonas flavigena NIAB 441	7.3	16			[46]
Cellulomonas sp. (GS2)	–	9.33	–	0.72	[47]
Micrococcus sp. (DG10)	–	3.33		3.11	[47]
Rhodothermus marinusa	7.1	1.8–4.03	0.05	0.025	[11,48]
Streptomyces roseiscleroticus NRRL-B-11019a,c	7.0	16.2	–	0.21	[49]
SSP-34	6.74	100.72–260	0.24	0.43	[50–52]
SSP-32	6.77	2.38	0.52	1.04	[50–52]
SSP-35	6.63	0.78	1.38	0.88	[50–52]
SSP-20	6.85	1.4	0.66	0.28	[50–52]
SSP-27	6.86	0.43	0.54	0.23	[50–52]
SSP-1	10.3	0.12	0.25	0.34	[50–52]
SSP-2	10.35	0.17	0.44	0.46	[50–52]
SSP-3	10.35	1.26	0.12	0.08	[50–52]
SSP-30	10.58	0.07	0.51	0.10	[50–52]
SSP-29	10.64	0.85	1.2	0.81	[50–52]

^aMicroorganisms reported to be producing ‘virtually’ cellulase-free xylanases.

^bCellulase assay was performed using hydroxyethyl cellulose.

^cCellulase assay carried out using 1% acid swollen cellulose prepared from Solca floc SW 40 wood pulp cellulose (Brown Co. Berlin, N.H.).

The optimal pH for bacterial xylanases is, in general, slightly higher than the pH optima of fungal xylanases [41,53,54]. In most industrial applications, especially in the paper and pulp industries, the low pH optima for growth and production of xylanase activity necessitates additional steps in the subsequent stages, which make fungal xylanases less suitable. Although high levels of xylanase activity are produced by several fungi, the presence of a considerable amount of cellulase activity and lower pH optimum make them less suitable for pulp and paper industries. Gomes et al. [39] reported xylanase activity (188.1 U ml⁻¹, optimum pH 5.2) and FPase activity (0.55 U ml⁻¹, optimum pH 4.5) from Trichoderma viride. Trichoderma reesei has been shown to produce even higher levels of xylanase (approximately 960 IU ml⁻¹ for 180 ml of culture filtrate) but this is associated with a corresponding increase in cellulase production (9.6 IU ml⁻¹) [37]. Like Trichoderma spp., Schizophillum commune is also one of the high xylanase producers with a xylanase activity of 1244 U ml⁻¹, CMCase activity 65.3 U ml⁻¹ and FPase activity 5.0 U ml⁻¹[27]. Among white rot fungi, a potent plant cell wall degrading fungus, Phanerochaete chrysosporium, produced a xylanase activity of 15–20 U ml⁻¹ in the culture medium, but the medium also contains high levels of cellulase activity, measuring about 12% of maximum xylanase activity [25]. Reports of fungal isolates with negligible cellulolytic activity (0.01 U ml⁻¹) like Thermomyces lanuginosus [32,55] are very rare. All other fungal strains produce considerable levels of cellulase activities (Table 1). Another major problem is the reduced xylanase yield when fungi are grown in a fermentor. The shearing forces in the fermentor originating from agitation causes disruption of fungal biomass. This results in a decreased yield of xylanase [56,57]. Even though there are differences in the growth conditions including pH, agitation and aeration, and the optimum conditions needed for xylanase production [18,25,27,32,37,39,40,49–57], there are little differences regarding molecular biology and biochemistry among prokaryotic and fungal xylanases [58].

3 Panorama of bacterial xylanases

A search for microorganisms producing high levels of xylanase activity at alkaline pH resulted in the isolation of several Bacillus spp. Bacillus circulans [40] produced 400 IU ml⁻¹ of xylanase with a pH optimum of 7 and 40% of this activity was retained at pH 9.2. Importantly, the culture supernatant contained low levels of cellulolytic activities, with 1.38 IU ml⁻¹ of endoglucanase (CMCase, EC 3.2.1.4) and 0.05 U ml⁻¹ of cellobiohydrolase (FPase, EC 3.2.1.91). Bacillus stearothermophilus strain T6 produced xylanases; however, the absolute levels were low and there was the associated production of detectable cellulase activity [1,41,42]. Lundgren et al. [42] even tried a Mill trial TCF (total chlorine-free) bleaching of pulp with xylanase from Bacillus stearothermophilus strain T6 which has optimum activity at pH 6.5 [41,42]. Rhodothermus marinus was found to produce thermostable xylanases (approximately 1.8–4.03 IU ml⁻¹), but there were also detectable amounts of thermostable cellulolytic activities [11,48]. Most of the other bacteria which degraded hemicellulosic materials are reported to be potent cellulase producers including Cellulomonas sp. (xylanase, 9.33 U ml⁻¹ and CMCase, 0.94 U ml⁻¹), Micrococcus sp. (xylanase, 3.33 U ml⁻¹ and CMCase, 3.11 U ml⁻¹) [47] and Streptomyces roseiscleroticus NRRL-B-11019 (xylanase, 16.2 IU ml⁻¹ and cellulase, 0.21 IU ml⁻¹) [49]. A detailed description of all other organisms producing cellulases along with xylanases are given in Table 1.

4 Xylanases from newly isolated Bacillus SSP-34

Ten potent xylanase producers obtained after extensive screening [50–52] were tested for cellulolytic activities. A filter paper assay was used to detect the activity of cellobiohydrolase (FPase or cellulose 1,4-β-cellobiosidase, EC 3.2.1.91) and carboxymethylcellulose was used to detect endocellulase activity (CMCase or 1,4-(1,3:1,4)-β-D-glucan-4-glucano hydrolase, EC 3.2.1.4)) [11]. In comparison with the other nine isolates, endocellulase (E.C.) or carboxymethyl cellulase (CMCase) activity of Bacillus SSP-34 was very low, 0.43 IU ml⁻¹ at pH 7 (Table 1) and no activity was observed at pH 9.2. Furthermore, while the cellobiohydrolase (FPase) activity of strain SSP-34 was 0.24 U ml⁻¹ at pH 7, it was undetectable at pH 9.2. The Bacillus SSP-34 xylanases showed 100 times greater activity than the other isolates studied with up to 260 IU ml⁻¹ in optimized medium [50,51] (Table 1). When SSP-34 was grown in a modified Horikoshi medium [59], enzyme production reached maximum when the culture pH shifted to 8.5 [51]. There was an initial increase in reducing sugar concentration due to xylan hydrolysis which was followed by a gradual decrease, possibly due to metabolic utilization by the culture. The optimum pH for xylanases from Bacillus SSP-34 ranged from 6.0–8.0 and the optimum temperature was 50°C. The xylanases were stable up to 2 h at pH values ranging from 4.5–9.0 and at temperatures 30–45°C while at 50°C they were stable for 20 min [50]. The culture SSP-34 attained stationary phase by 36 h and only low amounts of polysaccharases were detected at that time [50,51]; similar results have also been reported for Bacillus sp. [60]. The xylanase activity in the culture supernatant reached maximum at 96–102 h. A similar lack of xylanase activity during log phase has been reported in Aspergillus nidulans [61] and Bacillus sp. strain 41M-1 [62].

5 Trace cellulolytic activities associated with xylanases

The low amounts of cellulolytic activities in the culture supernatant for most of the bacteria may be due either to the presence of traces of cellulases or to the hydrolysis of xylan present in the cellulosic substrates used for cellulose assays [61,62]. It has been observed that commercial celluloses like Solka floc cellulose contain approximately 10% w/w xylose [64,65]. Thus, trace cellulolytic activity may be due to the release of xylose from the commercial cellulose substrates (Fig. 1). There could be other possibilities for the trace amounts of cellulolytic activities by the SSP-34 culture supernatant. Since the cellulose binding domains (CBDs) present in some endoglucanases play an important role in cellulose hydrolysis, negligible cellulolytic activities by some xylanases can also be attributed to the presence of CBDs in these xylanases [66–72]. Although it is quite unnatural for one enzyme to have a high affinity towards a non-specific substrate, CBDs are widely distributed in xylanases [69,73]. Apart from the hydrolytic activity against xylan, some of the microbial xylanases produced from Cellulomonas fimi [73], Clostridium stercorarium [74], Pseudomonas fluorescens [75], Streptomyces halstedii JM8 [76] and Fusarium oxysporum F3 [77] are also reported to contain cellulose binding domains. The reason for the presence of CBDs on plant cell wall hydrolases is possibly due to the use of cellulose as a general receptor for plant cell wall hydrolases [69]. It is the only non-variable structural polysaccharide in the cell wall of all plant species, although there are some marginal changes in the degree of crystallinity of cellulose [58]. Some exo-β-1,4-glycanases (e.g. exoglucanase/xylanase from Cellulomonas fimi) hydrolyse not only cellulose but also xylan since they share a common catalytic mechanism as evidenced by sequence homology of the family of β-1,4-glycanases showing the conservation of Glu-127, the acid/base catalyst in Cellulomonas fimi glycanase [78]. It is conceivable that some of the xylanases in the family retain glycosidase activity which can be attributed to a common catalytic mechanism [78,79]. Thus, it seems likely that the strain Bacillus SSP-34 produces high levels of xylanase with little or no cellulase.

1

Proposed mechanism of reducing sugar release from impure cellulose substrate by xylanase based on the reports of Bailey and Poutanen, 1989 [63] and Royer et al., 1992 [64]. → Point of attack of xylanase on xylose residues present in impure cellulose resulting in the ‘pseudo-cellulase’ activity.

6 Conclusion

The degree of environmental pollution caused by the large scale discharge of chlorine compounds from pulp and paper industry can potentially be remedied by the application of enzyme biotechnology. Cellulase-free xylanases are promising agents which might eventually lead to reduced chlorine usage in the biobleaching of paper pulps. However, care is needed to prevent the unwanted hydrolysis of cellulose on a large scale. The selective hydrolysis of xylan, especially the reprecipitated xylan, without decreasing the viscosity of the pulp facilitates the removal of lignin by mild oxidative agents.

In general it is evident that fungal xylanases with rare exception, are less suitable than bacterial xylanases, especially with respect to cellulases, operational pH and growth requirements. Most of the reported xylanases claimed to be free of cellulases actually have detectable amounts of cellulolytic activities. But the small amounts of cellulolytic activities in cultures of xylanase-producing organisms, including Bacillus SSP-34 [50–52], may be an artefact due to contaminant xylose residues present in commercial cellulose substrates, such as solca floc, used for the assay. There are also reports of endoglycanases, such as cellulase/xylanase cex from Cellulomonas fimi, which can break both xylan and cellulose. Another possibility is that cellulose binding domains present in xylanases exhibit some level of cellulose disruptions.

There are numerous reports of promising xylanase-producing bacteria including Bacillus. Only a few of them have high activities, like Bacillus circulans [40] and Bacillus SSP-34 [50–52]. Enzyme from Bacillus SSP-34 has negligible cellulolytic activities, good temperature and pH optima, and stability suitable for the pulp and paper industries. The negligible amounts of cellulases makes them suitable for producing good quality pulps for Rayon grade textile fibres with minimum damage to cellulose.

Acknowledgements

Authors are grateful to Director, Regional Research Laboratory, Trivandrum, for providing all facilities for the above work and thankful to CSIR for the fellowship given to S.S.