Proceedings of the 6th International Conference on Biotechnology in the Pulp and Paper Industry: Advances in Applied and Fundamental Research Mechanism of Streptomyces xylanases in prebleaching of kraft pulp the G. Elegir l *, M. Sykes 2, and T.W. Jeffries 2 1 Stazione Sperimentale per la Cellulosa Carta e Fibre Tessili Vegetali ed Artificial, Piazza L. da Vinci 26,20133 Milano, Italy, and 2 Institute for Microbial and Biochemical Technology, USDA, Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI 53705, USA * Corresponding author SUMMARY The action of endoxylanases purified from Streptomyces B-12-2 was compared in the pretreatment of hardwood and softwood kraft pulp. Xyl 3 gave the highest brightness following a standard bleaching sequence (C/DEDED) with hardwood, whereas xyl 1b was the most effective with softwood pulp. Xyl 1a and xyl 3 exerted a slight synergism in the hydrolysis of hardwood pulp xylan without increasing the final brightness. In contrast, the combination of the two enzymes substantially enhanced the brightness of softwood pulp. The degree of polymerization (DP) of the residual xylan decreased only slightly after 3 h treatment with purified xylanases. INTRODUCTION Pretreatment with xylanases has been shown to decrease chemicals needed for bleaching kraft pulp (1). However, the mechanism of xylanase-aided bleaching is not yet fully understood. One hypothesis is that xylanases degrade the redeposited xylan on the surface of the fibers thereby opening up pulp structure to access by bleaching chemicals (2). Xylanases might also release chromophores associated with lignin-carbohydrate complexes (3). The structure of such chromophores or possible linkages with xylan are not completely understood (4.5). It is also unclear whether or not different xylanases produced by xylanolytic organisms can affect bleaching differently (6). Streptomyces B- 12-2 produces two different classes of xylanases with remarkably different physiochemical and kinetic properties (7). They can be classified according to their molecular mass (Mr) and pi as type 1 [low-mr/ basic pi (xyl 1 and xyl 1b)] and type 2 [high Mr/acidic pi (xyl 2, xyl 3 and xyl 4)]. In this work the action of the three major xylanases 87
(xyl 1a, xyl 1b and xyl 3) from Streptomyces B- 12-2 was compared in the treatment of hardwood and softwood kraft pulp. MATERIALS AND METHODS Pulps. Hardwood kraft pulp (aspen) with kappa number of 9 and softwood kraft pulp (mixture of spruce, red pine and jack pine) with kappa number of 26 were obtained from Potlatch. Corp. (Cloquet Mn). The acid hydrolysate (corrected to 100%) composition of the hardwood pulp was: glucose 80.1%, xylose 19.0%, mannose 0.3% and arabinose 0.03% whereas for softwood it was: glucose 79.3%, xylose 9.9%, mannose 5.9%, arabinose 0.8% and galactose 0.5%. Xylanase treatment. Xylanase prebleaching was carried out at 10% consistency (w/v) in 50 mm potassium phosphate buffer at ph 7.0 and 60 C for 3 h Enzyme dosage was 3 IU/g oven-dried pulp. After the treatment the pulps were washed with distilled water. Chlorine bleaching. Pulp samples and controls were bleached for 30 min at 45 C and 3.5% consistency using a conventional bleaching sequence (C70/D30)EDED. The chlorine charge was 0.18 multiple or 1.62 active chlorine on oven-dried hardwood pulp and 0.2 multiple, 5.2 active chlorine for softwood pulp. The first extraction (E1) was performed at 10% consistency using 1% NaOH for 90 min at 65 C, whereas the second extraction (E2) was carried out for 1h at 60 C using 1% NaOH and 12% consistency. Pulp was bleached with 0.5% chlorine dioxide for 3 h at 65 C and 12% consistency (D1) and for 2 h under the same conditions (D2). Brighness was measured at 457 nm on handsheets using a Technidyne brightness meter (Tappi standard method T452 om 92). Chemical analyses. The carbohydrate composition of pulp samples was determined by HPLC after acid hydrolysis (8) using a Carbopac PA1 column equipped with a Dionex pulsed amperometric detector. Carbohydrates released by enzyme treatment were analyzed both as reducing sugars (arsenomolybdate method) (9) and total sugars (phenol/h2so4) (10). Chromophore release was determined spectrophotometically as previously reported (3). Extraction of xylan and DP determination. Pulps after enzymatic treatment (10 g) were delignified with NaClO2 according to Paice et al.(l1). Xylan was then extracted by a two step extraction procedure using sodium hydroxide and barium hydroxide as a completing agent to block mannan solubilization (12). Intrinsic viscosity data in 1 M cupriethylenediamine were used to calculate the degree of polymerization (DP) of xylan according to the following equation (13): DP xylan= 208 [intrinsic viscosity (dl g -1 )] 1.19 RESULTS AND DISCUSSION Treatment of pulps. Release of reducing sugars and the amount of solubilized xylan by treatment with purified xylanases from Streptomyces B- 12-2 were very similar for hardwood and softwood pulps. The amount of solubilized pulp xylan was calculated by HPLC after 88
acid hydrolysis of the oligosaccharides liberated during enzymatic treatment. Xylose and arabinose were the only sugars detected. Pulp xylan solubilization was also measured by total sugar release (H2SO4/phenol method); results were consistent with the HPLC data Xyl la produced 11.3 mg of sugars per g of softwood pulp, which corresponded to 11.4% of the initial amount of softwood xylan (Table 1). In contrast, xyl 1b produced 15.8 mg of sugars compared with 17.3 mg of sugars produced by xyl 3. These values corresponded to 16.0% and 17.5% respectively of the initial xylan in the softwood pulp. Xyl 1b was the most efficient enzyme in enhancing the brightness of softwood pulp despite the amount of solubilized xylan was smaller than that obtained with xyl 3. Furthermore, the combination of xyl 1a and xyl 3 increased substantially the brightness of softwood pulp although the hydrolysis yield was slightly smaller than when xyl 3 was used alone. With respect to hardwood pulp, xyl 3 solubilized a greater amount of xylan in comparison to xyl 1b and xyl 1a (Table 2). This greater solubilization corresponded to a higher brightness only after the D- 1 stage. In the case of hardwood pulp the combination of xyl 1a and xyl 3 showed a synergistic effect in solubilizing pulp xylan. However, the final brightness was not enhanced Chromophore release (as measured at 237 nm) was generally greater in the case of hardwood pulp (Tables 1 and 2). Xyl 3 and xyl 1b liberated a greater amount of chromophores both from hardwood and softwood pulp than xyl 1a did. An additive effect on chromophore release was observed when a combination of xyl 1a and xyl 3 or xyl 1b and xyl 3 was used. In most cases a greater chromophore release corresponded to higher brightness. These results suggest that the hydrolysis yield of pulp xylan required to obtain high brightness values is limited and xylan solubilization is probably not the only mechanism involved. After enzymatic prebleaching (3 h) the DP of the residual xylan did not decrease significantly. Xyl 1a and xyl 1b caused a drop by only 10% of softwood xylan (Table 1) whereas no drop was detected when xylanases were applied to hardwood pulp (Table 2). The absence of an extensive depolymerization of the residual xylan along with the increase in brightness indicates that only the hydrolysis of "specific" xylans is necessary to achieve a target brightness. 89
Table 2. Effect of purified endoxylanases from Streptomyces B- 12-2 on hardwood kraft pulp Hydrolysis Products. The HPLC analysis of the hydrolysis products from kraft pulps showed that xyl 3 liberated mostly xylobiose plus a significant amount of xylose and xylotriose (Table 3). On the contrary, xylose was not detected among hydrolysis products released by xyl 1a or xyl 1b. The major product liberated by the latter enzymes was xylotriose. Neither enzyme formed significant amounts of xylopentaose but significant amounts of oligosaccharides with DP>5 were produced. Xyl 1a and xyl 1b formed a greater amount of oligosaccharides with DP>5 from hardwood pulp than xyl 3. The hydrolysis pattern using a combination of xyl 1a and xyl 3 was very similar to that obtained using xyl 3 alone. Therefore, it is not possible to conclude that the synergistic action observed treating softwood pulp is due to a different action pattern with respect to carbohydrate products. Table 3. Hydrolysis products from reaction of purfied Streptomyces B- 12-2 xylanases with kraft pulp at ph 7.0. Substrate Enzyme Total recovered sugars (mole %) Xl X2 X3 X4 >X5 90
The hydrolysis products from hardwood kraft pulp obtained by the action of xyl 1a and xyl 3 depended upon the ph of the reaction At ph 7.0, the two enzymes showed a different oligosaccharides pattern but at ph 9.0 their patterns were almost identical (Table 3) Chromophore release. Xyl 1b was used to verify the correlation between chromophore release and brightness values. Figure 1 shows the behavior of brightness as a function of chromophore release when different enzyme dosages (0.5, 1.0, 2.0 and 4.0 IU/g oven-dried pulp) were applied to hardwood kraft pulp. Brightness was measured at the D1-stage. Given the difficulty in measuring pulp bleachability, chromophore release might be an easy way to monitor enzyme efficacy. Fig. 1. Correlation between brightness and chromophore release at different enzyme dosages. ACKNOWLEDGEMENTS The authors thank Mark Davis of the Analytical Chemistry and Microscopy Laboratory at the Forest Products Laboratory for his assistance in analyzing action patterns by HPLC. This research was supported by USDA competitive grant no. 92-37103-7984 from the program on Improved Utilization of Wood and Wood Fiber. and by Italian National Council of Research (CNR). 91
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BIOTECHNOLOGY IN THE PULP AND PAPER INDUSTRY R ECENT A DVANCES IN A PPLIED AND F UNDAMENTAL R ESEARCH Proceedings of the Sixth International Conference on Biotechnology in the Pulp and Paper Industry E DITED BY E WALD S REBOTNIK University of Technology Vienna, Austria K URT M ESSNER University of Technology Vienna, Austria Facultas-Universitätsverlag Vienna, Austria