Chapter 24
Improvement of Wheat-Straw Pulp Properties with an Alkali-Tolerant Xylanase from Pseudomonas sp. G6-2 Downloaded by NORTH CAROLINA STATE UNIV on October 7, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch024
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Jiachuan Chen , Jianyun Yang , Yinbo Qu , Peiji Gao , Baomin Wang , Qi Zhang , and Quihua Yang 1
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State Key Laboratory of Microbial Technology, Institute of Microbiology, Shandong University, Jinan 250100, China Department of Chemical Engineering, Shandong Institute of Light Industry, Jinan 250100, China 2
A n alkali-tolerant bacterium, which produced an extracellular alkali-stable xylanase, was isolated from soil and identified as a strain of Pseudomonas. The conditions of treating hypochlorite-bleached wheat straw pulps with this xylanase were investigated. Results showed an obvious improvement of brightness, water filterability and fragility of the pulp after enzymatic treatment. Hemicellulases from microorganisms have been investigated because of the possibility of degrading hemicellulose from agricultural residues into xylose and other monosaccharides. However, there is an increasing interest in applying hemicellulases, mainly xylanase, to the pulp bleaching process to reduce the amount of chlorine used in bleaching processes as well as improve the quality of pulp produced (1-4). Wheat straw is a major source of fibers for paper industry in China and many other developing countries. The main problems of straw pulps are poor water filterability, pasting the nets and rolls of paper machine and resultant paper products with low opacity, high fragility and low strength. As a result, straw pulps can only be used to produce paper of low grades. A part of wood pulps must be added in order to produce paper of high grades. The problems are probably related to high content of hemicellulose in the straw pulps. Therefore, straw pulp properties may be improved when the hemicellulose on the surface of fibers is partially removed by xylanase. Alkaline xylanases are preferable for this process because of the alkaline nature of most pulps. In this paper, the isolation of an alkalophilic bacterial strain, Pseudomonas sp. G6-2, the optimization of its alkali-tolerant xylanase producing conditions and the effects of treating hypochlorite-bleached wheat straw pulps with the xylanase on pulp properties were reported. The results showed that the papermaking properties of nonwood-based pulp can be improved distinctly by xylanase treatment. This gives the hope that the high grade paper may be produced by this technique from agricultural residues instead of wood. 0097-6156/96/0655-0308$15.00/0 © 1996 American Chemical Society
In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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This is very important for ecosystem protection in a world view, and is important for economic balance in the forest-inadequate countries such as China.
Isolation and Identification of Alkalophilic Xylanase Producing Bacteria Isolation of the Bacteria. A small amount of soil was suspended in sterilized water and inoculated into an flask containing enriching media (peptone, 0.2%; xylan, 2%; NaCl, 0.5%; pH 9.0). After incubation at 37 C , 120 rpm for 2 days, the culture broth was spread on alkaline xylan agar plates (xylan, 1%; K N 0 , 0.1%; M g S 0 , 0.05%; NaCl, 0.05%; K H P 0 , 0.05%; F e ( S 0 ) , 0.001; pH 9.0) which were then incubated at 37 °C for 2 days. From about 2000 colonies isolated from soil samples, an alkalophilic strain, designated as strain G6-2, was selected for xylanase production accordding to its high extracellular xylanase activity.
Downloaded by NORTH CAROLINA STATE UNIV on October 7, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch024
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Characterization and Identification of the Bacteria. Morphological properties and other taxonomic characteristics of the bacteria were studied according to the methods described in Bergey's Manual of Determinative Bacteriology (5). The strain G6-2 was an aerobic, gram-negative, motile and rod-shaped bacterium with polar flagellation. It grew well under pH 5.0-pH 10.0 and the growth rate was the highest at pH 8.5. The maximum temperature for growth was 42 C , and optimal temperature was about 37 C at pH 8.5. The strain reduced nitrates, hydrolyzed starch and gelatin, utilized D-xylose, DL-arginine and phenylalanine, synthesized fructan from sucrose and demonstrate activities of arginine dihydrolase and oxidase. It is clear that the bacterium belongs to the genus Pseudomonas, and is quite similar to Pseudomonas pseudomallei. e
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Optimization of Xylanase Production Media and Culture Conditions. The medium used for xylanase production was as following unless stated: 4% wheat bran, 0.25% urea, 0.25% peptone, 0.5% NaCl, pH 8.5. Cultivation was carried out at 37°C., with a shaking speed of 130 rpm. Assays of Enzymes. The culture supernatant was used as crude xylanase solution. Activity was assayed by measuring the amount of reducing sugars liberated from xylan by the 3,5-dinitrosalicilic acid method (6). The reaction mixture contained enzyme and 1.0% xylan (from oat spelt, Sigma Chemical Company) in 40 m M sodium barbital-0.4M HC1, pH 7.2, which was treated according to Bailey et al. (7). The reaction was carried out at 50 C for 30 minute unless stated. Cellulase activity was assayed by incubating 0.5 ml crude enzyme solution with 0.5%) CMC-Na, or with 50 ± 5 mg filter paper (Whatman No. 1) in 2 ml 40 m M sodium barbital-0.4M HC1, pH 7.2 for 30 min. After incubation, the reducing sugar released from the reaction was measure by DNS method as described above. e
In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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ENZYMES FOR PULP AND PAPER PROCESSING
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Fig. 1 Effects of concentration of wheat bran on the xylanase production
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Improvement of Wheat-Straw Pulp Properties
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One unit of activity corresponds to the equivalent of 1 μ mol xylose or glucose liberated per minute. Effects of Culture Conditions. Effects of carbon sources, concentrations of wheat bran, nitrogen sources and aeration conditions on the xylanase production of Pseudomonas G6-2 were investigated (Table I, Fig. 1 and Table II). Since the isolate is strictly aerobic, aeration may have a strong effect on xylanase production. In this study, the aeration condition was changed by using a three-baffled flask instead of a normal flask. The results showed that the enzyme production was greatly improved. Under the optimal conditions, Pseudomonas sp. G6-2 can produce more than 300 IU/ml xylanase within 96 hr. Table I Effects of Carbon Sources on the Xylanase Production 1% wheat bran 1% wheat bran 1% wheat bran 1% wheat bran 0.5% glucose 0.5% xylose 0.5% starch 100 Relative activity (%) 0 0 23.2 8.5 Final pH
