The present invention is directed to a modified xylanase, which shows an improved thermostability when compared to the naturally occurring xylanase. Specifically the present invention is directed to a modified xylanase, wherein said xylanase has increased thermostability and wherein said xylanase is modified through either the introduction of a non-native disulfide bridge, introduction of an N-terminal mutation, or both.
Current strategies for the production of paper use a chemical bleaching step, which is essential for the colour and quality of the paper. Chemical bleaching uses chlorine or chlorine dioxide and produces substantial amounts of by-products, which are environmental pollutants. The bleaching process can be enhanced by using an enzymatic pre-treatment with xylanase (Paice and Jurasek, 1984 Journal of Wood Chemistry and Technology, 4(2):187-198), which lowers the chlorine charge needed to affect bleaching, thereby reducing pollutants. In addition there is less bleaching chemical used, which lowers the chemical costs. New bleaching technology using, oxygen or peroxides with xylanase, are also as effective in brightening the pulp.
The step in the process where the enzyme is applied is after a hot alkali treatment, so that the pulp is very basic and hot. Both of these conditions are sub-optimal for xylanase enzymatic activity. Many pulp mills have the capability to acidify the pulp to a pH which is closer to the pH optimum for the enzyme; however, cooling the pulp would be too energy intensive (expensive) to be used in the mill setting. Therefore, the intrinsic thermostability of the enzymes is a critical parameter for their use in the bio-bleaching processes.
It is therefore desirable, to increase the thermostability of xylanases so that they may find wider application in the pre-treatment of kraft pulp.
Xylanase also has uses in non-pulp applications. Xylanases have been reported to be useful in clarifying juice and wine (Zeikus. J. G., Lee, Y.-E., and Saha, B. C. 1991. ACS Symp. Ser. 460:36-51; Beily, P. 1991. ACS Symp. Ser. 460:408-416; Woodward J. 1984. Top Enzyme Ferment. Biotechnol. 8:9-30), extracting coffee, plant oils and starch (McCleary, B. V. 1986. Int. J. Biol. Macromol. 8:349-354; Beily, P. 1991. ACS Symp. Ser. 460:408-416; Woodward J. 1984. Top Enzyme Ferment. Biotechnol. 8:9-30), for the production of food thickeners (Zeikus. J. G., Lee, Y.-E., and Saha, B. C. 1991. ACS Symp. Ser. 460:36-51), altering texture in bakery products (Maat, J., Roza. M., Verbakel, J., Stam, H., Santos da Silva, M. J., Bosse, M., Egmond, M. R., Hagemans, M. L. D., v. Gorcom, R. F. M., Hessing, J. G. M., v.d. Hodel, C.A.M.J.J., and Rotterdam, C. 1992. In Xylans and xylanases. Visser, J., Beldman, G., Kusters-van Someren, M. A. and Voragen, A. G. J., eds. Elsevier Sci pub., Amsterdam. ISBN 0-444-894-772; McCleary, B. V. 1986. Int. J. Biol. Macromol. 8:349-354), and in the washing of super precision devices and semiconductors (Takayuki, I., Shoji, S. U.S. Pat. No. 5,078,802, issue date 92 Jan. 07). Several of these application could benefit from a thermostable xylanase, for example, food processing at elevated temperatures.
A thermostable xylanase from Thermoascus aurantiacus was produced in U.S. Pat. No. 4,966,850 (Yu et al.) from a particular strain of T. aurantiacus, while culturing the strain at high temperature culturing conditions, however this enzyme is not a member of the family G xylanases (Gilkes et al. 1991, Microbiol. Reviews 55(2):303-315), which is the subject of this patent.
Arase et al. (FEBS 316:123-127, 1993) report improvements in thermostability of Bacillus pumilus xylanase through random mutagenesis of the gene by chemical mutagens. Their improvements in thermostability are minor in comparison to that of the present invention. The prior art most stable mutant maintained 40% residual activity after a short period of 20 minutes at 57.degree. C. This mutant had a low specific activity, equivalent to 19% of the wild type B. pumilus xylanase.
Site directed mutagenesis has been used to produce more stable proteins. Disulfide (SS) bonds in proteins restrict the degree of freedom for the unfolded state and thereby stabilize the folded state. The first type of protein stabilization performed by genetic manipulation was the introduction of disulfide bonds. One or two amino acids in the protein are replaced with cysteines; a disulfide bond forms in vivo or in vitro. If the introduced disulfide bond causes no or little tertiary structural change, the cross-links stabilizes the protein. Disulfide bonds have been engineered into T4 lysozyme (T4L) (Perry, L. J. and Wetzel, R. (1984) Science 226, 555-557; Wetzel, R., Perry, L. J. Baase, W. A. and Becktel, W. J. (1988) Proc. Nat. Acad. Sci. USA 85, 401-405), subtilisin (Wells, J. A. and Powers, D. B. (1986) J. Biol. Chem. 261, 6564-6570; Mitchinson, C. and Wells, J. A. (198), dihydrofolate reductase (DHFR) (Villafranca, J. E., Howell, E. E., Oatley, S. J., Xuong, N. and Kraut, J. (1987) Biochemistry 26, 2182-2189), and the Phage .lambda. repressor (C 1) (Sauer, R. T., Hehir, K., Stearman, R. S. et al. (1986) Biochemistry 25, 5992-5998) with stabilization occurring in some cases but not others.
For example, in T4L the introduction of SS bonds showed an increase in thermostability of between 6.degree. and 11.degree. C. based on reversible denaturation at pH 2 (Matsumura et al, 1989, Nature 342:291-293). This data does not show how much activity remains after heating the sample, which in a functional sense is what is important for an industrial enzyme. The RNASE H SS bond mutant is also stabilized by 11.8.degree. C. as measured by reversible thermal denaturation, but has no enzymatic activity (Kanaya et al, 1991, Journal of Biological Chemistry 226(10):6038-6044). In DHFR the artificial SS bond contributes to stability of the protein against chemical denaturation, but does not confer thermostability (Villafranca et al, 1987, Biochemistry 26:2182-2189). A similar situation occurs with subtilisin, where 5 different engineered SS bonds do not confer thermostability (Mitchinson and Wells, 1989, Biochemistry 28:4807-4815).
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