Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1994-07-15
2001-01-30
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S201000, C435S220000, C435S222000
Reexamination Certificate
active
06180382
ABSTRACT:
The present invention relates to heterologous enzymes produced by recombinant strains of
Bacillus licheniformis
and, in particular, to a xylanase derived from
Bacillus pumilus
PRL B12 which is efficient for use in the biobleaching of lignocellulosic pulp, expression vectors and recombinant
Bacillus licheniformis
hosts for the expression of the xylanase, and the use of the xylanase in the biobleaching of lignocellulosic pulp.
A common objective in the manufacture of products (such as paper products) from lignocellulosic pulps is to provide a pulp from which the product produced has as high a final brightness as is possible. A major factor that limits such final brightness is the quantity of lignin present in the pulp.
Lignin in such lignocellulosic pulps is mostly bound up-with hemicellulose in a lignocellulosic complex. A major interface between the lignin and the remainder of the carbohydrates of the lignocellulosic complex is formed by xylan (1,4-&bgr;-D-xylan) which is bonded thereto. To remove the lignin from the pulp, these bonds must first be broken.
Conventionally, lignocellulosic pulps, such as wood pulp, are delignified by being chemically-cooked. While being extremely useful for its purposes, (up to ninety-five per cent of the lignin present may be removed therefrom by such chemical-cooking), chemical-cooking cannot, by itself, reach higher levels of delignification without severly attacking the carbohydrates in the pulp. Thus, cooking must be stopped before the loss of carbohydrates becomes too important and before further delignification has occurred. This remaining lignin imparts a brown color to the cellulosic fibers in the pulp, thereby preventing the product fabricated therefrom from having as high a final brightness as possible.
To obtain further delignification of the pulp after chemical-cooking, various “bleaching” sequences are used. Conventionally, such bleaching sequences include “Classical” (or conventional) bleaching sequences and Elemental Chlorine Free bleaching sequences. These bleaching sequences include a delignification stage followed by a series of bleaching stages. In the delignification stage, the pulp is first subjected to a chlorine and/or chlorine dioxide treatment step, usually followed by an alkaline extraction step.
While being effective for facilitating the removal (“delignification”) of substantial quantities of lignin from the chemically-cooked pulp, treatment of the pulp nonetheless suffers from drawbacks. Perhaps the most troublesome of these drawbacks (especially in the case of classical sequences) is that their use results in discharges of chlorinated compounds which have been linked to the formation of absorbable organic halides (AOX). AOX compounds have been associated with environmental toxicity. It is believed that the AOX concentration in effluents is directly linked to the quantity of chlorine and chlorine dioxide compounds that are used in the process. Thus, AOX levels have become commonly used as a standard in the industry for determining the content of chlorinated organics in the bleaching plant's effluents. As such, industries, such as the wood pulp and paper industry, which make products from such pulp have come under increasing pressure and regulation to reduce both AOX production and the consumption of both chlorine and chlorine dioxide used during bleaching.
To reduce or eliminate AOX production and the consumption of Cl
2
and ClO
2
while still achieving acceptable levels of deligninification, it has been proposed to utilize various hemicellulolytic enzymes to facilitate lignocellulosic pulp delignification (in a process commonly referred to as “biobleaching”). In particular, it has been proposed to use of a wide variety of diverse xylanases which are secreted by a range of various fungi and bacteria, including bacteria of the genus Bacillus, for treating the pulp. However, these attempts have had varying degrees of success depending upon the precise characteristics of the xylanase which has been employed therefor. To be successfully employed in commercial biobleaching applications, a xylanase should be efficient over the pH range naturally possessed by the pulp when the xylanase is utilized during biobleaching. Furthermore, the xylanase should be devoid of any residual cellulase activity.
In biobleaching, xylanase may be added to the pulp after it has exited the chemical-cooker, but before it has been chemically-treated. At that point, the pulp typically has a pH in the range of about 7.0 to 9.5. The identification and utilization of a xylanase which is efficient over this alkaline pH range would greatly reduce the control that must be maintained over that aspect of the process conditions as well as reduce the quantity of chlorine and reactive oxidants needed to chemically-treat the chemically-cooked pulp.
It has been known for some years that microorganisms of the species
Bacillus pumilus
extracellularly secrete xylanases. Indeed, as early as 1960 it was disclosed that the culture media of
B. pumilus
contains xylanases which make this culture broth useful for food processing applications (see Canadian Letters Patent No. 603,953 and U.S. Pat. No. 2,821,501). However, nowhere do those patents either disclose, teach or suggest the isolation and/or purification of the enzymes (including the xylanases) from the culture broth into which they are secreted. Furthermore, it was reported therein that in pH's above 8, those xylanases will generally be inactivated.
To the best of our knowledge, the xylanase of only two strains of
Bacillus pumilus
have ever been isolated and/or purified:
Bacillus pumilus
IPO and
Bacillus pumilus
DSM 6124. However, there is nothing to indicate whether the xylanase from
B. pumilus
PRL B12 would have potential usefulness in biobleaching applications.
Only one xylanase from any strain of
B. pumilus
has ever been proposed for use in biobleaching, and that xylanase was from a specially designed mutant—
B. pumilus
DSM 6124. The isolation and purification of the xylanase secreted from
B. pumilus
DSM 6124, as well as its use in biobleaching has been disclosed in International Publication No. WO 91/02839 and International Publication No. WO 92/03540. However, as reported therein, the xylanase of strain DSM 6124 has an optimum pH of only 5-7 and appears to be of limited efficiency for the delignification of pulps having pH's of up to only about 8.5.
The presence, in the culture broth, of extracellular xylanases secreted by
Bacillus pumilus
PRL B12 and
B. pumilus
PRL B92 has also long been known, being reported as early as 1954 (1). It was further reported therein that, when in the milieu of the culture broth, the xylanase of
B. pumilus
PRL B12 is stable up to pH 11. However, nowhere does that reference either disclose, teach or suggest the isolation and/or purification of the xylanase from the culture broth into which it is secreted, nor is there any indication whatsoever in (1) as to what physical characteristics such an isolated and/or purified xylanase would have when not in the milieu of the culture broth. Furthermore, those xylanases have never been proposed for use in the biobleaching of chemical pulp.
It is known that, when in the culture broth into which they are secreted, xylanases are often contaminated by other enzymes. This can make the isolation and purification of the xylanase difficult and costly. This is particularly significant in that there is no information in (1) as to whether or not those xylanases ever were, or ever could be, isolated and/or purified, nor is there any information in (1) as to how difficult or successful one could expect such a task to be.
Furthermore, it is known that, in the culture broth, the contamination of the xylanase by other enzymes can effect the apparent physical characteristics of the xylanase, including its efficiency over different pH ranges. This aspect is particularly notable in light of the fact that xylanases exhibit a wide variety of characteristics, even when secreted from extensively homologous microor
Amory Antoine
Andre Christophe
De Buyl Eric
Detroz Ren{acute over (e)}
Lahaye Andr{acute over (e)}e
Achutamurthy Ponnathapu
Rao Manjunath
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