National Research Council of Canada

Wing L Sung

Makoto Yaguchi

Fang Huang

Mark Wood

Kazuhiko Ishikawa

Jeffrey S Tolan

Warren W Wakarchuk

Saran A Narang

Witold A Neugebauer

James F Whitfield

Patrick T Wong

Gordon E Willick

David R Rose

Robert L Campbell

Witold Surewicz

A modified Family 11 xylanase enzyme comprising cysteine residues at positions 99 and 118 to form an intramolecular disulfide bond is provided. The modified xylanase is produced by substitution of an amino acid at position 99, 118 or both positions 99 and 118 with a cysteine to produce the intramolecular disulfide bond. Xylanases of the invention display improved thermophilicity, alkalophilicity or thermostability relative to wild-type xylanases. Such xylanases find use in a variety of applications in industry that require enzyme activities at temperatures and/or pH values above that of the native enzyme.

Modification of xylanases to increase thermophilicity, thermostability and alkalophilicity

A method of extracting fibers from decorticated plant bast skin involves pre-treating decorticated plant bast skin of a fiber plant with an aqueous solution containing trisodium citrate having a pH in a range of about 8-14 at a temperature of about 90&#xb0; C. or less; and subsequently treating recovered fibers with a protease at alkaline pH.

Enzymatic preparation of plant fibers

A method of extracting hemp fibers from decorticated hemp bast skin involves pre-treating the decorticated hemp bast skin with an aqueous solution containing di-sodium citrate, tri-sodium citrate or a mixture thereof having a pH of from about 6-13 at temperature of about 90&#xb0; C. or less; and subsequently treating recovered fiber with a enzyme. Determining the extent of completion of a plant fiber degumming process involves treating degummed fiber with a recombinant pectinase expressed in an organism that produces neither cellulose nor xylanase, to release reducing sugar from any residua pectin on the degummed fiber, and, quantifying the released reducing sugar.

Extraction of hemp fibers

The present invention pertains to modified xylanase enzymes that exhibit increased thermostability and alkalophilicity, when compared with their native counterparts. Several modified xylanases exhibiting these properties are disclosed including xylanases with at least one modification at amino acid position 10, 27, 29, 75, 104, 105, 125, 129, 132, 135, 144, 157, 161, 162 or 165, or a combination thereof. Also included within the present invention is a modified xylanase that comprises at least one substituted amino acid residue and that may be characterized as having a maximum effective temperature (MET) between about 69&#xb0; C. and about 78&#xb0; C., wherein the modified xylanase is a Family 11 xylanase obtained from a Trichoderma sp. The present invention also includes a modified Family 11 xylanase obtained from a Trichoderma sp. characterized as having a maximum effective pH (MEP) between 5.8 and about 7.6. Modified xylanases characterized as having a MET between about 69&#xb0; C. and about 78&#xb0; C. and a MET between about 5.8 and 7.6 are also disclosed.

Modified xylanases exhibiting increased thermophilicity and alkalophilicity

The present invention provides a xylanase, or a modified xylanase enzyme comprising at least one substituted amino acid residue at a position selected from the group consisting of amino acid 11, 116, 118, 144 and 161, the position determined from sequence alignment of the modified xylanase withxylanase II amino acid sequence. The xylanases described herein exhibit improved thermophilicity, alkalophilicity, expression efficiency, or a combination thereof, in comparison to a corresponding native xylanase.

Xylanases with enhanced thermophilicity and alkalophilicity

The present invention is directed to thermostable xylanase enzymes are suitable for feed pelleting applications. The novel xylanase enzymes comprise at least 40% of their optimal activity from a pH range from about pH 3.5 to about pH 6.0, and from about 40 to about 60&#xb0; C., and exhibit at least 30% of their optimal activity after a pre-incubation step for 30 minutes at 70&#xb0; C. in the presence of 40% glycerol. Also disclosed are modified xylanase molecules comprising either a basic amino acid at position 162 (TrX numbering), or its equivalent position in other xylanase molecules, at least one disulfide bridge, or a combination thereof. The thermostable xylanase molecules of the present invention have a physiological temperature and pH optima and are useful as animal feeds additives since they can withstand the heat associated with feed sterilization and pellet formation, yet they exhibit optimal activity within an animal to aid in breakdown of ingested feed.

Thermostable xylanases

Producing a xylanase enzyme of superior performance in the bleaching of pulp. More specifically, a modified xylanase of Family 11 that shows improved thermophilicity, alkalophilicity, and thermostability as compared to the natural xylanase. The modified xylanases contain any of three types of modifications: (1) changing amino acids 10, 27, and 29 of Trichoderma reesei xylanase II or the corresponding amino acids of another Family 11 xylanase, where these amino acids are changed to histidine, methionine, and leucine, respectively; (2) substitution of amino acids in the N-terminal region with amino acids from another xylanase enzyme. In a preferred embodiment, substitution of the natural Bacillus circulans or Trichoderma reesei xylanase with a short sequence of amino acids from Thermomonospora fusca xylanase yielded chimeric xylanases with higher thermophilicity and alkalophilicity; (3) an extension upstream of the N-terminus of up to 10 amino acids. In a preferred embodiment, extension of the N-terminus of the xylanase with the tripeptide glycine-arginine-arginine improved its performance.

Modification of xylanase to improve thermophilicity, alkophilicity and

Modification of xylanase to improve thermophilicity, alkalophilicity and

Certain analogues of human parathyroid hormone (hPTH) have been found to be effective for the treatment of osteoporosis, while showing decreased side effects. Analogues showing this effect include all sequences from hPTH-(1-28)-NH.sub.2 to hPTH-(1-31)-NH.sub.2 and all sequences from [Leu.sup.27 ]-hPTH-(1-28)-NH.sub.2 to [Leu.sup.27 ]-hPTH-(1-33)-NH.sub.2. Also included are cyclic analogues cyclo(Lys.sup.26 -Asp.sup.30) [Leu.sup.27 ]-hPTH-(1-34)NH.sub.2 and cyclo (Lys.sup.27 -Asp.sup.30 )-hPTH-(1-34)-NH.sub.2. Analogues in the form of the carboxyl terminal amide are particularly effective.

Parathyroid hormone analogues for the treatment of osteoporosis

The thermostability of the 20,396 dalton Bacillus circulans xylanase was increased by site-directed mutagenesis. The thermostability was conferred by the presence of non-native disulfide bridges, and selected N-terminal mutations. The introduction of these non-native disulfide bridges was accomplished by the examination of the three-dimensional structure of the enzyme, and choosing sites where a favorable geometry for a bridge existed. The N-terminal mutations were constructed on the basis of primary sequence comparison with other family G xylanases. The mutant proteins were examined for their ability to retain enzymatic activity after heating as an indication of increased thermostability. These thermotolerant variants are useful as an alternative to chemical bleaching of Kraft pulp in a pre-bleaching step (bio-bleaching). The pre-bleaching involves temperatures higher than that normally used for these enzymes and accordingly these thermotolerant variants can be advantageously used at this step. Thermotolerant xylanases are also of use in the food processing industry.

Construction of thermostable mutants of a low molecular mass xylanase

A process is described for monitoring the production of recombinant proteins. It is based on the discovery that infrared spectra of E. coli strains and transformants which overproduce recombinant proteins can be measured as a function of pressure, and a pressure-induced distinct shifting pattern can be observed in specific spectral parameters of transformants. In particular, the difference in the pressure-induced shift of the amide III band, between the E. coli cells and the transformants over-producing the recombinant proteins provides an efficient and non-intrusive technique for on-line monitoring of the production of recombinant proteins in E. coli.

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Gloucester, Canada

Wing L Sung