National Research Council of Canada

Her Majesty the Queen in Right of Canada, As Represented by the National

Makoto Yaguchi

Wing L Sung

Kazuhiko Ishikawa

Paul R Carey

Timothy Lessard

Warren W Wakarchuk

Marianne Pusztai-Carey

Marianne Pusztai

David R Rose

Robert L Campbell

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

A process for identifying proteinaceous protoxins expressed by Bacillus thuringiensis genes is disclosed. According to the process, daughter toxins are first generated by subjecting a protoxin-containing material, such as parasporal crystals of Bacillus thuringiensis, to limited proteolysis with a proteolytic enzyme in an aqueous suspension having a pH above 9.5. The daughter toxins are then separated by high performance anion-exchange liquid chromatography at a constant pH in excess of 10 in an increasing gradient of a salt, preferably sodium chloride. The gradient conditions, which are specific for the column used, are achieved by employing a series of buffers having increasing concentration of the salt and introduced at a predetermined time and rate. The procedure provides a chromatogram showing clearly identifiable peaks of toxins and permits therefore the qualitative and quantitative characterization of the original mixture and isolation of the individual toxins. By this it provides a way of screening and testing new Bacillus thuringiensis isolates, both single--and multigene, and monitoring the level of expression of known genes from a known strain. The digestion and isolation conditions permit the production of the toxins in a biologically fully active state.

Identification, quantitation and purification of insecticidal proteins

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 for identifying proteinaceous protoxins expressed by Bacillus thuringiensis genes is disclosed. According to the process, daughter toxins are first generated by subjecting a protoxin-containing material, such as parasporal crystals of Bacillus thuringiensis, to limited proteolysis with a proteolytic enzyme in an aqueous suspension having a pH above 9.5. The daughter toxins are then separated by high performance anion-exchange liquid chromatography at a constant pH in excess of 10 in an increasing gradient of a salt, preferably sodium chloride. The gradient conditions, which are specific for the column used, are achieved by employing a series of buffers having increasing concentration of the salt and introduced at a predetermined time and rate. The procedure provides a chromatogram showing clearly identifiable peaks of toxins and permits therefore the qualitative and quantitative characterization of the original mixture and isolation of the individual toxins. By this it provides means of screening and testing new Bacillus thuringiensis isolates, both single - and multigene, and monitoring the level of expression of known genes from a known strain. The digestion and isolation conditions permit the production of the toxins in a biologically fully active state.

Isolation, quantitation and purification of insecticidal proteins from

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Ottawa, Canada

Makoto Yaguchi