The C₁ enzyme required for the microbial degradation of crystalline cellulose, has been isolated from Cellulase Meiji, a commercial cellulase derived from culture filtrates of Trichoderma viride. Initial purification was accomplished with a substrate affinity column, using Avicel as the crystalline cellulose substrate. The resulting Avicel Water Fraction contained essentially all of the recovered C₁ activity and yielded an 8.2 fold purification.
The C₁ component was then further purified by chromatography on a weak anion exchange column (Bio-Gel DM). Elution with a pH 4.8 sodium citrate buffer resolved three cellulase fractions as well as a noncellulolytic fraction which remained on the column. The first peak of the elution pattern contained 97 per cent of the recovered C<sub>x</sub> activity. The C<sub>x</sub> enzymes are those which hydrolyze derivatized or soluble cellulose such as carboxymethylcellulose, but cannot, by themselves, degrade crystalline cellulose. Recovery of the C<sub>x</sub> activity was 103 per cent and Peak II and Peak III proteins from Bio-Gel DM chromatography had little of this activity. These latter two protein peaks possessed very little ability alone to degrade crystalline cellulose but, when the C<sub>x</sub> (Peak I) and either C₁ protein fraction (Peak II or III) were recombined, the specific activity of the mixture was 16 per cent higher than that of the starting material (Avicel Water Fraction) placed on the column. Analytical disc gel electrophoresis (stacking at pH 5.9 and running at pH 8.5 in the separating gel) separated the C<sub>x</sub> (five bands) and C₁ enzymes (three bands). Peak III contained only the three C₁ protein bands.
The three C₁ proteins (termed A, B and C) were purified from the Avicel Water Fraction by preparative disc gel electrophoresis. These proteins contained low C activity X . and little C₁ activity when assayed alone, but showed synergism with a C<sub>x</sub>-rich fraction (Peak I from Bio-Gel DM) in the degradation of crystalline cellulose.
These proteins formed a single band when they were individually rerun on analytical disc gel electrophoresis. They have been referred to as A, B and C in decreasing order of their mobilities on disc gel. Proteins A, B and C are glycoproteins and contain 10.4, 13.8 and 15.2 per cent carbohydrate, respectively. The molecular weights (estimated by their mobilities in different per cent acrylamide gels) are 34,500, 51,000 and 55,000 for A, B and C respectively.
Electrofocusing in polyacrylamide gels confirmed the conclusion drawn from anion exchange chromatography that these C₁ proteins are very acidic; the isoelectric points of these proteins are about 3.5. Preparative electrofocusing of Avicel Water Fraction proteins in a sucrose density gradient failed to resolve even the C<sub>x</sub> enzymes from the C₁ enzymes.
In conclusion, anion exchange chromatography and disc gel electrophoresis have been found to be useful techniques for purifying the C₁ enzymes. Multiple C₁ enzymes have been obtained which are homogeneous by the criterion of disc gel electrophoresis. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/106294 |
| Date | January 1970 |
| Creators | Lang, John Andrew |
| Contributors | Biochemistry and Nutrition |
| Publisher | Virginia Polytechnic Institute and State University |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | 130, 3 leaves, 22 unnumbered leaves of plates, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 41147564 |
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