An acidic and thermostable carboxymethyl cellulase from the yeast Cryptococcus sp. S-2: purification, characterization and improvement of its recombinant enzyme production by high cell-density fermentation of Pichia pastoris.
The extracellular carboxymethyl cellulase (CSCMCase) from the yeast, Cryptococcus sp. S-2, was produced when grown on cellobiose. It was purified to homogeneity from the supernatant by ultrafiltration, DEAE-5PW anion exchange column and TSK-Gel G3000SW gel filtration. The purified enzyme was monomeric protein with molecular mass of approximately 34kDa. The optimum temperature and pH for the action of the enzyme were at 40-50 degrees C and 3.5, respectively. It was stable at pH range of 5.5-7.5 and retained approximately 50% of its maximum activity after incubating at 90 degrees C for 1h. Moreover, it could able to hydrolyze carboxymethyl cellulose sodium salt higher than insoluble cellulose substrate such as Avicel, SIGMACELL and CM cellulose. Due to its action at acidic pH and moderately stable at high temperature, the gene encoding carboxymethyl cellulase (CSCMCase) was isolated and improved the enzyme yield by high cell-density fermentation of Pichia pastoris. The CSCMCase cDNA contains 1023 nucleotides and encodes a 341-amino acid. It was successfully expressed under the control of alcohol oxidase I promoter using methanol induction of P. pastoris fermentation in a 2L ABLE bioreactor. The production of the recombinant carboxymethyl cellulases was higher than that from Cryptococcus sp. S-2 of 657-fold (2.75 and 4.2 x 10(-3) mg protein L(-1), respectively) indicating that the leader sequence of CSCMCase has been recognized and processed as efficiently by P. pastoris. Furthermore, the recombinant enzyme was purified in two-step of ultrafiltration and hydrophobic interaction chromatography which would be much more convenient for large-scale purification for successful industrial application.
Thongekkaew
Jantaporn
J
Department of Biological Science, Faculty of Science, Ubon-Rajathanee University, Warinchumrab, Ubon-Ratchathani 34190, Thailand. jantaporn_25@yahoo.com
Ikeda
Hiroko
H
Masaki
Kazuo
K
Iefuji
Haruyuki
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003454:Cryptococcus / Q000201:enzymology*; D017931:DNA Primers; D018076:DNA, Complementary; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D002480:Cellulase; C019826:carboxymethylcellulase