Characterization and molecular cloning of a novel endoglucanase from Trichoderma sp. C-4.
A fungal strain, C-4, was isolated from etiolated leaves. Based on taxonomic studies, the fungus C-4 can be classified as a strain of Trichoderma species. When strain C-4 was cultured in Mandels' medium at 28 degrees C for 6 days, the enzyme activities detected in the broth corresponded to 8.2 U/ml (28.1 U/mg) carboxymethylcellulase activity. An endoglucanase (EG; F-I-II) was purified from the culture filtrate of the strain through a four-step procedure-chromatography on Sephacryl S-200, DEAE-Sephadex A-50, Con A-Sepharose, and Chromatofocusing on Mono-P (HPLC). The molecular weight of this EG, which was called C4endoII, was determined to be about 51 kDa. The optimum temperature and pH of C4endoII were 50 degrees C and 5.0, respectively. Incubation at 50 degrees C for 24 h did not destroy the cellulose degradation activity. Amino acid sequence analysis revealed the N-terminal sequence of an internal peptide of C4endoII to be Phe-Ala-Gly-Ile-Asn-Ile-Ala-Gly-Phe-Asp-Phe, which is homologous to EGII from Trichoderma reesei. A C4endoII cDNA (C4endoII) was cloned from a cDNA library constructed using the mRNA of the strain cultivated in a cellulase-induction medium. The deduced protein sequence of C4endoII was 417 amino acids long and had a putative signal sequence of 21 amino acids with a predicted cleavage site after Ala-21. A single potential N-glycosylation site was present in the amino acid sequence.
Department of Biological Science, University of Ulsan, 680-749 Ulsan, South Korea.
Ok-Ju
OJ
Sul
Ji-Hyun
JH
Kim
Sun-Ju
SJ
Park
Young-Jun
YJ
Son
Bo-Ryung
BR
Park
Dae Kyun
DK
Chung
Choon-Soo
CS
Jeong
In-Seob
IS
Han
D000595:Amino Acid Sequence; D002480:Cellulase / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013045:Species Specificity; D013696:Temperature; D013997:Time Factors; D014242:Trichoderma / Q000201:enzymology*
D002480:Cellulase