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T T Teeri

Publications and source records attributed to T T Teeri.

46 records · Page 3Linked to original sources
PubMed1992

Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.

The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.

Adsorption↗
PubMed1992

Saccharomyces cerevisiae cells secreting an Aspergillus niger beta-galactosidase grow on whey permeate.

We describe the construction of a lactose-utilizing Saccharomyces cerevisiae that expresses the cDNA for a secreted, thermostable beta-galactosidase (lacA) from Aspergillus niger. Yeast cells expressing the lacA gene from the yeast ADH1 promotor on a multicopy plasmid secrete up to 40% of the total beta-galactosidase activity into the growth medium. The secreted product is extensively N-glycosylated, and cells expressing the lacA gene grow on whey permeate (4% w/v lactose) with a doubling time of 1.6 hours. Such strains may offer a solution to the increasing problem of waste whey disposal.

Alcohol Dehydrogenase↗
PubMed1991

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli.

Single-chain antibodies consist of the variable, antigen-binding domains of antibodies joined to a continuous polypeptide by genetically engineered peptide linkers. We have used the flexible interdomain linker region of a fungal cellulase to link together the variable domains of an anti-2-phenyloxazolone IgG1 and show here that the resulting single-chain antibody is efficiently secreted and released to the culture medium of Escherichia coli. The yield of affinity-purified single-chain antibody is 1-2 mg/l of culture medium and its affinity and stability are comparable to those of the corresponding native IgG.

Amino Acid Sequence↗
PubMed1990

Site-directed mutagenesis of the putative catalytic residues of Trichoderma reesei cellobiohydrolase I and endoglucanase I.

Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesei cellobiohydrolase I and endoglucanase I. It is shown that mutagenesis of the residue E126, previously proposed to be the proton donor in CBHI, did not totally inactivate the enzyme while mutagenesis of the residue E127 in the homologous enzyme EGI resulted in complete loss of activity. These results are compared with those obtained in similar studies of other glucanases and the effects on enzymatic activity of hyperglycosylation of the yeast produced cellulases are discussed.

Catalysis↗
PubMed1990

Molecular modelling study of antigen binding to oxazolone-specific antibodies: the Ox1 idiotypic IgG and its mature variant with increased affinity to 2-phenyloxazolone.

Structural models of the variable domains of the murine anti-2-phenyloxazolone IgG (Ox1 idiotype) and its somatic variant, which has higher affinity to the hapten 2-phenyloxazolone, were constructed by computer-aided model building using known structures of highly homologous immunoglobulins as templates. Molecular dynamics simulations were used to dock the hapten between the VL and VH domains. The hapten is predicted to bind to slightly different sites in the two models. Hypotheses concerning the role of a number of preferred mutations in anti-oxazolone variants are presented. These can be tested by mutagenesis and crystallography. In particular, the higher binding affinities of the different antibody variants are shown to correlate with better complementarity of electrostatics. The molecular dynamic simulations also suggest that two mobile tryptophans at the mouth of the pocket may play an important role in the binding of hapten.

Amino Acid Sequence↗
PubMed1988

EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme.

A novel endoglucanase from Trichoderma reesei, EGIII, has been purified and its catalytic properties have been studied. The gene for that enzyme (egl3) and cDNA have been cloned and sequenced. The deduced EGIII protein shows clear sequence homology to a Schizophyllum commune enzyme (M. Yaguchi, personal communication), but is very different from the three other T. reesei cellulases with known structure. Nevertheless, all the four T. reesei cellulases share two common, adjacent sequence domains, which apparently can be removed by proteolysis. These homologous sequences reside at the N termini of EGIII and the cellobiohydrolase CBHII, but at the C termini of EGI and CBHI. Comparison of the fungal cellulase structures has led to re-evaluation of hypotheses concerning the localization of the active sites.

Amino Acid Sequence↗
PubMed1988

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae.

Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose. Cellulolytic strains of the yeast Saccharomyces cerevisiae were constructed by transferring cDNAs coding for these enzymes into yeast on an expression plasmid. These cellulolytic yeasts were able to secrete efficiently the large, heterologous proteins to the culture medium. The recombinant cellulases were observed to be heterogeneous in Mr due, at least partly, to variable N-glycosylation. Recombinant CBHII was able to bind to crystalline cellulose, although slightly less efficiently than the native enzyme. Both of the two recombinant cellulases were able to degrade amorphous cellulose. In a fermenter cultivation, around 100 micrograms/ml of CBHII was secreted into the yeast growth medium.

Cellulose↗
PubMed1987

Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi.

A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to 10(6) clones/micrograms mRNA, is described. Routine synthesis of transcripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma reesei cellulases.

Bacteriophage lambda↗
PubMed1987

Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II.

Fungal cellobiohydrolases are unique enzymes capable of degrading highly ordered crystalline cellulose. We present here the isolation and complete sequence analysis of the chromosomal and cDNA copies of the structural gene (cbh2) coding for one of the major cellobiohydrolases (CBH II) of Trichoderma reesei. We also present data on expression of the cbh2 gene and show that the transcription start points of the cbh2 gene are heterogeneous and are located 32 to 52 bp downstream from a putative TATA box. The derived CBH II protein sequence is 471 amino acids long and the coding region is interrupted by three short introns. Most of the CBH II protein bears no apparent resemblance to CBH I and endoglucanase I. However, a short region of extensive homology is found in all Trichoderma cellulases characterized so far, suggesting that this region is important for cellulose hydrolysis. The implications of this information with regard to the evolution of fungal cellulase genes and the enzymology of cellulose hydrolysis are discussed.

Amino Acid Sequence↗