ABSTRACT
Endoglucanase I (EGI) secreted from Trichoderma viride HK-75 has a unique transglycosylation activity. The genomic and cDNA clones encoding EGI (egl1) of T. viride HK-75 were isolated and characterized. The coding region of egl1, composed of 1392 bp, was found to encode a polypeptide of 464 amino acids that has extensive similarity (93.8%) with EGI of T. reesei. Expression of the egl1 gene in E. coli as a fusion protein (with N-terminal thioredoxin and C-terminal histidine tag) led to a large production of a nonglycosylated protein of 62.5 kDa. However, it formed an insoluble inclusion body. Upon denaturation with 8 M urea followed by dialysis and successive purification, the enzymatically active recombinant EGI (rEGI) was obtained at a level as high as 18.3 mg/l of 1,000 ml of culture. The rEGI had 67.8% activity for carboxymethyl cellulose (CMC), compared to native EGI (nEGI). The optimum pH and optimum temperature of rEGI were lower than those of nEGI by 0.5 and 5 degrees C, respectively. The rEGI also had narrower CMCase ranges than nEGI in pH and temperature stabilities. However, the catalytic and transglycosylation abilities against cellotriose of rEGI were comparable to those of nEGI. These results suggest that the glycosylation is important for the stabilities of EGI but not critical for the essential enzymatic capacity.
Subject(s)
Cellulase/metabolism , Gene Expression Regulation, Fungal , Trichoderma/enzymology , Amino Acid Sequence , Base Sequence , Cellulase/chemistry , Cellulase/genetics , Cellulase/isolation & purification , Cellulose 1,4-beta-Cellobiosidase , Chromatography, Thin Layer , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Escherichia coli/metabolism , Gene Expression Regulation, Enzymologic , Glycosylation , Hydrogen-Ion Concentration , Inclusion Bodies/enzymology , Models, Genetic , Molecular Sequence Data , Plasmids/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Temperature , Trichoderma/geneticsABSTRACT
Saccharomyces cerevisiae wy2 exhibits a novel life cycle, with delayed homothallism caused by a defective HO gene. In this strain, gradual diploidization occurs during successive subcultures. Three amino acids of wy2 HO were different from those of wild-type (wt) HO, which included a nonsense mutation (TAG) from Trp-292 and two amino acid changes of His-475 to Leu and Glu-530 to Lys. The ho gene of heterothallic strain CG379 was also sequenced in this study. Four amino acids of ho were different from those of HO. Among different amino acids in wy2 HO and ho, the alteration of His-475 to Leu was common between them. His-475 in HO was previously suggested to be involved in the DNA binding. We constructed a variety of chimeric HO genes by exchanging the corresponding restriction fragments generated from the wt HO, wy2 HO and ho genes. These results and the site-directed mutagenesis studies allowed us to draw the following conclusions: (a) Gly-223 is essential for HO activity; (b) mutation of His-475 to Leu significantly reduces the HO activity; (c) amber mutation (TAG) in wy2 HO car be suppressed inefficiently.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/genetics , Genes, Fungal/physiology , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , DNA Mutational Analysis , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , Gene Expression Regulation, Fungal , Genes, Fungal/genetics , Mutagenesis, Site-Directed , Mutation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins , Structure-Activity Relationship , Time FactorsABSTRACT
The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was heterologously expressed in mannosyltransferase mutants of Saccharomyces cerevisiae, in which the pmt1 gene and the kre2 gene were disrupted. The GAI enzymes expressed in these yeast mutant cells exhibited a lesser extent of O-glycosylation. Secretion of GAI expressed in the pmt1-disruptant and in the kre2-disruptant, respectively, was almost the same as that of GAI expressed in wild type (wt) strains. The number of O-linked mannose in GAI from wt yeast strain ranged in size from one (Man1) to five (Man5). On the other hand, the O-linked oligosaccharides of GAI from the pmt1-disruptant ranged in size from Man1 to Man4. Man5 was not detected and Man2-Man4 were reduced in proportion to the reduction of Man1. The O-linked oligosaccharides of GAI from the kre2-disruptant ranged from Man1 to Man4, and the molar amount of Man4 was reduced to 27.3%, compared to that of the wt strain. The hydrolyzing abilities for soluble starch and the adsorbing abilities on raw starch were comparable between both disruptants and wt strains. However, the digesting abilities for raw starch of the disruptants were decreased to 70% of those of the wt strains. Stabilities of GAI of the disruptants were reduced toward extreme pH and high temperature, compared to those of the wt strains. These results demonstrate that the O-linked oligosaccharides of GAI are responsible for the enzyme stability and activity toward insoluble substrates but not for secretion.
Subject(s)
Aspergillus/enzymology , Glucan 1,4-alpha-Glucosidase/metabolism , Oligosaccharides/metabolism , Aspergillus/metabolism , Cloning, Molecular , Enzyme Stability , Glucan 1,4-alpha-Glucosidase/chemistry , Glucan 1,4-alpha-Glucosidase/genetics , Glycosylation , Mannosyltransferases/genetics , Saccharomyces cerevisiae/genetics , Serine/metabolism , Threonine/metabolism , TransfectionABSTRACT
A modified glucoamylase gene (glaA) with an extra Thr- and Ser-rich Gp-I domain (T. Semimaru, M. Goto, K. Furukawa, and S. Hayashida, Appl. Environ. Microbiol. 61:2885-2890, 1995) was introduced into a mutant parental host, Aspergillus awamori var. kawachi, in which the original glaA gene had been completely deleted and replaced with the hygromycin phosphotransferase gene. The modified glaA was successfully expressed and secreted. The modified glucoamylase possessed higher digestibility of raw corn starch and higher stabilities in response to heat and extreme pH.
Subject(s)
Aspergillus/genetics , Aspergillus/metabolism , Glucan 1,4-alpha-Glucosidase/genetics , Glucan 1,4-alpha-Glucosidase/metabolism , Chromosome Mapping , Cloning, Molecular , Gene Expression Regulation, Fungal , Mutagenesis , Phosphotransferases (Alcohol Group Acceptor)/genetics , Plasmids/genetics , Recombination, Genetic , Sequence Deletion , Serine/genetics , Threonine/genetics , Zea mays/metabolismABSTRACT
Saccharomyces cerevisiae wy2 segregated to 2 mater and 2 non-mater in relation to mating ability. The non-mater segregants behaved as the normal type of homothallic life cycle. On the other hand, the mater segregants gradually formed spores during successive subcultures, indicating that slow interconversion of mating-type happened to occur during subcultures. We termed this novel type of life cycle "delayed homothallism". The results of complementation tests with standard ho strains and introduction of a wild type HO gene showed that delayed homothallism was caused by a defective HO gene. The amino acid sequence deduced from the nucleotide sequence of the wy2 HO gene differed from the wild type HO gene in three amino acid residues. In the carboxy terminus of HO protein, there are three repeats of cysteine and histidine that are postulated to play a role in binding of HO protein to DNA. However, wy2 HO protein lacked one such repeat at residues Cys470-His475, where His was replaced by Leu.
