Shou Takashima

Characterization of Mouse Sialyltransferase Genes: Their Evolution and Diversity

Sialic acids are negatively charged acidic sugars, and sialylglycoconjugates often play important roles in various biological phenomena. Sialyltransferases are involved in the synthesis of sialylglycoconjugates, and 20 members of the mammalian sialyltransferase family have been identified to date. These sialyltransferases are grouped into four families according to the carbohydrate linkages they synthesize: β-galactoside α2,3-sialyltransferases (ST3Gal I-VI), β-galactoside α2,6-sialyltransferases (ST6Gal I and II), GalNAc α2,6-sialyltransferases (ST6GalNAc I-VI), and α2,8-sialyltransferases (ST8Sia I-VI). Analysis of the amino acid sequence similarities, substrate specificities, and gene structures of mouse sialyltransferases has revealed that they can be further divided into seven subfamilies. The genomic structural resemblance of members of the same subfamily suggests that they arose from a common ancestral gene through gene duplication events. These multiple sialyltransferase genes are needed for fine control of the expression of sialylglycoconjugates, resulting in a variety of developmental stage- and tissue-specific glycosylation patterns.

Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties

We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host. In this system, the expression of T. reesei cellulase genes were regulated under the control of A. oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer. The production of recombinant cellulases by A. oryzae transformants reached a maximum after 3-4 days of cultivation. In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation. The recombinant cellulases were purified and characterized. The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes. The optimal temperatures and pHs of recombinant cellulases were 50-70 degrees C and 4-5, respectively. Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here.

Cloning, sequencing, and expression of the cellulase genes of Humicola grisea var. thermoidea

We have cloned an endoglucanase (EGI) gene and a cellobiohydrolase (CBHI) gene of Humicola grisea var. thermoidea using a portion of the Trichoderma reesei endoglucanase I gene as a probe, and determined their nucleotide sequences. The deduced amino acid sequence of EGI was 435 amino acids in length and the coding region was interrupted by an intron. The EGI lacks a hinge region and a cellulose-binding domain. The deduced amino acid sequence of CBHI was identical to the H. grisea CBHI previously reported, with the exception of three amino acids. The H. grisea EGI and CBHI show 39.8% and 37.7% identity with T. reesei EGI, respectively. In addition to TATA box and CAAT motifs, putative CREA binding sites were observed in the 5 upstream regions of both genes. The cloned cellulase genes were expressed in Aspergillus oryzae and the gene products were purified. The optimal temperatures of CBHI and EGI were 60 degrees C and 55-60 degrees C, respectively. The optimal pHs of these enzymes were 5.0. CBHI and EGI had distinct substrate specificities: CBHI showed high activity toward Avicel, whereas EGI showed high activity toward carboxymethyl cellulose (CMC).

Comparison of gene structures and enzymatic properties between two endoglucanases from Humicola grisea

We have cloned two endoglucanase genes (egl3 and egl4) from a thermophilic fungus, Humicola grisea. The coding region of the egl3 gene was interrupted by an intron of 56-bp, and the deduced amino acid sequence of the egl3 gene was 305 amino acids in length and showed 98.4% identity with Humicola insolens EGV. The coding region of the egl4 gene was also interrupted by an intron of 173-bp, which contains 34 TTC repeated sequence units, and the deduced amino acid sequence of the egl4 gene was 227 amino acids in length and showed 61.5% identity with H. grisea EGL3. The typical hinge and the cellulose-binding domain were observed in the C-terminal region of EGL3, but they were not observed in EGL4. In the 5 upstream region of both genes, there were a TATA box or its similar sequence, CAAT motifs, and 6-bp sites which are identical or similar to the consensus sequence for binding a catabolite repressor CREA in Aspergillus nidulans. The egl3 and the egl4 genes were expressed in Aspergillus oryzae, and the translation products were purified. The fusion protein, EGL4CBD, which consists of a catalytic domain of EGL4 and the C-terminal region of EGL3, was also constructed and produced by A. oryzae, and purified. These enzymes showed relatively high activity toward carboxymethyl cellulose (CMC) and could not hydrolyze p-nitrophenyl-beta-D-glucoside and p-nitrophenyl-beta-D-cellobioside. The positive effect of substituting the C-terminal region of EGL4 with that of EGL3 was observed in the hydrolysis of CMC.

Correlation between cellulose binding and activity of cellulose-binding domain mutants of Humicola grisea cellobiohydrolase 1

The cellulose‐binding domains (CBDs) of fungal cellulases interact with crystalline cellulose through their hydrophobic flat surface formed by three conserved aromatic amino acid residues. To analyze the functional importance of these residues, we constructed CBD mutants of cellobiohydrolase 1 (CBH1) of the thermophilic fungus Humicola grisea, and examined their cellulose‐binding ability and enzymatic activities. High activity on crystalline cellulose correlated with high cellulose‐binding ability and was dependent on the combination and configuration of the three aromatic residues. Tyrosine works best in the middle of the flat surface, while tryptophan is the best residue in the two outer positions.