We previously cloned three endoglucanase genes, rce1, rce2, and rce3, that were isolated from Rhizopus oryzae as the first cellulase genes from a member of the subdivision Zygomycota. In this study, two cDNAs homologous to the rce1 gene, designated the mce1 and mce2 cDNAs, were cloned from Mucor circinelloides, a member of the subdivision Zygomycota. The mce1 cDNA encoded an endoglucanase (family 45 glycoside hydrolase) having one carbohydrate-binding module (CBM), designated MCE1, and the mce2 cDNA encoded the same endoglucanase having two tandem repeated CBMs, designated MCE2. The two cDNAs contained the same sequences but with a 147-bp insertion. The corresponding genomic mce gene consisted of four exons. The mce1 cDNA was created from exons 1, 3, and 4, and the mce2 cDNA was created from exons 1, 2, 3, and 4. These results indicate that the mce1 and mce2 cDNAs were created from one genomic mce gene by alternative splicing. MCE1 and MCE2, purified to apparent homogeneity from the culture supernatant of M. circinelloides, had molecular masses of 43 and 47 kDa, respectively. The carboxymethyl cellulase specific activity of MCE2 was almost the same as that of MCE1, whereas the Avicelase specific activity of MCE2 was two times higher than that of MCE1. Furthermore, MCE2, whose two tandem CBMs might be more effective for degradation of crystalline cellulose than one CBM, was secreted only at an early culture stage when crystalline cellulose was abundant.Cellulose is the major plant cell wall polysaccharide and is degraded by cellulases. This degradation is considered to be due to the synergistic action of three types of cellulase components: endoglucanases (EC 3.2.1.4; endo--D-1,4-glucanases), cellobiohydrolases (EC 3.2.1.91), and -glucosidases (EC 3.2.1.21) (11).The endoglucanases, a large, ubiquitous family of enzymes that hydrolyze -1,4 linkages adjacent to unsubstituted glucose residues (10), are produced by a broad range of organisms, including fungi (23), bacteria (2), plants (17), and insects (termites) (12). In the textile and detergent industries, endoglucanases have been used for removing microfibrils from the surface of cellulosic fabrics, which enhances the softness and brightness of the cellulosic fabrics (1,3,4,8). Because of the importance of the endoglucanases in these industries, many fungal endoglucanase genes from members of the subdivision Deuteromycotina, such as Aspergillus sp., Fusarium sp., Humicola sp., Penicillium sp., and Trichoderma sp., have been cloned and characterized (21). However, there were no reports of the isolation of endoglucanase genes from members of the subdivision Zygomycota until we isolated two endoglucanases, RCE1 and RCE2, from Rhizopus oryzae as the first cellulases from a member of the subdivision Zygomycota (16) and cloned the encoding genes, rce1, rce2, and rce3 (15). We also cloned the pce1 gene, which is homologous to the codon usage-optimized rce1 gene, from Phycomyces nitens, a member of the same subdivision, and we isolated and characterized endoglucanase ...
In the detergent industry, fungal endoglucanases have been used to release microfibrils (defibrillation) from the surface of dyed cellulosic fabrics to enhance color brightness. Although endoglucanases for laundry use must have various properties, such as a neutral or alkaline optimum pH, resistance to anionic surfactants and oxidizing agents (main components in detergents), and high defibrillation activity, all-purpose endoglucanases have not been obtained yet. As a result of screening of endoglucanases, a new family 45 endoglucanase (family 45 glycoside hydrolase), designated STCE1, was obtained and purified to apparent homogeneity from the culture supernatant of Staphylotrichum coccosporum NBRC 31817. The molecular mass of STCE1 was 49 kDa. The optimum pH for the carboxymethyl cellulase activity of STCE1 was 6.0, and the optimum temperature was 60°C. STCE1 was highly resistant to an anionic surfactant and an oxidizing agent. Furthermore, the defibrillation activities on dyed cotton and lyocell fabrics of STCE1 were higher than those of the other representative endoglucanases tested. These results indicate that STCE1 is an all-purpose enzyme for laundry use. A gene encoding STCE1, designated the stce1 gene, was cloned from S. coccosporum, and the complete sequence was determined. STCE1 consisted of three distinct domains: an N-terminal catalytic domain (family 45), a linker domain, and a C-terminal carbohydrate-binding module (family 1). The amino acid sequences of the catalytic domain of STCE1 were phylogenetically close to those of the family 45 endoglucanases EGL3, EGL4, and EGV from a Humicola sp. Hence, the stce1 gene was transferred into Humicola insolens and expressed. As a result, extremely high levels (0.90 mg protein per ml of culture supernatant, 27% of the total proteins) of the recombinant STCE1 were secreted as a mature form in the culture supernatant.
We examined the characteristics of family 45 endoglucanases (glycoside hydrolases family 45; GH45) from Mucorales belonging to Zygomycota in the use of textiles and laundry. The defibrillation activities on lyocell fabric of family 45 endoglucanases from Mucorales, such as RCE1 and RCE2 from Rhizopus oryzae, MCE1 and MCE2 from Mucor circinelloides, and PCE1 from Phycomyces nitens, were much higher than those of the other family 45 endoglucanases. By contrast, family 45 endoglucanases from Mucorales were less resistant to anionic surfactant and oxidizing agent, main components in detergents, than the other family 45 endoglucanases. RCE1 consists of two distinct modules, a catalytic module and a carbohydrate-binding module family 1 (CBM1), and these common specific characteristics were considered to due to the catalytic module, but not to the CBM1.Key words: endoglucanase; cellulase; glycoside hydrolase family 45; Zygomycota; Mucorales Cellulose is the major plant cell wall polysaccharide and is degraded by cellulases. This degradation is considered to be achieved by the synergistic action of three types of cellulase components: endoglucanases (EC 3.2.1.4, endo--D-1,4-glucanases), cellobiohydrolases (EC 3.2.1.91), and -glucosidases (EC 3.2.1.21). 1)Most cellulosic fabrics consisting of cellulosic fibers, such as cotton, linen, ramie, viscose, and lyocell, have a tendency to form microfibrils. The accumulation of microfibrils on the surface of the fabrics make the fabrics look hairy and scatters incident light, thereby lessening the brightness of the original colors. These phenomena are considered to be negative features of cellulosic fabrics. Hence, the removal of the microfibrils (defibrillation) is necessary to increase the commercial value of cellulosic fabrics. In the textile and detergent industries, endoglucanases are used to remove microfibrils from the surface of cellulosic fabrics, enhancing color brightness. [2][3][4][5] Although lyocell fabrics possess many positive attributes for high-quality fabrics, they have the negative characteristic to form larger quantities of microfibrils than other cellulosic fabrics in the dyeing, finishing, and washing processes. Hence, high defibrillation of lyocell fabrics by endoglucanase treatment is in demand for textile use. 6)Family 45 endoglucanases (glycoside hydrolases family 45; GH45) are the main endoglucanases used in the textile and detergent industries because of their high defibrillation activities, 6,7) and many fungal family 45 endoglucanases have been purified and characterized. The fungal kingdom is classified into four phylums: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Zygomycota is classified into two classes: Zygomycetes and Trichomycetes.8) Furthermore, Zygomycetes is classified into 12 orders: Mucorales, Entomophthorales, Zoopagales, Endogonales, Amoebidiales, Eccrinales, Harpellales, Asellariales, etc. In our previous studies, six endoglucanases, RCE1, RCE2, and RCE3 from Rhizopus oryzae, 9,10) MCE1 and MCE2 from Mucor circinelloid...
We previously cloned three endoglucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota. In this study, an endoglucanase gene, designated a pce1 gene, was cloned by plaque hybridization with the codon usage-optimized rce1 gene as a probe from Phycomyces nitens, a member of the subdivision Zygomycota. The pec1 gene had an open reading frame of 1,038 nucleotides encoding an endoglucanase (PCE1) of 346 amino acid residues. The amino acid sequence deduced from the pce1 gene consisted of a cellulose-binding domain (CBD) at the N terminus and of a catalytic domain belonging to family 45 glycoside hydrolase at the C terminus. PCE1 was purified to apparent homogeneity from the culture supernatant of P. nitens and the molecular mass was found to be 45 kDa. The optimum pH for the CMCase activity of PCE1 was 6.0, and the optimum temperature was 50 degrees C, the lowest among the family 45 endoglucanases.
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