Tomoko Nishimura

Purification and characterization of new endo-1,4-β-d-glucanases from Rhizopus oryzae

Abstract New extracellular endoglucanases, designated RCE1 and RCE2, produced by Rhizopus oryzae isolated from the soil, were purified to apparent homogeneity from the culture supernatant. The molecular mass of RCE1 and that of RCE2 were found to be 41 kDa and 61 kDa, respectively. The N-terminal amino acid sequences of RCE1 and RCE2 showed high homology with those of the family I cellulose-binding domains. Internal amino acid sequences of RCE1 and RCE2 showed homology with that of the catalytic domain of EGV from Humicola insolens belonging to family 45 endoglucanase. The cellooligosaccharide hydrolysis patterns of RCE1 and RCE2 were similar to that of EGV from H. insolens. These results indicate that RCE1 and RCE2 are family 45 endoglucanases having a cellulose binding domain at their N-terminus. RCE1 and RCE2 hydrolyzed carboxymethylcellulose (CMC), insoluble cellooligosaccharide (G33), cellohexaose, and cellopenpaose, but not Avicel, xylan, galactan, arabinan, mannan, or laminarin. The CMCase activity of both enzymes was inhibited by Cu 2+ , Zn 2+ , Co 2+ , and Pb 2+ . The optimum pH for the CMCase activity of both enzymes was found to be between pH value 5.0 and 6.0, and the optimum temperature was 55°C, the lowest among the family 45 endoglucanases. These results indicate that RCE1 and RCE2 represent a new type of endoglucanases having the lowest optimum temperature among the family 45 endoglucanases.

Purification and Characterization of a New Family 45 Endoglucanase, STCE1, from Staphylotrichum coccosporum and Its Overproduction in Humicola insolens

ABSTRACT 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.

Specific characteristics of family 45 endoglucanases from Mucorales in the use of textiles and laundry

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.

Exploring Amino Acids Responsible for the Temperature Profile of Glycoside Hydrolase Family 45 Endoglucanase EGL3 from Humicola grisea

EGL3 and RCE1 are glycoside hydrolase family 45 endoglucanases isolated from Humicola grisea and Rhizopus oryzae respectively. The amino acid sequences of the two endoglucanases are homologous; on the other hand, the optimum temperature of EGL3 is higher than that of RCE1. In this study, four chimeric endoglucanases, named ER1, ER2, ER3 and ER4, in which one of four sequential amino acid regions of the EGL3 catalytic domain (CAD) was replaced by the corresponding RCE1 amino acids, were constructed to explore the region responsible for the EGL3 temperature profile. Then their temperature profiles were compared with that of the recombinant EGL3. Replacement of the N-terminal region of EGL3 with that of RCE1 caused the EGL3 temperature profile to shift to a lower temperature. These results suggest that the N-terminal amino acids of the EGL3 are responsible for the EGL3 temperature profile.

Amino Acid Regions of Family 45 Endoglucanases Involved in Cotton Defibrillation and in Resistance to Anionic Surfactants and Oxidizing Agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.