Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

Abstract

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.The rumen microbial ecosystem is composed of anaerobic microorganisms, such as bacteria, fungi, and protozoa. Some of these rumen microorganisms, the cellulolytic bacteria, are able to digest cellulosic material of plants and produce energy for the host animals. Many cellulolytic enzymes have been isolated from rumen microorganisms, and the genes encoding these enzymes have been cloned and sequenced (5, 9, 17). However, the precise mechanisms of lignocellulose degradation in the rumen are not yet fully understood. In order to clarify these mechanisms, it is necessary to study the microbial cellulolytic enzymes biochemically and genetically.The anaerobic cellulolytic bacterium Eubacterium cellulosolvens is sporadically dominant in the rumen (18). It is known that E. cellulosolvens 5 adheres tightly to cellulose, so studies of this adhesion have been performed (13,14,(23)(24)(25)(26). Some cellulose-binding proteins (CBPs) have been found in culture supernatant and cell lysate of the organism (14, 26). A gene encoding cellulose-binding protein A (CBPA), which is one of these CBPs, has been cloned and characterized (23-25). Additionally, the presence of some proteins exhibiting carboxymethyl cellulase (CMCase) activity in culture supernatant and cell lysate of E. cellulosolvens 5 was revealed by zymogram analysis (26). In order to advance res...

Processivity and Enzymatic Mode of a Glycoside Hydrolase Family 5 Endoglucanase from Volvariella volvacea

“…Supernatants were analyzed for monosaccharides, disaccharides, and oligosaccharides by thin-layer chromatography (TLC) according to a method described by Zhang et al (23). Hydrolysis of cellooligosaccharides (cellotriose, cellotetraose, cellopentaose) by EG1 and its derivatives was carried according to a method described by Yoda et al (24). Products were analyzed by TLC after 2 h of incubation at 50°C.…”

Isolation and Identification of Cellulose-Binding Proteins from Sheep Rumen Contents

“…Furthermore, among the seven endoglucanases in F. succinogenes S85, only EGF and endoglucanase G have CBMs (18). Since it is known that the removal of CBMs from endoglucanase remarkably reduces catalytic activities with various polysaccharides, endoglucanses with CBMs may play an important role in the digestion of cellulosic materials in the rumen (2,56).…”

“…Although these studies have provided valuable information about many of the cellulases and hemicellulases of F. succinogenes, the actual contribution of these enzymes to the degradation of feedstuff in the rumen remains unclear. Some fibrolytic enzymes possess carbohydrate-binding modules (CBMs), which play an important role in assisting the enzymes to hydrolyze cellulosic materials (2,56). Additionally, the adhesion of bacterial cells to cellulose appears to be a prerequisite for efficient cellulose hydrolysis by bacteria (27).…”

Soluble Carbohydrates Repress the Cellulolytic Activity of Clostridium cellulovorans and Eubacterium cellulosolvens

“…Thus, our study included Clostridium cellulovorans, which has served as a model system for the study of mesophilic cellulolytic clostridia [15]. We also studied Eubacterium cellulosolvens, which has been a valuable model system for non-clostridial cellulolytic bacteria [12,20,21], and its inclusion provided a phenotypic range for comparison.…”