In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex. Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a "designer" cellulosome, or a recombinant cellulosome with a specific function. We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units. The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli. The assembly of recombinant EngB with the miniCbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome) containing a variety of cellulolytic subunits attached to the nonenzymatic component termed CbpA (2). All cellulosomal enzymatic subunits contain a twice-repeated sequence, usually at their C termini, called the dockerin domain. These dockerin domains are considered to bind to the hydrophobic domains termed cohesins (19,26), which are repeated nine times in CbpA (14). So far, we have cloned and sequenced eight cellulosomal cellulase genes from C. cellulovorans (engB [3] [17], and exgS [9]). In addition, the genes for cellulosomal mannanase (manA) (15) and pectate lyase (pelA) (17) have also been cloned and sequenced.In order to understand the hydrolytic mechanism of the cellulosome and to use cellulosomes for industrial purposes, it is very important to make recombinant cellulosomes, or "designer" cellulosomes, with specific functions. To prepare recombinant cellulosomes, it is necessary to express the cellulosomal enzyme subunits that retain their dockerin domains. Escherichia coli is a very efficient host for expression of heterologous genes. However, during the expression of cellulosomal subunits from C. cellulovorans by E. coli, we found that almost all recombinant enzyme proteins were partially degraded between the catalytic and dockerin domains by the proteases of E. coli. Thus, this problem prevented us from preparing recombinant cellulosomes.As a host for heterologous expression, Bacillus subtilis has many attractive features (21). These features include the ability to secrete extracellular proteins, ease of genetic manipulation, and fast growth. However, one major problem is the presence of high levels of extracellular proteases. Aminov et al. (1) tried to express the celE gene, encoding one of the cellulosomal subunits from Clostridium thermocellum, ...
An anaerobic mesophile, Clostridium cellulovorans, produces a multienzyme complex called the cellulosome and actively degrades polysaccharides in the plant cell wall. C. cellulovorans also changes cellulosomal subunits to form highly active combinations dependent on the carbon substrate. A previous study reported on the synergistic effects of exoglucanase S (ExgS) and endoglucanase H (EngH) that are classified into the glycosyl hydrolase (GH) families 48, and 9, respectively. In this study, we investigated synergistic effects of ExgS and EngK, a GH9 cellulase different from EngH. In addition, since EngK was known to produce cellobiose as its main product, the inhibition on cellulase activity of EngK with cellobiose was examined. As a result, the effect of cellobiose inhibition on EngK coexistent with ExgS was found to be much lower than that with EngH. Thus, although EngH and EngK are in the same GH9 family, enzymatic activity in the presence of cellobiose was significantly different.
The Keap1-Nrf2 system serves as a defense mechanism against oxidative stress and electrophilic toxicants by inducing more than one hundred cytoprotective proteins, including antioxidants and phase 2 detoxifying enzymes. Since induction profiles of Nrf2 target genes have been studied exclusively in cultured cells, and not in animal models, their tissue-specificity has not been well characterized. In this paper, we examined and compared the tissue-specific expression of several Nrf2 target genes in zebrafish larvae by whole-mount in situ hybridization (WISH). Seven zebrafish genes (gstp1, mgst3b, prdx1, frrs1c, fthl, gclc and hmox1a) suitable for WISH analysis were selected from candidates for Nrf2 targets identified by microarray analysis. Tissue-restricted induction was observed in the nose, gill, and/or liver for all seven genes in response to Nrf2-activating compounds, diethylmaleate (DEM) and sulforaphane. The Nrf2 gene itself was dominantly expressed in these three tissues, implying that tissue-restricted induction of Nrf2 target genes is defined by tissue-specific expression of Nrf2. Interestingly, the induction of frrs1c and gclc in liver and nose, respectively, was quite low and that of hmox1a was restricted in the liver. These results indicate the existence of gene-specific variations in the tissue specificity, which can be controlled by factors other than Nrf2.
Cellulose comprises a major portion of biomass on the earth, and the turnover of this material contributes to the CO 2 cycle. Cellulases, which play a major role in the turnover of cellulosic materials, have been found either as free enzymes that work synergistically, or as an enzyme complex called the cellulosome. This review summarizes some of the general properties of cellulosomes, and more specifically, the properties of the Clostridium cellulovorans cellulosome. The C. cellulovorans cellulosome is an extracellular enzyme complex with a molecular weight of about 1 × 10 6 , and is comprised of at least ten subunits. The major subunit is the scaffolding protein CbpA, with a molecular weight of 189,000. This nonenzymatic subunit contains a cellulose binding domain (CBD) that binds the cellulosome to the substrate, nine conserved cohesins or enzyme binding domains, and four conserved surface layer homologous (SLH) domains. It is postulated that the SLH domains help to bind the cellulosome to the cell surface. The cellulosomal enzymes include cellulases (family 5 and 9 endoglucanases and a family 48 exoglucanase), a mannanase, a xylanase, and a pectate lyase. The cellulosome is capable of converting Arabidopsis and tobacco plant cells to protoplasts. One of the endoglucanases, EngE, contains three tandemly repeated SLHs at its N-terminus, and therefore appears capable of binding to the scaffolding protein CbpA as well as to the cell surface. Cellulosomes can attack crystalline cellulose, but the free cellulosomal enzymes can attack only soluble and amorphous celluloses. Nine genes for the cellulosome are found in a gene cluster cbpA-exgS-engH-engK-hbpA-engL-manA-engM-engN. Other cellulosomal genes such as engB, engE, and engY are not linked to the major gene cluster or to each other. By determining the structure and function of the cellulosome, we hope to increase the efficiency of the cellulosome by genetic engineering techniques.
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