Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85

Abstract

Two endoglucanases designated EG1 and EG2 were purified by column chromatography from the nonsedimentable extracellular culture fluid of Bacteroides succinogenes S85. They accounted for approximately 32 and 11%, respectively, of the total endoglucanase present in the nonsedimentable fraction. The most active enzyme (EG1) had a molecular weight of 65,000, pl of 4.8, and temperature and pH optima of 39°C and 6.4, respectively. The Km for carboxymethyl cellulose was 3.6 mg/ml, and the V.., was 84 U/mg. The major products of cellulose hydrolysis catalyzed by EG1 were cellotriose and cellobiose. EG2 was present as two components with molecular weights of 118,000 and 94,000. The two components had nearly identical cyanogen bromide peptide maps, thereby indicating that the 94,000-dalton component was a proteolytic degradation product of the 118,000-dalton enzyme. The larger component, which was more abundant in the culture fluid than the smaller form was, had a Km of 12.2 mg/ml and a V.x of 10.4 U/mg. It was a basic protein with a, pl of 9.4, a temperature optimum of 39°C, and a pH optimum of 5.8. The major product of cellulose hydrolysis was cellotetraose. EG2 exhibited specific binding to acid-swollen cellulose, whereas EG1 did not, and neither of them had affinity for crystalline cellulose. Based on the substrate specificities and the affinities of the two enzymes for cellulose, we postulated that EG2 is involved in the early stages of cellulose hydrolysis and that EG1 is active primarily on the products arising from EG2.Bacteroides succinogenes is a predominant cellulolytic bacterium in the bovine rumen (6,20). However, neither nongrowing cells nor cell-free culture fluid from B. succinogenes affects the extensive hydrolysis of crystalline cellulose (16). Therefore, our research was centered on determining the types of cellulolytic activities possessed by the bacterium, with the ultimate objective being to identify the enzymes responsible for the extensive hydrolysis of cellulose by growing cultures.During growth of B. succinogenes in continuous culture with cellulose as the carbon source, endoglucanase and chloride-stimulated cellobiosidase activities have been shown to be present in both the cells and the extracellular culture fluid (18). A cellodextrinase was detected in the periplasmic space, while cellobiase activity was membrane associated (18,19). Greater than 70% of the endoglucanase activity was present in the extracellular culture fluid of cells grown in either a chemostat culture or a batch culture after all of the cellulose was digested (11,16,19). Results of the batch culture study revealed that 50 to 62% of the extracellular endoglucanase was associated with sedimentable membrane fragments, 9 to 13% was associated with nonsedimentable material with a molecular weight greater than 4 x 106, and 28 to 38% was associated with molecules with a molecular weight of approximately 45,000, as determined by exclusion chromatography (15,16,31). The endoglucanase activities in these various fractions were further ...

Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

“…Endoglucanases which do not have homology to EngB and EngD probably also exist in C. cellulovorans, since an endoglucanase gene, engC, has also been sequenced, and it has no homology to either engB or engD (28). The multiplicity of endoglucanases has been the subject of investigation, as it is not known whether this variety is due to expression of different genes or to posttranslational modification of the same gene product (20)(21)(22). Gene engB has high homology to engD and perhaps to other endoglucanases in C. cellulovorans, as evidenced by their cross-reactivities.…”

“…The degradative process can be different for different species, especially between anaerobic and aerobic organisms. With cellulolytic organisms such as Tichoderma reesei, Neocallismastix frontalis, Pseudomonas fluorescens, Cellulomonas fimi, Fibrobacter succinogenes, and Ruminococcus flavefaciens, it is not certain whether the enzymes essential for the degradation of crystalline cellulose function individually or whether they are present on the cell or substrate surface as a complex (1,15,20). There is evidence that in Clostridium thernocellum as well as in Clostridium strain C7, the enzymes are present in a complex, termed the "cellulosome" (3, 18,19,22,23).…”

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

“…Several other cloned gene products have been shown to possess activities on multiple substrates Gilkes et al, 1984;Hamamoto et al, 1990;McGavin & Forsberg, 1988;Taylor et al, 1987). The substrates that EngB is capable of using all have p-1,4 glycosyl linkages.…”

Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase

“…Nevertheless, the mechanism by which the bacterium degrades cellulose is still unknown (18,44). To elucidate the physiological mechanism of cellulose degradation by the bacterium, three endoglucanases, endoglucanase 1 (EGI), EG2 (46), and EG3 (49), a chloride-stimulated cellobiosidase (31), and a cellodextrinase (28,29) were purified and characterized. EG I was released from cells during growth, and EG2 was mainly cell associated, while EG3 was released late during growth (48).…”