Purification and characterization of two .beta.-1,4-endoglucanases from Thermomonospora fusca

Calza, R. E.; Irwin, Diana C.; Wilson, David B.

doi:10.1021/bi00347a044

Cited by 129 publications

(65 citation statements)

References 32 publications

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“…Second, the endoglucanases are subject to proteolytic cleavage, giving more than one component of some of the endoglucanases in the extracellular culture fluid. Growth on cellulose has been shown to induce extracellular protease activity in Cellulomonas fimi (23), and culture supernatants of Thermomonospora fusca possess protease activity which contributes to endoglucanase multiplicity (5 Apart from the fact that endoglucanase multiplicity is caused by the factors listed above, it has also been attributed to posttranslational modification by chemical substitution, such as glycosylation. Two of three endoglucanases purified from Cellulomonas sp.…”

Section: Discussionmentioning

confidence: 99%

“…Two of three endoglucanases purified from Cellulomonas sp. are glycosylated (2), as is one from Clostridium thermocellum (29) and at least one from T. fusca (5). In contrast, neither of the two endoglucanases from B. succinogenes was a glycoprotein, although periodic acidSchiff staining of nonsedimentable culture fluid revealed glycosylated proteins (unpublished data), and the extracellular cellobiosidase is a glycoprotein (19).…”

Section: Discussionmentioning

confidence: 99%

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

McGavin

Forsberg

1988

J Bacteriol

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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 ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

McGavin

Forsberg

1988

J Bacteriol

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“…The role of a serine protease in the processing of cellulases has been described in Thermomonospora fusca (now Thermobifida fusca), where the protease encoded by the tfpA gene cleaves its cellulases into many different isoenzymes (Calza et al, 1985;Lao & Wilson, 1996). A BLAST search (Altschul et al, 1997) Chitin-binding domains in proteases have been previously described for protease C from Streptomyces griseus (Sidhu et al, 1994).…”

Section: Cloning Of Two Serine Proteases From S Lividans Involved Inmentioning

confidence: 99%

Posttranslational processing of the xylanase Xys1L from Streptomyces halstedii JM8 is carried out by secreted serine proteases

et al. 2003

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The xylanase Xys1L from Streptomyces halstedii JM8 is known to be processed extracellularly, to produce a protein of 33?7 kDa, Xys1S, that retains catalytic activity but not its cellulose-binding capacity. This paper demonstrates that at least five serine proteases isolated from Streptomyces spp. have the ability to process the xylanase Xys1L. The genes of two of these extracellular serine proteases, denominated SpB and SpC, were cloned from Streptomyces lividans 66 (a strain commonly used as a host for protein secretion), sequenced, and overexpressed in S. lividans; both purified proteases were able to process Xys1L in vitro. Three other previously reported purified Streptomyces serine proteases, SAM-P20, SAM-P26 and SAM-P45, also processed Xys1L in vitro. The involvement of serine proteases in xylanase processing-degradation in vivo was demonstrated by co-expression of the xylanase gene (xysA) and the gene encoding the serine protease inhibitor (SLPI) from S. lividans. Co-expression prevented processing and degradation of Xys1L and resulted in a threefold increase in the xylanase activity present in the culture supernatant. SpB and SpC also have the capacity to process other secreted proteins such as p40, a cellulose-binding protein from S. halstedii JM8, but do not have any clear effect on other secreted proteins such as amylase (Amy) from Streptomyces griseus and xylanase Xyl30 from Streptomyces avermitilis.

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“…In fact, Thermomonospora strains have attracted considerable attention as sources of highly active and thermostable enzymes for lignocellulose saccharification in general (McCarthy, 1987). Some progress has been made towards biochemical characterization of the cellulose-degrading system in Thermomonospora fusca (Calza et al, 1985) and we are adopting a similar approach to identification of the enzymes involved in xylan degradation. In this paper, purification and characterization of the thermostable intracellular /I-xylosidase of T. fusca is described.…”

Section: Introductionmentioning

confidence: 99%

Purification and Characterization of a Thermostable -Xylosidase from Thermomonospora fusca

Bachmann

McCarthy

1989

Microbiology

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Xylan-degrading enzymes, including 8-xylosidase (EC 3.2.1 .37), were induced when Thermomonospora fusca was grown at 50 "C in liquid medium containing 0.2% xylan. The intracellular P-xylosidase activity was concentrated and characterized by fast protein liquid chromatography and gel electrophoresis. A zymogram technique was developed to identify 8-xylosidase directly on polyacrylamide gels. A single enzyme (168 kDa; PI 4.37) was identified and purified to homogeneity. The consistent detection of a single band on denaturing SDS gels suggested that the enzyme was composed of identical subunits; since the subunit molecular mass was 56 kDa, a trimeric structure is suggested. High activity against p-nitrophenyl P-Dxylopyranoside (pNPX) occurred in the pH range 5.0-9-0 and temperature range 40-60 "C. The enzyme was stable at room temperature at pH 6.0-8-0; it had a half-life of 8 h at 65 "C, and of 1.5 h at 70 "C. The purified enzyme did not exhibit any detectable activity against arabinoxylan, carboxymethylcellulose orp-nitrophenyl P-D-glucopyranoside. The enzyme had a K, of 0.89 mM (pNPX) and was inhibited by D-XylOSe (Ki 19 mM) but not D-glucose. The size of the T. fusca enzyme is in the range reported for the few other bacterial P-xylosidases described, but the acidic nature of the protein and its affinity for the substrate have more in common with some of the monomeric P-xylosidases described in fungi.

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Purification and characterization of two .beta.-1,4-endoglucanases from Thermomonospora fusca

Cited by 129 publications

References 32 publications

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

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

Posttranslational processing of the xylanase Xys1L from Streptomyces halstedii JM8 is carried out by secreted serine proteases

Purification and Characterization of a Thermostable -Xylosidase from Thermomonospora fusca

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