Purification and properties of a novel type of exo‐1,4‐β‐glucanase (Avicelase II) from the cellulolytic thermophile Clostridium stercorarium

Abstract

Avicelase II was purified to homogeneity from culture supernatants of Clostridium stercorarium. A complete separation from the major cellulolytic enzyme activity (avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective retardation of avicelase II. The enzyme has an apparent molecular mass of 87 kDa and a pI of 3.9. Determination of the N-terminal amino acid indicates that avicelase II is not a proteolytically processed product of avicelase I. Maximal activity of avicelase II is observed between pH 5 and 6. In the presence of Ca2+, the enzyme is highly thermostable, exhibiting a temperature optimum around 75 degrees C. Hydrolysis of avicel occurs at a linear rate for three days at 70 degrees C. Avicelase II is active towards unsubstituted celluloses, cellotetraose and larger cellodextrins. It lacks activity towards carboxymethylcellulose and barley beta-glucan. Unlike other bacterial exoglucanases, avicelase II does not hydrolyze aryl-beta-D-cellobiosides. Avicel is degraded to cellobiose and cellotriose at a molar ratio of approximately 4:1. With acid-swollen avicel as substrate, cellotetraose is also formed as an intermediary product, which is further cleaved to cellobiose. The degradation patterns of reduced cellodextrins differ from that expected for a cellobiohydrolase attacking the non-reducing ends of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while cellohexaitol is initially cleaved into cellobiitol and cellotetraose. These findings, taken together, indicate that avicelase II represents a novel type of exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of the substrate, releases cellotetraose, cellotriose, or cellobiose from the non-reducing end of the cellulose chains.

Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’ with separate glycosyl hydrolase family 9 and 48 catalytic domains

“…Remarkably, this Avicelase activity is significantly higher than that reported for fungal and bacterial cellobiohydrolases (Tomme et al, 1988 ;Bronnenmeier et al, 1991 ;Kruus et al, 1995). The activity towards the cellulosic substrate is of the same On: Fri, 11 May 2018 07:36:05…”

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure‐guided recombination

“…In the chimeras, both these measured properties extend beyond the range of the parents. Many of the chimeras are very stable: indeed, this experiment has added 35 new Cel48 enzymes with a T opt of > 60°C to the six natural thermostable cellulases that have been characterized to date: C. thermocellum ATCC 27405 CelS [24], C. thermocellum F7 CelS [25], C. thermocellum ATCC 27405 CelY [26], Thermobifida fusca YX CelF [27], C. stercorarium CelY [28], and Anaerocellum thermophilum DSM 6725 CelA [29].…”

Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and Technologies and Future Perspectives

“…Several cellulose degrading enzymes from various thermophilic organisms have been investigated. These include cellulases mainly isolated from anaerobic bacteria such as Anaerocellum thermophilum (Zverlov et al, 1998), Clostridium thermocellum (Romaniec et al, 1992), Clostridium stercorarium (Bronnenmeier et al, 1991;Bronnenmeier & Staudenbauer, 1990) and Caldocellum saccharolyticum (Te'o V et l., 1995), Pyrococcus furiosus (Ma & Adams, 1994), Pyrococcus horikoshi (Rahman et al,1998), Rhodothermus strains (Hreggvidsson et al, 1996), Thermotoga sp., (Ruttersmith et al, 1991), Thermotoga marittima (Bronnenmeier et al, 1995), Thermotoga neapolitana (Bok et al, 1998). Xylanase have been detected in Acidothermus cellulolyticus in different Thermus, Bacillus, Geobacillus, Alicyclobacillus and Sulfolobales species (Sakon et al, 1996).…”