Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans

Abstract

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHDeltaCBM and EngYDeltaCBM devoid of the CBM lost activity toward all substrates including CMC. EngKDeltaCBM and EngMDeltaCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.

Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae)

“…HSH-810 (Kim et al 2005) isolated from other environments, whose endo-1,4-β-glucanases also manifest optimal activity at pH 7.0 and notable activity at a wide pH ranging from 4.5 to 10. However, these characteristics may apparently constitute an exception, because the rule is that bacterial endo-1,4-β-glucanases work at an optimal pH, but do not show activity at wide intervals of pH, as has been documented in Sporocytophaga myxococcoides (Goksoyr 1988), Pseudomonas fluorescens (Yamane et al 1970); Ruminiclostridium thermocellum (Romaniec et al 1992), Aquifex aeolicus (Kim et al 2000), Ruminococcus albus (Ohara et al 2000), Cellulomonas biazotea (Rajoka et al 2004), Clostridium cellulovorans (Arai et al 2006), Bacillus mycoides (Balasubramanian et al 2012), and Bacillus sp. (Harshvardhan et al 2013).…”

Microbial enzyme systems for biomass conversion: emerging paradigms

“…The GH9 enzymes include several different modular themes and consequent alterations in activity patterns [52]. The versatility in the repertoire of the cellulosomal GH9 enzymes may be a necessary and advantageous adaptation of cellulosomes [53,54]. In addition to the latter cellulosomal enzymes, additional components, both enzymatic and structural, seem to be affected by the carbon source on which the bacterium is grown [43,48,50,[55][56][57][58].…”

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

“…These have complementary cohesins, called dockerins, which are specific for each bacterial species (Fig. 4) (Gilligan & Reese, 1954;Lynd et al, 2002;Arai et al, 2006;). For the bacterial cell, the biosynthesis of a cellulosome enables a specific adhesion to the substrate of interest without competition with other microorganisms.…”