Synergistic Hydrolysis of Carboxymethyl Cellulose and Acid-Swollen Cellulose by Two Endoglucanases (CelZ and CelY) from Erwinia chrysanthemi

Abstract

Erwinia chrysanthemi produces a battery of hydrolases and lyases which are very effective in the maceration of plant cell walls. Although two endoglucanases (CelZ and CelY; formerly EGZ and EGY) are produced, CelZ represents approximately 95% of the total carboxymethyl cellulase activity. In this study, we have examined the effectiveness of CelY and CelZ alone and of combinations of both enzymes using carboxymethyl cellulose (CMC) and amorphous cellulose (acid-swollen cellulose) as substrates. Synergy was observed with both substrates. Maximal synergy (1.8-fold) was observed for combinations containing primarily CelZ; the ratio of enzyme activities produced was similar to those produced by cultures of E. chrysanthemi. CelY and CelZ were quite different in substrate preference. CelY was unable to hydrolyze soluble cellooligosaccharides (cellotetraose and cellopentaose) but hydrolyzed CMC to fragments averaging 10.7 glucosyl units. In contrast, CelZ readily hydrolyzed cellotetraose, cellopentaose, and amorphous cellulose to produce cellobiose and cellotriose as dominant products. CelZ hydrolyzed CMC to fragments averaging 3.6 glucosyl units. In combination, CelZ and CelY hydrolyzed CMC to products averaging 2.3 glucosyl units. Synergy did not require the simultaneous presence of both enzymes. Enzymatic modification of the substrate by CelY increased the rate and extent of hydrolysis by CelZ. Full synergy was retained by the sequential hydrolysis of CMC, provided CelY was used as the first enzyme. A general mechanism is proposed to explain the synergy between these two enzymes based primarily on differences in substrate preference.The hydrolysis of cellulose into soluble sugars by microbial systems offers the potential to provide a renewable feedstock for the production of fuels and chemicals (10,17,22). However, the crystalline structure and insoluble nature of cellulose represents a formidable challenge for enzymatic hydrolysis. Interactions between different cellulase enzymes and substrates are quite complex (2,5,20,24,36). The solubilization of crystalline cellulose by the fungus Trichoderma longibranchiatum has been extensively studied as a model primarily due to its commercial utility. Cellulases produced by T. longibranchiatum can be divided into three classes: endoglucanases (carboxymethyl cellulases [CMCases]), which hydrolyze amorphous regions of cellulose; exoglucanases (cellobiohydrolases), which progressively cleave cellobiose units from the ends of crystalline or amorphous cellulose; and ␤-glucosidases (cellobiases), which hydrolyze soluble cellooligosaccharides into glucose (5,20). Multiple enzymes of each type are produced by T. longibranchiatum. Combinations of these fungal enzymes function in a synergistic fashion (23,24,32,35,36). Bacteria also produce multiple enzymes for cellulose hydrolysis (5, 21, 25). Synergy has been demonstrated for combinations of bacterial exoglucanases and endoglucanases (3,12,23,28) and for combinations of bacterial endoglucanases and fungal exoglucanases (2,18,33). In...

Microbial Cellulose Utilization: Fundamentals and Biotechnology

“…Another cellulase gene that has been successfully expressed in Z. mobilis was cloned from Acetobacter xyli- (ii) Enteric bacteria. Two E. chrysanthemi endoglucanases, encoded by celY and celZ, and the A. xylinum cellulase gene have been expressed in both E. coli as well as the related enteric bacterium K. oxytoca (230,502,755,779,780,781,782). Initially the expression of celY in E. coli was poor due to promoter construction (230).…”

Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii

“…The organisms which are predominantly responsible for the degradation of lignocellulosic biomass are fungi, highlight the basidiomycetes (Rabinovich et al, 2004). The distribution of the biomass involves the formation of long polysaccharides chain, primarily cellulose, hemicellulose, and subsequent hydrolysis, which can be converted into ethanol by fermentation (Zhou and Ingram, 2000).…”

Utilization of White Grape Pomace for Lactic Acid Production

“…In this method, cellulose and hemicellulose was hydrolyzed to corresponding monomers, which were then quantified in HPLC. In the first stage, dried pomace (300 mg) free of stems and seeds was suspended in 3 mL of 12 M H2SO4 in a test tube and the tube was kept at room temperature for 3 h. In the second stage, water was added to the suspension to a final H2SO4 concentration of 0.80 M and the tube was kept at 100 C in the water bath for 4 h (Zhou and Ingram, 2000). The pH was neutralized by CaCO3 and insoluble materials were removed by centrifugation at 3024 g. Supernatants were diluted with water and analyzed for glucose, xylose, and arabinose and the concentrations were multiplied by anhydro factors (0.90 for glucose; 0.88 for xylose and arabinose) to calculate cellulose and hemicellulose contents of the pomace, respectively.…”