Synergism Between Purified Bacterial and Fungal Cellulases

Baker, John O.; Adney, William S.; Thomas, Steven R.; Nieves, Rafael A.; Chou, Yat‐Chen; Vinzant, Todd B.; Tucker, Melvin P.; Laymon, Robert A.; Himmel, Michael E.

doi:10.1021/bk-1995-0618.ch009

Cited by 28 publications

(23 citation statements)

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“…Although there are several factors that may have prevented complete cellulose conversion, one limitation may be the absence of synergistic cellulase enzymes, such as the endoglucanases and b-glucosidases typically co-secreted by the fungus. Additional experiments have shown that adding an endoglucanase to the reactions can result in complete cellulose hydrolysis (Baker et al, 1995;Baker et al, 1998). More detailed investigations using binary cellulase systems are currently underway.…”

Section: Discussionmentioning

confidence: 99%

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

Jeoh¹,

Ishizawa²,

Davis³

et al. 2007

Biotech & Bioengineering

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Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.

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Section: Discussionmentioning

confidence: 99%

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

Jeoh¹,

Ishizawa²,

Davis³

et al. 2007

Biotech & Bioengineering

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460

321

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“…The enzyme activity assays were based on a high-throughput microplate method as described in previous work [22]. A 2.2 ml deep-well microplate (Greiner, Monroe, NC, USA) was used to add 250 μl of 1% (w/v) stock substrate (CMC, Avicel, oat spelt xylan, cellobiose, xylobiose), 50 μl of 1 M citrate buffer (pH 5.0) and 200 μl of appropriately diluted enzyme samples (20 ng to 100 μg/well).…”

Section: Enzyme Activity Assaysmentioning

confidence: 99%

“…Few studies have investigated the synergism among catalytic domains of various bacterial enzymes, and the synergistic interactions between bacterial and fungal hydrolases acting on pretreated lignocellulosic biomass. Some reports have shown exo/exo and exo/endo synergism between fungal and bacterial enzymes hydrolyzing crystalline cellulose [21,22]. Recent publications have reported synergy between Trichoderma and Serratia/Streptomyces based on chitin-degrading hydrolases completely hydrolyzing untreated crab shells [23].…”

Section: Introductionmentioning

confidence: 99%

Strategy for Identification of Novel Fungal and Bacterial Glycosyl Hydrolase Hybrid Mixtures that can Efficiently Saccharify Pretreated Lignocellulosic Biomass

et al. 2010

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A rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, was applied in order to develop tailored enzyme cocktails to efficiently hydrolyze pretreated lignocellulosic biomass. The fungal cellulases include cellobiohydrolase I (CBH I; GH family 7A), cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B), and β-glucosidase (βG; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), β-glucosidase (LβG; GH family 1), endoxylanases (LX1 and LX2; GH family 10), and β-xylosidase (LβX; GH family 52) from multiple sources were cloned, expressed, and purified. Enzymatic hydrolysis for varying enzyme combinations was carried out on ammonia fiber expansion (AFEX)-treated corn stover at three total protein loadings (i.e., 33, 16.5, and 11 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary to maximize both glucan and xylan yields was determined using a suitable design of experiments. The optimal hybrid enzyme mixtures contained fungal cellulases (78% of total protein loading), which included CBH I (loading ranging between 9-51% of total enzyme), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LX1 (13%) and LβX (9%). The hybrid mixture was effective at 50°C, pH 4.5 to maximize saccharification of AFEX-treated corn stover resulting in 95% glucan and 65% xylan conversion. This strategy of screening novel enzyme mixtures on pretreated lignocellulose would ultimately lead to the development of tailored enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.

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“…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.…”

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confidence: 99%

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

Zhou

Ingram

2000

J Bacteriol

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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...

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Synergism Between Purified Bacterial and Fungal Cellulases

Cited by 28 publications

References 0 publications

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

Strategy for Identification of Novel Fungal and Bacterial Glycosyl Hydrolase Hybrid Mixtures that can Efficiently Saccharify Pretreated Lignocellulosic Biomass

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

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