Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw

Abstract

BackgroundAn efficient hydrolysis of lignocellulosic substrates to soluble sugars for biofuel production necessitates the interplay and synergistic interaction of multiple enzymes. An optimized enzyme mixture is crucial for reduced cost of the enzymatic hydrolysis step in a bioethanol production process and its composition will depend on the substrate and type of pretreatment used. In the present study, an experimental design was used to determine the optimal composition of a Trichoderma reesei enzyme mixture, comprising the main cellulase and hemicellulase activities, for the hydrolysis of steam-exploded wheat straw.MethodsSix enzymes, CBH1 (Cel7a), CBH2 (Cel6a), EG1 (Cel7b), EG2 (Cel5a), as well as the xyloglucanase Cel74a and the xylanase XYN1 (Xyl11a) were purified from a T. reesei culture under lactose/xylose-induced conditions. Sugar release was followed in milliliter-scale hydrolysis assays for 48 hours and the influence of the mixture on initial conversion rates and final yields is assessed.ResultsThe developed model could show that both responses were strongly correlated. Model predictions suggest that optimal hydrolysis yields can be obtained over a wide range of CBH1 to CBH2 ratios, but necessitates a high proportion of EG1 (13% to 25%) which cannot be replaced by EG2. Whereas 5% to 10% of the latter enzyme and a xylanase content above 6% are required for highest yields, these enzymes are predicted to be less important in the initial stage of hydrolysis.ConclusionsThe developed model could reliably predict hydrolysis yields of enzyme mixtures in the studied domain and highlighted the importance of the respective enzyme components in both the initial and the final hydrolysis phase of steam-exploded wheat straw.

The Accessible Cellulose Surface Influences Cellulase Synergism during the Hydrolysis of Lignocellulosic Substrates

“…[12,13] Previous work has also shown that cellulase synergism is more apparent during the hydrolysis of more crystalline cellulosic substrates, with little or no synergy observed on amorphous cellulose (e.g., phosphoric acid swollen cellulose (PASC)). [20][21][22][23][24][25][26] We [27] and other workers [28,29] have shown that the pretreatment and nature of the cellulosic substrate used resulted in significant variations in substrate characteristics, such as cellulose accessibility, degree of polymerization (DP), and crystallinity. [11,15,16] These sometimes contradictory observations indicate that the relationship between enzyme synergism and the nature of the cellulosic substrate has not been fully resolved.…”

“…[17][18][19] As mentioned earlier, only a limited number of studies have explored the extent of synergistic cooperation among cellulases and other enzymes during hydrolysis of pretreated lignocellulosic substrates. [20][21][22][23][24][25][26] We [27] and other workers [28,29] have shown that the pretreatment and nature of the cellulosic substrate used resulted in significant variations in substrate characteristics, such as cellulose accessibility, degree of polymerization (DP), and crystallinity. The amount and nature of the non-cellulosic components, such as residual hemicellulose; lignin; and the type of lignin-carbohydrate complexes remaining after pretreatment have also been shown to impede the effectiveness of enzymatic hydrolysis.…”

Use of Cellulases from Trichoderma reesei in the Twenty-First Century—Part II

“…Including beta-xylosidase and xylanase in the mixture (CBH1/ CBH2/EG1/BGL/BXL/XYN) led to higher conversion of AFEX-pretreated corn stover (above 80% in 24 h) than the industrial enzyme complexes Spezyme or Accelerase (Gao et al, 2009(Gao et al, , 2011. The importance of xylanase addition was also demonstrated for steam-pretreated wheat straw although only low levels of xylan are left after this pretreatment (Billard et al, 2011). Addition of other auxiliary enzymes (β-xylosidase, α-arabinofuranosidase and α-glucuronidase) increased xylose yields, but without increasing glucose yields further.…”

“…EG1 levels, on the contrary, had to be increased to about 35%, compared to 5-10% in a T. reesei cocktail. Higher EG1 ratios were also necessary for optimized hydrolysis of ammonia-fiber expansion pretreated corn stover and steamexploded wheat straw (Billard et al, 2011). Including beta-xylosidase and xylanase in the mixture (CBH1/ CBH2/EG1/BGL/BXL/XYN) led to higher conversion of AFEX-pretreated corn stover (above 80% in 24 h) than the industrial enzyme complexes Spezyme or Accelerase (Gao et al, 2009(Gao et al, , 2011.…”

Engineering cellulosic bioreactors by template assisted DNA shuffling and in vitro recombination (TADSir)

“…The incompatibility of bacterial and fungal derived enzyme mixtures with bio-feedstock cell walls results in limited access to cellulose structural targets and an inability to harvest sequestered cellulosic energy stores (Billard et al, 2012;Banerjee et al, 2010;Arantes and Saddler, 2010). Additionally, their exogenous expression produces difficulties such as die codons, codon usage mismatches, and aberrant splicing (US 2002/0062502 A1, May 23, 2002.…”