Techno-economic analysis of recombinant Endo-β-1,4-Glucanase production from Escherichia coli Eg-RK2 culture using oil palm empty fruit bunch

A techno-economic analysis of recombinant cellulase production from E. coli Eg-RK2 was conducted to support the fulfilling of Indonesia’s energy roadmap for ethanol production. The plant utilizes OPEFB as a primary substrate in cellulase production, with an expected lifetime of 12 years. The plant is assumed to be built in Indonesia and it will fulfill 1% of the total market demand. The effect of different pretreatment processes (alkaline, steam explosion, and sequential acid-alkaline) on the profitability parameter was also studied. A simulation using SuperPro Designer was used to calculate the mass and energy balance based on the kinetic parameters of E. coli EgRK2. A technology evaluation showed that alkaline pretreatment provides the highest yield with no known inhibitors formed. The steam explosion pretreatment offers the lowest rate of lignin and hemicellulose removal, and it is understood to form known fermentation inhibitors. The NPVs of the alkaline, steam explosion and sequential acid-alkaline pretreatments are USD 32,121,000, USD -36,841,000, and USD 384,000, respectively, which means the alkaline pretreatment is economically very feasible for the production of cellulase.

Enzymatic Hydrolysis of Softwood Derived Paper Sludge by an In Vitro Recombinant Cellulase Cocktail for the Production of Fermentable Sugars

Paper sludge is an attractive biomass feedstock for bioconversion to ethanol due to its low cost and the lack of pretreatment required for its bioprocessing. This study assessed the use of a recombinant cellulase cocktail (mono-components: S. cerevisiae-derived PcBGL1B (BGL), TeCel7A (CBHI), ClCel6A (CBHII) and TrCel5A (EGII) mono-component cellulase enzymes) for the efficient saccharification of softwood-derived paper sludge to produce fermentable sugars. The paper sludge mainly contained 74.3% moisture and 89.7% (per dry mass (DM)) glucan with a crystallinity index of 91.5%. The optimal protein ratio for paper sludge hydrolysis was observed at 9.4: 30.2: 30.2: 30.2% for BGL: CBHI: CBHII: EGII. At a protein loading of 7.5 mg/g DW paper sludge, the yield from hydrolysis was approximately 80%, based on glucan, with scanning electron microscopy micrographs indicating a significant alteration in the microfibril size (length reduced from ≥ 2 mm to 93 µm) of the paper sludge. The paper sludge hydrolysis potential of the Opt CelMix (formulated cellulase cocktail) was similar to the commercial Cellic CTec2® and Celluclast® 1.5 L cellulase preparations and better than Viscozyme® L. Low enzyme loadings (15 mg/g paper sludge) of the Opt CelMix and solid loadings ranging between 1 to 10% (w/v) rendered over 80% glucan conversion. The high glucose yields attained on the paper sludge by the low enzyme loading of the Opt CelMix demonstrated the value of enzyme cocktail optimisation on specific substrates for efficient cellulose conversion to fermentable sugars.

Selection of purification condition for recombinant endoglucanase originated from goat rumen bacteria in Escherichia coli

Cellulase is an important enzyme that plays a role in cleaving β-1,4 glucoside on cellulose to release glucose, which is of economic value and can be applied in many different fields. The 1545 bp endoglucanase gene mined from goat rumen's bacterial metagenomic data was expressed in Escherichia coli Rosetta2. In this study, the recombinant endoglucanase was purified by his-tag affinity chromatography with differrent processes, such as using phosphate buffer with or without sodium cloride, pretreatment of samples with ammonium sulphate before supplying into affinity column, using various concentration of imidazole for washing... Finally the endoglucanse was sucessfully purified by his-tag affinity column using sodium chloride-free phosphate buffer of which 150 mM and 400 mM imidazole were used for washing and enzyme elution, respectively. The resulting enzyme showed its high purity of 99%. CMC plate assay confirmed that although less active than commercial cellulase (Sigma), the recombinant cellulase hydrolyzed CMC to form a clear zone (halo) around the well. The purified enzyme is capable of using as material for further analysis.