Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris.

OBJECTIVE: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. RESULTS: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg L-1), and were secreted at moderate to high percentages of the total extracellular protein (51-94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U L-1) that could allow their direct application.

Application of endo-ß-1,4,D-mannanase and cellulase for the release of mannooligosaccharides from steam-pretreated spent coffee ground.

Spent coffee ground (SCG), a present waste stream from instant coffee production, represents a potential feedstock for mannooligosaccharides (MOS) production. MOS can be used in nutraceutical products for humans/animals or added to instant coffee, increasing process yield and improving product health properties. The SCG was evaluated for MOS production by steam pretreatment and enzymatic hydrolysis with a recombinant mannanase and a commercial cellulase cocktail (Acremonium, Bioshigen Co. Ltd, Japan). The mannanase was produced using a recombinant strain of Yarrowia lipolytica, used to produce and secrete endo-1,4-ß,D-mannanase from Aspergillus aculeatus in bioreactor cultures. Endo-1,4-ß,D-mannanase was produced with an activity of 183.5 U/mL and 0.23 mg protein/mL. The enzyme had an optimum temperature of 80 °C, and the activity in the supernatant was improved by 150 % by supplementation with 0.2 % sodium benzoate and 35 % sorbitol as a preservative and stabiliser, respectively. The steam pretreatment of SCG improved the enzymatic digestibility of SCG, thus reducing the required enzyme dosage for MOS release. Combined enzymatic hydrolysis of untreated or steam-pretreated (150, 190 and 200 °C for 10 min) SCG with mannanase and cellulase cocktail resulted in 36-57 % (based on mannan content) of MOS production with a degree of polymerization of up to 6. The untreated material required at least 1 % of both mannanase and cellulase loading. The optimum mannanase and cellulase loadings for pretreated SCG hydrolysis were between 0.3 and 1 and 0.4 and 0.8 %, respectively. Statistical analysis suggested additive effect between cellulase cocktail and mannanase on MOS release, with no indication of synergism observed.

Enzymatic hydrolysis of spent coffee ground.

Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol). Wild fungi were isolated from both SCG and coffee beans and screened for enzyme production. The enzymes produced from the selected wild fungi and recombinant fungi were then evaluated for enzymatic hydrolysis of SCG, in comparison to commercial enzyme preparations. Out of the enzymes evaluated on SCG, the application of mannanase enzymes gave better yields than when only cellulase or xylanase was utilised for hydrolysis. The recombinant mannanase (Man1) provided the highest increments in soluble solids yield (17 %), even when compared with commercial preparations at the same protein concentration (0.5 mg/g SCG). The combination of Man1 with other enzyme activities revealed an additive effect on the hydrolysis yield, but not synergistic interaction, suggesting that the highest soluble solid yields was mainly due to the hydrolysis action of mannanase.