Abstract
The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked BG was entrapped in calcium alginate gel particles. More than 60% of the enzyme activity could be recovered under optimized conditions, and glutaraldehyde cross-linking decreased leakage of BG from the calcium alginate particles. The immobilized BG aggregates were visualized by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized BG were recycled for cellulase catalyzed hydrolysis of Avicel. No significant loss in BG activity was observed for up to 20 rounds of reaction recycle steps of the BG particles of 48 h each, verifying a significant stabilization of the BG by immobilization. Similar high glucose yields were obtained by one round of enzymatic hydrolysis of hydrothermally pretreated barley straw during a 72 h reaction with immobilized BG and free BG.
Highlights
In order to produce bioethanol from lignocellulosic biomass, the 1,4-β-D-glycosidic linkages in cellulose must be cleaved to release glucose for the subsequent fermentation
Glutaraldehyde treated BG aggregates were successfully entrapped in 3.75% calcium alginate
In this way, 60% of BG residual activity could be recovered in the calcium alginate particles
Summary
In order to produce bioethanol from lignocellulosic biomass, the 1,4-β-D-glycosidic linkages in cellulose must be cleaved to release glucose for the subsequent fermentation. Cellulases (and hemicellulases) produced by the fungus Trichoderma reesei (an anamorph of Hypocrea jecorina) (Rut. C-30) are currently the main cellulases used in the industrial development of second generation bioethanol [1]. T. reesei secretes three main types of cellulose-degrading enzymes for catalyzing this enzymatic hydrolysis process: (1) endo-1,4-β-D-glucanase (EG, EC 3.2.1.4), which catalyzes random cleavage the internal β-1,4 bonds in the cellulosic polymers; (2) exo-1,4-β-D-glucanase or cellobiohydrolase (CBH, EC 3.2.1.91), which catalyzes hydrolysis of the second outermost β-1,4 bonds by attacking the cellulose from the ends only, releasing mainly cellobiose; CBH1 (or Cel7A). For the sake of completion, it should be mentioned that a new type of lytic polysaccharide monooxygenases (EC 1.-.-.-), categorized as auxiliary family activity 9, AA9, which catalyze oxidative cleavage of 1,4-β-linkages in cellulose are known [5]; the endo-β-1,4-glucanase IV (“Cel61A”) of T. reesei Rut C-30 has been categorized in family AA9 [6].
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