Abstract
Backgroundβ-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance.ResultsLight activated cassette YF1/FixJ and the SRRz lysis system were successfully constructed to produce Bgl1A(A24S/F297Y), a mutant β-glucosidase tolerant to both glucose and ethanol. By optimizing the parameters for light induction, Bgl1A(A24S/F297Y) activity reached 33.22 ± 2.0 U/mL and 249.92 ± 12.25 U/mL in 250-mL flask and 3-L fermentation tank, respectively, comparable to the controls of 34.02 ± 1.96 U/mL and 322.21 ± 10.16 U/mL under similar culture conditions with IPTG induction. To further simplify the production of our target protein, the SRRz lysis gene cassette from bacteriophage Lambda was introduced to trigger cell autolysis. As high as 84.53 ± 6.79% and 77.21 ± 4.79% of the total β-glucosidase were released into the lysate after cell autolysis in 250 mL flasks and 3-L scale fermentation with lactose as inducer of SRRz. In order to reduce the cost of protein purification, a cellulose-binding module (CBM) from Clostridium thermocellum was fused into the C-terminal of Bgl1A(A24S/F297Y) and cellulose was used as an economic material to adsorb the fusion enzyme from the lysate. The yield of the fusion protein could reach 92.20 ± 2.27% after one-hour adsorption at 25 °C.ConclusionsWe have developed an efficient and inexpensive way to produce β-glucosidase for potential industrial applications by using the combination of light induction, cell autolysis, and CBM purification strategy.
Highlights
Background βGlucosidases (EC 3.2.1.21) are a heterogeneous group of enzymes that hydrolyze β-1,4-glycosidic bond in disaccharides, oligosaccharides, aryl, and alkyl β-glucosides, and release non-reducing terminal glucosyl residues [1, 2]
Similar trends were obtained in terms of enzyme activity and cell growth, regardless of either T7 promoter or light activated cassette was used for bgl1A(A24S/F297Y) expression (Fig. 2)
Our results indicated that the binding between Bgl1A(A24S/F297Y)-cellulose-binding module (CBM) and cellulose could be accomplished within 1 h (Fig. 6)
Summary
Background βGlucosidases (EC 3.2.1.21) are a heterogeneous group of enzymes that hydrolyze β-1,4-glycosidic bond in disaccharides, oligosaccharides, aryl-, and alkyl β-glucosides, and release non-reducing terminal glucosyl residues [1, 2]. Β-glucosidases have attracted considerable interests because of their potential applications in a variety of biotechnological processes, such as production of ethanol from agricultural wastes [3, 4], release of aromatic compounds from flavorless glycosidic precursors [5], and synthesis of useful β-glucosides [1], etc. Production of β-glucosidases from native sources has been a great challenge due to low level enzyme expression and high costs in protein purification [2, 7]. To overcome these disadvantages, heterologous host strains have been employed to produce β-glucosidases. A number of bottlenecks exist when large scale production of β-glucosidases (from E. coli) is needed. As E. coli and T7 promoter are the most frequently used prokaryotic expression combinations, the addition of chemical inducers such as isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of about 1 mM or more is needed to achieve maximal production of the desired
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