BackgroundMost β-glucosidases reported are sensitive to the end product (glucose), making it the rate limiting component of cellulase for efficient degradation of cellulose through enzymatic route. Thus, there are ongoing interests in searching for glucose-tolerant β-glucosidases, which are still active at high glucose concentration. Although many β-glucosidases with different glucose-tolerance levels have been isolated and characterized in the past decades, the effects of glucose-tolerance on the hydrolysis of cellulose are not thoroughly studied.ResultsIn the present study, a novel β-glucosidase (Bgl6) with the half maximal inhibitory concentration (IC50) of 3.5 M glucose was isolated from a metagenomic library and characterized. However, its poor thermostability at 50 °C hindered the employment in cellulose hydrolysis. To improve its thermostability, random mutagenesis was performed. A thermostable mutant, M3, with three amino acid substitutions was obtained. The half-life of M3 at 50 °C is 48 h, while that of Bgl6 is 1 h. The Kcat/Km value of M3 is 3-fold higher than that of Bgl6. The mutations maintained its high glucose-tolerance with IC50 of 3.0 M for M3. In a 10-h hydrolysis of cellobiose, M3 completely converted cellobiose to glucose, while Bgl6 reached a conversion of 80 %. Then their synergistic effects with the commercial cellulase (Celluclast 1.5 L) on hydrolyzing pretreated sugarcane bagasse (SCB) were investigated. The supplementation of Bgl6 or mutant M3 to Celluclast 1.5 L significantly improved the SCB conversion from 64 % (Celluclast 1.5 L alone) to 79 % (Bgl6) and 94 % (M3), respectively. To further evaluate the application potential of M3 in high-solids cellulose hydrolysis, such reactions were performed at initial glucose concentration of 20–500 mM. Results showed that the supplementation of mutant M3 enhanced the glucose production from SCB under all the conditions tested, improving the SCB conversion by 14–35 %.ConclusionsThese results not only clearly revealed the significant role of glucose-tolerance in cellulose hydrolysis, but also showed that mutant M3 may be a potent candidate for high-solids cellulose refining.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0383-z) contains supplementary material, which is available to authorized users.

A novel β-galactosidase (Bgal1-3) was isolated from a marine metagenomic library and then its cross-linked enzyme aggregates (CLEAs) were prepared. The enzymatic properties of Bgal1-3-CLEAs were studied and compared with that of the free enzyme. The thermostability and storage stability of Bgal1-3 were significantly improved after it was immobilized as CLEAs. The galactose-tolerance of the enzyme was also enhanced after the immobilization, which could relieve the inhibitory effect and then tends to be beneficial for the galacto-oligosaccharides (GOS) synthesis. Moreover, higher GOS yield was achieved (59.4 ± 1.5%) by Bgal1-3-CLEAs compared to the free counterpart (57.1 ± 1.7%) in an organic-aqueous biphasic system. The GOS content and composition of the syrups synthesized by the free enzyme and Bgal1-3-CLEAs were similar and they both contained at least seven different oligosaccharides with the degree of polymerization (DP) ranging between 3 and 9. Furthermore, Bgal1-3-CLEAs maintained 82.1 ± 2.1% activity after ten cycles of reuse; the GOS yield of the tenth batch was 52.3 ± 0.3%, which was still higher than that of the most former reports. To the best of our knowledge, this is the first report on the GOS synthesis using CLEAs of β-galactosidase in an organic-aqueous biphasic system. The study not only further expands the application scope of CLEA, but also provides a potential catalyst for the synthesis of GOS with low cost.

Widespread utilization of polyethylene terephthalate (PET) has caused critical environmental pollution. The enzymatic degradation of PET is a promising solution to this problem. In this study, PETase, which exhibits much higher PET-hydrolytic activity than other enzymes, was successfully secreted into extracellular milieu from Bacillus subtilis 168 under the direction of its native signal peptide (named SPPETase). SPPETase is predicted to be a twin-arginine signal peptide. Intriguingly, inactivation of twin-arginine translocation (Tat) complexes improved the secretion amount by 3.8-fold, indicating that PETase was exported via Tat-independent pathway. To the best of our knowledge, this is the first report on the improvement of Tat-independent secretion by inactivating Tat components of B. subtilis 168 in LB medium. Furthermore, PET film degradation assay showed that the secreted PETase was fully active. This study paves the first step to construct an efficient engineered strain for PET degradation.

In this study, the twin-arginine (Tat) signal peptide PhoD was used to direct the secretion of the β-galactosidase Bgal1-3 into the growth medium of an engineered strain of Bacillus subtilis 168. After 24 h of cultivation, the extracellular activity reached 1.15 U/mL, representing 78% of the total activity. Bgal1-3 was exported via both Tat-dependent and Tat-independent pathways. To improve the secretion amounts, two more copies of the target gene were inserted into the designated loci on the chromosome, further improving the extracellular enzymatic activity to 2.15 U/mL. The engineered strain with three copies of bgal1-3 was genetically stable after 150 generations. To the best of our knowledge, this is the first report on the functional secretion of a heterologous protein via both Tat-dependent and Tat-independent pathways mediated by a Tat signal peptide in B. subtilis. Furthermore, this study provides us with a markerless engineered strain for the production of β-galactosidase.