Enhancing the Thermostability of Feruloyl Esterase EstF27 by Directed Evolution and the Underlying Structural Basis

To improve the thermostability of EstF27, two rounds of random mutagenesis were performed. A thermostable mutant, M6, with six amino acid substitutions was obtained. The half-life of M6 at 55 °C is 1680 h, while that of EstF27 is 0.5 h. The <i>K</i>_cat/<i>K</i>_m value of M6 is 1.9-fold higher than that of EstF27. The concentrations of ferulic acid released from destarched wheat bran by EstF27 and M6 at their respective optimal temperatures were 223.2 ± 6.8 and 464.8 ± 11.9 μM, respectively. To further understand the structural basis of the enhanced thermostability, the crystal structure of M6 is determined at 2.0 Å. Structural analysis shows that a new disulfide bond and hydrophobic interactions formed by the mutations may play an important role in stabilizing the protein. This study not only provides us with a robust catalyst, but also enriches our knowledge about the structure–function relationship of feruloyl esterase

MOESM1 of Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration

Additional file 1. Figure S1. Multiple sequence alignment of Bgl6 with other glucose tolerant β-glucosidases from GH1 family. Figure S2. Positive clones with improved thermostability. Figure S3. SDS-PAGE analysis of the recombinant Bgl6 and the mutants. Figure S4. Thin-Layer Chromatography (TLC) analysis of the hydrolysis of cellobiose and cello-oligosaccharide by Bgl6. Figure S5. Positions of the mutations in Bgl6. Figure S6. High-Performance Liquid Chromatography (HPLC) analysis of the concentration of the celllobiose released from SCB. Figure S7. Time course of the cellobiose concentrations in the SCB hydrolysis. Figure S8. Thin layer chromatography (TLC) analysis of the hydrolysis of hydrolysis products from SCB. Figure S9. Effects of glucose on the hydrolysis of pretreated SCB (10 %, w/v) by Celluclast 1.5 L (♦) alone and supplemented with mutant M3 (●). Table S1. Primers used to construct the mutants of Bgl6

Characterization of the Cross-Linked Enzyme Aggregates of a Novel β‑Galactosidase, a Potential Catalyst for the Synthesis of Galacto-Oligosaccharides

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