An endo-β-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1–20, a catalytic domain of glycoside hydrolase family 7 (GH7), a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1). The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0–6.0) and excellent stability at pH3.0–8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley β-glucan (2020 ± 9 U mg–1), moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 μg) reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.
Glycosylation is an efficient strategy to modulate the solubility, stability, bioavailability and bioactivity of drug-like natural products. Biological methods, such as whole-cell biocatalyst, promise a simple but highly effective approach to glycosylate biologically active small molecules with remarkable regio- and stereo-selectivity. Herein, we use the entomopathogenic filamentous fungus Isaria fumosorosea ACCC 37814 to biotransform a panel of phenolic natural products, including flavonoids and anthraquinone, into their glycosides. Six new flavonoid (4-O-methyl)glucopyranosides are obtained and structurally characterized using high resolution mass and nuclear magnetic resonance spectroscopic techniques. These compounds further expand the structural diversity of flavonoid glycosides and may be used in biological study.
A highly N-phosphonomethylglycine (glyphosate)-resistant Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in Escherichia coli strain ER2799 and transgenic tobacco.
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