High level expression and biochemical characterization of an alkaline serine protease from Geobacillus stearothermophilus to prepare antihypertensive whey protein hydrolysate

Background Proteases are important for hydrolysis of proteins to generate peptides with many bioactivities. Thus, the development of novel proteases with high activities is meaningful to discover bioactive peptides. Because natural isolation from animal, plant and microbial sources is impractical to produce large quantities of proteases, gene cloning and expression of target protease are preferred. Results In this study, an alkaline serine protease gene (GsProS8) from Geobacillus stearothermophilus was successfully cloned and expressed in Bacillus subtilis. The recombinant GsProS8 was produced with high protease activity of 3807 U/mL after high cell density fermentation. GsProS8 was then purified through ammonium sulfate precipitation and a two-step chromatographic method to obtain the homogeneous protease. The molecular mass of GsProS8 was estimated to be 27.2 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 28.3 kDa by gel filtration. The optimal activity of GsProS8 was found to be pH 8.5 and 50 °C, respectively. The protease exhibited a broad substrate specificity and different kinetic parameters to casein and whey protein. Furthermore, the hydrolysis of whey protein using GsProS8 resulted in a large amount of peptides with high angiotensin-I-converting enzyme (ACE) inhibitory activity (IC50 of 0.129 mg/mL). Conclusions GsProS8 could be a potential candidate for industrial applications, especially the preparation of antihypertensive peptides.

Enhanced biohydrogen production from cotton stalk hydrolysate of Enterobacter cloacae WL1318 by overexpression of the formate hydrogen lyase activator gene

Background Biohydrogen production from lignocellulose has become an important hydrogen production method due to its diversity, renewability, and cheapness. Overexpression of the formate hydrogen lyase activator ( fhlA ) gene is a promising tactic for enhancement of hydrogen production in facultative anaerobic Enterobacter . However, little work has been reported in terms of the fhlA gene cloning and expression in Enterobacter cloacae as well as the engineered strain for lignocellulose-based hydrogen production.Results The formate hydrogen lyase activator ( fhlA ) gene was cloned and overexpressed in Enterobacter cloacae WL1318, and the cumulative hydrogen production and dynamics, glucose and xylose consumption, cell growth, and soluble metabolites were analyzed in the wild and recombinant strains. The results showed that the recombinant strain significantly enhanced cumulative hydrogen production by 188% following fermentation of cotton stalk hydrolysate for 24 h, and maintained improved production above 30% throughout the fermentation process compared to the wild strain. Accordingly, overexpression of the fhlA gene resulted in an enhanced hydrogen production potential ( P ) and maximum hydrogen production rate ( R m ), as well as a shortened lag phase time ( λ ) for the recombinant strain. Additionally, the recombinant strain also displayed improved glucose (12%) and xylose (3.4%) consumption and hydrogen yield Y(H 2 /S) (37.0%) compared to the wild strain. Moreover, the metabolites and specific enzyme profiles demonstrated that reduced flux in the competitive branch, including succinic, acetic, and lactic acids, and ethanol generation, coupled with increased flux in the pyruvate node and formate splitting branch, benefited hydrogen synthesis.Conclusions The results conclusively prove that overexpression of fhlA gene in E. cloacae WL1318 can effectively enhance the hydrogen production from cotton stalk hydrolysate, and reduce the metabolic flux in the competitive branch. It’s the first attempt to engineer the fhlA gene in the hydrogen producing bacterium E. cloacae . This work provides a highly efficient engineered bacterium for biohydrogen production from fermentation of lignocellulosic hydrolysate in the future.

The senses

“The senses” is the introductory chapter of the book Sensory Transduction and describes early studies in the anatomy of sense organs and physiology of the senses. It introduces methods in physiology and molecular biology, which were critical tools used to unlock the secrets of sensation, including intracellular and extracellular recording, patch-clamp recording, gene cloning and expression, and structural studies such as cryo-EM. It concludes by describing in detail the discovery of the piezo proteins, which are the stretch-sensitive channels now known to be responsible for much of mammalian touch and proprioception.