Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T5015, and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

Single-chain tandem macrocyclic peptides as a scaffold for growth factor and cytokine mimetics

Mimetics of growth factors and cytokines are promising tools for culturing large numbers of cells and manufacturing regenerative medicine products. In this study, we report single-chain tandem macrocyclic peptides (STaMPtides) as mimetics in a new multivalent peptide format. STaMPtides, which contain two or more macrocyclic peptides with a disulfide-closed backbone and peptide linkers, are successfully secreted into the supernatant by Corynebacterium glutamicum-based secretion technology. Without post-secretion modification steps, such as macrocyclization or enzymatic treatment, bacterially secreted STaMPtides form disulfide bonds, as designed; are biologically active; and show agonistic activities against respective target receptors. We also demonstrate, by cell-based assays, the potential of STaMPtides, which mimic growth factors and cytokines, in cell culture. The STaMPtide technology can be applied to the design, screening, and production of growth factor and cytokine mimetics.

Novel cysteine substitution p.(Cys1084Tyr) causes variable expressivity of qualitative and quantitative VWF defects

Von Willebrand factor (VWF) is an extremely cysteine-rich multimeric protein that is essential for maintaining normal hemostasis. The cysteine residues of VWF monomers form intra- and inter-molecular disulfide bonds that regulate its structural conformation, multimer distribution and ultimately its hemostatic activity. In this study we investigated and characterized the molecular and pathogenic mechanisms through which a novel cysteine variant p.(Cys1084Tyr) causes an unusual, mixed phenotype form of von Willebrand disease (VWD). Phenotypic data including bleeding scores, laboratory values, VWF multimer distribution and desmopressin response kinetics were investigated in 5 members (2 parents and 3 daughters) of a consanguineous family. VWF synthesis and secretion were also assessed in a heterologous expression system and in a transient transgenic mouse model. Heterozygosity for p.(Cys1084Tyr) was associated with variable expressivity of qualitative VWF defects. Heterozygous individuals had reduced VWF:GPIbM (<0.40IU/mL) and VWF:CB (<0.35IU/mL) as well as relative reductions in high-molecular weight multimers, consistent with type 2A VWD. In addition to these qualitative defects, homozygous individuals also displayed reduced FVIII:C/VWF:Ag leading to very low FVIII levels (0.03-0.1IU/mL) as well as reduced VWF:Ag (<0.40IU/mL) and VWF:GPIbM (<0.30IU/ml). Accelerated VWF clearance and impaired VWF secretion contributed to the fully expressed homozygous phenotype with impaired secretion arising due to disordered disulfide connectivity.

Reversible Protein Capture and Release by Redox-Responsive Hydrogel in Microfluidics

Stimuli-responsive hydrogels have a wide range of potential applications in microfluidics, which has drawn great attention. Double cross-linked hydrogels are very well suited for this application as they offer both stability and the required responsive behavior. Here, we report the integration of poly(N-isopropylacrylamide) (PNiPAAm) hydrogel with a permanent cross-linker (N,N′-methylenebisacrylamide, BIS) and a redox responsive reversible cross-linker (N,N′-bis(acryloyl)cystamine, BAC) into a microfluidic device through photopolymerization. Cleavage and re-formation of disulfide bonds introduced by BAC changed the cross-linking densities of the hydrogel dots, making them swell or shrink. Rheological measurements allowed for selecting hydrogels that withstand long-term shear forces present in microfluidic devices under continuous flow. Once implemented, the thiol-disulfide exchange allowed the hydrogel dots to successfully capture and release the protein bovine serum albumin (BSA). BSA was labeled with rhodamine B and functionalized with 2-(2-pyridyldithio)-ethylamine (PDA) to introduce disulfide bonds. The reversible capture and release of the protein reached an efficiency of 83.6% in release rate and could be repeated over 3 cycles within the microfluidic device. These results demonstrate that our redox-responsive hydrogel dots enable the dynamic capture and release of various different functionalized (macro)molecules (e.g., proteins and drugs) and have a great potential to be integrated into a lab-on-a-chip device for detection and/or delivery.

The Periplasmic Oxidoreductase DsbA Is Required for Virulence of the Phytopathogen Dickeya solani

In bacteria, the DsbA oxidoreductase is a crucial factor responsible for the introduction of disulfide bonds to extracytoplasmic proteins, which include important virulence factors. A lack of proper disulfide bonds frequently leads to instability and/or loss of protein function; therefore, improper disulfide bonding may lead to avirulent phenotypes. The importance of the DsbA function in phytopathogens has not been extensively studied yet. Dickeya solani is a bacterium from the Soft Rot Pectobacteriaceae family which is responsible for very high economic losses mainly in potato. In this work, we constructed a D. solani dsbA mutant and demonstrated that a lack of DsbA caused a loss of virulence. The mutant bacteria showed lower activities of secreted virulence determinants and were unable to develop disease symptoms in a potato plant. The SWATH-MS-based proteomic analysis revealed that the dsbA mutation led to multifaceted effects in the D. solani cells, including not only lower levels of secreted virulence factors, but also the induction of stress responses. Finally, the outer membrane barrier seemed to be disturbed by the mutation. Our results clearly demonstrate that the function played by the DsbA oxidoreductase is crucial for D. solani virulence, and a lack of DsbA significantly disturbs cellular physiology.

A New Practice to Monitor the Fabrication Process of Fab-Targeting Ligands from Bevacizumab by LC-MS: Preparation and Analytical Characterization

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as a model. The evaluation focused on the purification of fragments through a nonaffinity-based process using a centrifugal ultrafiltration technique and mild reduction conditions for the intact production of F(ab’) fragments with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, F(ab’) fragments with a defined chemical composition were successfully obtained via proteolytic digestion followed by a controlled reduction reaction process maintaining the integrity of the binding sites. The ultrafiltration purification technique appears to be suitable for the removal of the digestive enzyme but inefficient for the removal of Fc fragments, thus requiring additional processing. A suitable analytical strategy was developed, allowing us to demonstrate the reformation of disulfide bridges between the two reduced cysteines within F(ab’) fragments.

Molecular Identification of Keratinase DgokerA from Deinococcus gobiensis for Feather Degradation

Keratin is a tough fibrous structural protein that is difficult to digest with pepsin and trypsin because of the presence of a large number of disulfide bonds. Keratin is widely found in agricultural waste. In recent years, especially, the development of the poultry industry has resulted in a large accumulation of feather keratin resources, which seriously pollute the environment. Keratinase can specifically attack disulfide bridges in keratin, converting them from complex to simplified forms. The keratinase thermal stability has drawn attention to various biotechnological industries. It is significant to identify keratinases and improve their thermostability from microorganism in extreme environments. In this study, the keratinases DgoKerA was identified in Deinococcus gobiensis I-0 from the Gobi desert. The amino acid sequence analysis revealed that DgoKerA was 58.68% identical to the keratinase MtaKerA from M. thermophila WR-220 and 40.94% identical to the classical BliKerA sequence from B. licheniformis PWD-1. In vitro enzyme activity analysis showed that DgoKerA exhibited an optimum temperature of 60 °C, an optimum pH of 7 and a specific enzyme activity of 51147 U/mg. DgoKerA can degrade intact feathers at 60 °C and has good potential for industrial applications. The molecular modification of DgoKerA was also carried out using site-directed mutagenesis, in which the mutant A350S enzyme activity was increased by nearly 30%, and the results provide a theoretical basis for the development and optimization of keratinase applications.

Dual-Responsive Alginate Hydrogel Constructed by Sulfhdryl Dendrimer as an Intelligent System for Drug Delivery

Intelligent stimulus-triggered release and high drug-loading capacity are crucial requirements for drug delivery systems in cancer treatment. Based on the excessive intracellular GSH expression and pH conditions in tumor cells, a novel glutathione (GSH) and pH dual-responsive hydrogel was designed and synthesized by conjugates of glutamic acid-cysteine dendrimer with alginate (Glu-Cys-SA) through click reaction, and then cross-linked with polyethylene glycol (PEG) through hydrogen bonds to form a 3D-net structure. The hydrogel, self-assembled by the inner disulfide bonds of the dendrimer, is designed to respond to the GSH heterogeneity in tumors, with a remarkably high drug loading capacity. The Dox-loaded Glu-Cys-SA hydrogel showed controlled drug release behavior, significantly with a release rate of over 76% in response to GSH. The cytotoxicity investigation indicated that the prepared DOX-loaded hydrogel exhibited comparable anti-tumor activity against HepG-2 cells with positive control. These biocompatible hydrogels are expected to be well-designed GSH and pH dual-sensitive conjugates or polymers for efficient anticancer drug delivery.