Salt Mediated Modulation of Autolysis of Thermolysin-Like Proteinase, Salilysin, Isolated from a Moderate Halophile, Chromohalobacter salexigens DSM3043.

Halophilic salilysin is first synthesized as a pro-form, which has been shown autolysis activity to process pro-region (55 amino acids long) three times to form intermediate 1 (I1), intermediate 2 (I2) and final mature (M) salilysin. The autolysis of I1- to M-form salilysin in vitro was significantly accelerated with increasing NaCl concentration up to 4 M. Strong salting-out salts, (NH4)2SO4, Na2SO4 and MgSO4, were more effective, suggesting that autolysis is enhanced by inter-molecular association or structure compaction or both. However, MgCl2, a salting-in salt, was also effective, suggesting that other mechanisms, such as charge shielding and ionic binding to this halophilic protein, operated. Autolytic cleavage at site 3 resulted in mixed formation of correctly and incorrectly processed mature forms in the absence of salt, indicating that salt affected the accuracy of autolytic cleavage reaction. Far UV circular dichroism (CD) measurements indicated that E167A pro-salilysin showed an identical CD spectrum to the wild-type mature salilysin, suggesting pro-form has a proper fold for proteolytic activity. Thermal scanning indicated that E167A pro-salilysin was more heat-stable by ~ 10 °C than mature form. The CD spectra, thermal stability and modeling structure of salilysin clearly suggested that pro-salilysin is folded to the same structure as native form and is functional for autolysis.

Mechanically Strong, Scalable, Mesoporous Xerogels of Nanocellulose Featuring Light Permeability, Thermal Insulation, and Flame Self-Extinction.

Scalability is a common challenge in the structuring of nanoscale particle dispersions, particularly in the drying of these dispersions for producing functional, porous structures such as aerogels. Aerogel production relies on supercritical drying, which exhibits poor scalability. A solution to this scalability limitation is the use of evaporative drying under ambient pressure. However, the evaporative drying of wet gels comprising nanoscale particles is accompanied by a strong capillary force. Therefore, it is challenging to produce evaporative-dried gels or "xerogels" that possess the specific structural profiles of aerogels such as mesoscale pores, high porosity, and high specific surface area (SSA). Herein, we demonstrate a structure of mesoporous xerogels with high porosity (∼80%) and high SSA (>400 m2 g-1) achieved by exploiting cellulose nanofibers (CNFs) as the building blocks with tunable interparticle interactions. CNFs are sustainable, wood-derived materials with high strength. In this study, the few-nanometer-wide CNFs bearing carboxy groups were structured into a stable network via ionic inter-CNF interaction. The outline of the resulting xerogels was then tailored into a regular, millimeter-thick, board-like structure. Several characterization techniques highlighted the multifunctionality of the CNF xerogels combining outstanding strength (compression E = 170 MPa, σ = 10 MPa; tension E = 290 MPa, σ = 8 MPa), moderate light permeability, thermal insulation (0.06-0.07 W m-1 K-1), and flame self-extinction. As a potential application of the xerogels, daylighting yet insulating, load-bearing wall members can be thus proposed.

Salt-enhanced processing, proteolytic activity and stability of halophilic thermolysin-like proteinase, salilysin, isolated from a moderate halophile, Chromohalobacter salexigens DSM3043.

Moderately halophilic bacterium, Chromohalobacter salexigens DSM3043, has a gene Csal_2537 encoding thermolysin-like M4 proteinase. This gene was cloned to pET expression vectors, resulting in high expression of recombinant proteinase, named as salilysin (salinity-dependent thermolysin-like proteinase), in Escherichia coli cytoplasm. This gene encodes precursor form of salilysin containing 348 amino acid residues (Pro-salilysin) consisting of 55 amino acids pro-sequence and following mature proteinase. Pro-sequence was cleaved three times to form intermediate 1, intermediate 2 and final mature salilysin. The processing rate was greatly accelerated in a salt concentration-dependent manner. Purified inactive mutant Pro-E167A-salilysin was correctly processed by purified mature salilysin, indicating that autolysis and inter-molecular processing occurred in its maturation processes. Proteolytic activity of mature salilysin against both peptide and protein substrates was also enhanced along with the addition of higher concentration of salt, 0-3.2 M NaCl, consistent with its halophilic origin. Mature salilysin was stabilized by ~8 °C in the presence of 1 M NaCl by thermal scanning using circular dichroism. One of the precursor form, intermediate 1, showed ~20 °C higher denaturation temperature than mature form, suggesting rigid and stable structure of this precursor form.

Nanocellulose Xerogels With High Porosities and Large Specific Surface Areas.

Xerogels are defined as porous structures that are obtained by evaporative drying of wet gels. One challenge is producing xerogels with high porosity and large specific surface areas, which are structurally comparable to supercritical-dried aerogels. Herein, we report on cellulose xerogels with a truly aerogel-like porous structure. These xerogels have a monolithic form with porosities and specific surface areas in the ranges of 71-76% and 340-411 m2/g, respectively. Our strategy is based on combining three concepts: (1) the use of a very fine type of cellulose nanofibers (CNFs) with a width of ~3 nm as the skeletal component of the xerogel; (2) increasing the stiffness of wet CNF gels by reinforcing the inter-CNF interactions to sustain their dry shrinkage; and (3) solvent-exchange of wet gels with low-polarity solvents, such as hexane and pentane, to reduce the capillary force on drying. The synergistic effects of combining these approaches lead to improvements in the porous structure in the CNF xerogels.