Cryomilled electrospun nanofiber mats containing d-mannitol exhibit suitable for aerosol delivery of proteins.

Dry powder inhalers (DPIs) are the first choice for inhalation drug development. However, some conventional DPI formulation processes require heating, which may damage high molecular weight drugs such as proteins and nucleic acids. In this study, we propose a novel DPI preparation process that avoids the use of heat. Dry powders were prepared by cryomilling nanofiber mats composed of polyvinyl alcohol, D(-)-mannitol (Man), and α-chymotrypsin (α-Chy) as the model drug using the electrospinning method. The addition of Man conferred high dispersibility and excellent in vitro aerosol performance to the nanofiber mat powder in a very short milling time (less than 0.5 min) as assessed using the Andersen cascade impactor. Powders were classified according to the degree of friability, and among these, nanofiber mats containing 15 % Man and milled for 0.25 min exhibited the highest aerosol performance. Nanofiber mats containing Man milled for less than 0.5 min also exhibited greater α-Chy enzymatic activity than a nebulized α-Chy solution. Furthermore, single inhalation induced no significant lung tissue damage as evidenced by lactate dehydrogenase activity assays of mouse bronchoalveolar lavage fluid. This novel DPI formulation process may facilitate the safe and efficient inhalational delivery of therapeutic proteins.

Identification and Characterization of a Pepsin- and Chymotrypsin-Resistant Peptide in the α Subunit of the 11S Globulin Legumin from Common Bean (Phaseolus vulgaris L.).

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

Chemical modification of α-chymotrypsin enabling its release from alginate hydrogel by electrochemically generated local pH change.

This study investigates the controlled release of α-chymotrypsin from an alginate hydrogel matrix. When protein molecules entrapped in the hydrogel matrix have a size smaller than the hydrogel pores, their hold/release from the polymer matrix are controlled by the electrostatic interaction between the guest molecules and host polymer. α-Chymotrypsin, as a model protein, was chemically modified with negatively charged species to change its pI and to convert its attractive interaction with a negatively charged alginate hydrogel matrix to a repulsion interaction allowing its release by pH-triggered signal. Then, bulk pH changes and electrochemically controlled local pH changes resulting from oxygen reduction were used for the controlled release of the enzyme from the alginate hydrogel. Three batches of modified α-chymotrypsin with different linker/enzyme ratios were synthesized, and their release profiles were investigated. The activity of both unmodified and modified α-chymotrypsin was evaluated using a UV-visible spectrophotometer following the standard procedure for the enzymatic assay of α-chymotrypsin (EC 3.4.21.1) and compared across all batches. Direct infusion electrospray ionization mass spectrometry (DI ESI-MS) was used to analyze the protein modifications and their impact on the isoelectric point values.

Enhancing Chymotrypsin Activity and Stability of Capillary Immobilized Enzyme Microreactors Using Zeolitic Imidazolate Frameworks as Encapsulation Materials.

Open-tubular immobilized enzyme microreactors (OT-IMERs) are some of the most widely used enzyme reaction devices due to the advantages of simple preparation and fast sample processing. However, the traditional approaches for OT-IMERs preparation had some defects such as limited enzyme loading amount, susceptibility to complex sample interference, and less stability. Here, we report a strategy for the preparation of highly active and stable OT-IMERs, in which the single-stranded DNA-enzyme composites were immobilized in capillaries and then encapsulated in situ in the capillaries via zeolitic imidazolate frameworks (ZIF-L). The phosphate groups of the DNA adjusted the surface potential of the enzyme to negative values, which could attract cations, such as Zn2+, to promote the formation of ZIF-L for enzyme encapsulation. Using chymotrypsin (ChT) as a model enzyme, the prepared ChT@ZIF-L-IMER has higher activity and better affinity than the free enzyme and ChT-IMER. Moreover, the thermal stability, pH stability, and organic solvent stability of ChT@ZIF-L-IMER were much higher than those of free enzyme and ChT-IMER. Furthermore, the activity of ChT@ZIF-L-IMER was much higher than that of ChT-IMER after ten consecutive reactions. To demonstrate the versatility of this preparation method, we replaced ChT with glucose oxidase (GOx). The stability of GOx@ZIF-L-IMER was also experimentally demonstrated to be superior to that of GOx and GOx-IMER. Finally, ChT@ZIF-L-IMER was used for proteolytic digestion analysis. The results showed that ChT@ZIF-L-IMER had a short digestion time and high digestive efficiency compared with the free enzyme. The present study broadened the synthesis method of OT-IMERs, effectively integrating the advantages of metal-organic frameworks and IMER, and the prepared OT-IMERs significantly improved enzyme stability. All of the results indicated that the IMER prepared by this method had a broad application prospect in capillary electrophoresis-based high-performance enzyme analysis.

Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

A Multicomponent Mannich Reaction Catalyzed by Hydrolases Immobilized on Titanate Nanotubes.

This study presents an innovative method for synthesizing ß-amino carbonylated compounds, specifically 2-[phenyl(phenylamino)methyl] cyclohexanone, achieving high conversions and diastereomeric ratios. Using trypsin or α-chymotrypsin in both free and immobilized forms on titanate nanotubes (NtsTi), synthesized through alkaline hydrothermal methods, successful immobilization yields were attained. Notably, α-chymotrypsin, when free, displayed a diastereoselective synthesis of the anti-isomer with 97 % conversion and 16 : 84 (syn : anti) diastereomeric ratio, which slightly decreased upon immobilization on NtsTi. Trypsin, in its free form, exhibited diastereoselective recognition of the syn-isomer, while immobilization on NtsTi (trypsin/NtsTi) led to an inversion of diastereomeric ratio. Both trypsin/NtsTi and α-chymotrypsin/NtsTi demonstrated significant catalytic efficiency over five cycles. In conclusion, NtsTi serves as an effective support for trypsin and α-chymotrypsin immobilization, presenting promising prospects for diastereoselective synthesis and potential industrial applications. Furthermore, it offers promising prospects for the diastereoselective synthesis of 2-[phenyl(phenylamino)methyl] cyclohexanone through multicomponent Mannich reaction and future industrial application.

Thrombin has dual trypsin-like and chymotrypsin-like specificity.

BACKGROUND: The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates. OBJECTIVES: To establish if thrombin can cleave at Trp residues. METHODS: The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage. RESULTS: Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond. CONCLUSION: The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.

Native top-down mass spectrometry for monitoring the rapid chymotrypsin catalyzed hydrolysis reaction.

Enzymes play crucial roles in life sciences, pharmaceuticals and industries as biological catalysts that speed up biochemical reactions in living organisms. New catalytic reactions are continuously developed by enzymatic engineering to meet industrial needs, which thereby drives the development of analytical approaches for real-time reaction monitoring to reveal catalytic processes. Here, taking the hydrolase- chymotrypsin as a model system, we proposed a convenient method for monitoring catalytic processes through native top-down mass spectrometry (native TDMS). The chymotrypsin sample heterogeneity was first explored. By altering sample introduction modes and pHs, covalent and noncovalent enzymatic complexes, substrates and products can be monitored during the catalysis and further confirmed by tandem MS. Our results demonstrated that native TDMS based catalysis monitoring has distinctive strength on real-time inspection and continuous observation, making it a promising tool for characterizing more biocatalysts.