Adaptability to the environment of protease by secondary structure changes and application to enzyme-selective hydrolysis.

The reactions involving enzymes are significantly influenced by various environmental factors. Clarity of how the activity and structure of proteases impact their function is crucial for more efficient application of enzymes as a tool. The impact of temperature, pH, and ionic strength on changes in protease activity, secondary structure, and protein conformation during enzymatic hydrolysis were investigated in this study. The enzymatic activity and secondary structure of acid-base protease were found to undergo significant modifications under different physical conditions, as demonstrated by UV spectrophotometry and FTIR spectroscopy analysis. Specifically, variations in α-helix and ß-fold content were observed to correlate with changes in enzyme activity. Molecular simulation analysis revealed that physical conditions have varying effects on the protease, particularly influencing enzyme activity and secondary structure. Evaluation of the proteases indicated alterations in both enzyme activity and structure. This treatment selectively hydrolyzed ß-lactoglobulin and reduced sensitization. These findings offer novel perspectives on the functionalities and regulatory mechanisms of proteases, as well as potential industrial applications.

Characterization of proteases from Irpex lacteus grown on minimally denatured soybean meal.

BACKGROUND: Acid and thermal stabilities are important properties for the preparation of acidic protein beverage. It is an important method for enzymatic modification to improve the functional properties of protein. Irpex lacteus protease showed a selective hydrolysis to soy proteins. The purpose of this study was to investigate the mechanism of enzymatic hydrolysis and its effects on acid and thermal stabilities of soy proteins. RESULTS: The I. lacteus protease selectively hydrolyzed the α and α' subunits of the native soybean ß-conglycinin (7S globulin) to produce products that presented as the 55 kDa band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences of 55 kDa polypeptides were analyzed in gel multi-enzyme digestion followed by liquid chromatography-mass spectrometry. By matching the multi-enzyme digestion peptides with the published polypeptide chain sequences of the α and α' subunits, it was confirmed that the 55 kDa polypeptides were formed by eliminating amino acid residues on both sides of the N- and C-terminals. From the published protein structure database (https://www.uniprot.org/), it is known that the cleaved peptide bonds were in extension regions. Non-selective enzyme hydrolysis of both ß-conglycinin (7S globulin) and glycinin (11S globulin), with corresponding drastic increases in the degree of hydrolysis, was observed when the substrates were preheated to the denaturation degree of 40% and above. However, 55 kDa hydrolyzed products and B polypeptides showed some extent of resistance to the proteolysis by I. lacteus protease even if denaturation degree was 100%. Both selective and non-selective hydrolysis of soy proteins by I. lacteus protease improved the acid and heat stabilities under the same hydrolysis conditions (enzyme/substrate ratio, time, and temperature). CONCLUSION: Enzymatic hydrolysis of soybean proteins by the I. lacteus protease can effectively improve the acid and thermal stabilities of proteins. This discovery is significant to avoid aggregation during processing in the beverage industry. In the near future, the protease has potential application value for modification of other proteins. © 2022 Society of Chemical Industry.

Optimization of Selective Hydrolysis of Cruciferins for Production of Potent Mineral Chelating Peptides and Napins Purification to Valorize Total Rapeseed Meal Proteins.

Preventing oxidation and microbial spoilage are both major concerns in food industries. In this context, this study aimed to valorize the total rapeseed meal proteins with controlled enzymatic proteolysis to generate potent mineral-chelating peptides from cruciferins while keeping intact the antimicrobial napins. Implementation of proteolysis of total rapeseed protein isolate with the Prolyve® enzyme highlighted an interesting selective hydrolysis of the cruciferins. Hence, the mechanism of this particular hydrolysis was investigated through a Design of Experiments method to obtain a model for the prediction of kinetics (cruciferin degradation and napin purity) according to the operating conditions applied. Then, multicriteria optimization was implemented to maximize the napin purity and yield while minimizing both enzymatic cost and reaction time. Antioxidant assays of the peptide fraction obtained under the optimal conditions proved the high metal-chelating activity preservation (EC50 = 247 ± 27 µg) for more than three times faster production. This fraction might counteract lipid oxidation or serve as preventing agents for micronutrient deficiencies, and the resulting purified napins may have applications in food safety against microbial contamination. These results can greatly help the development of rapeseed meal applications in food industries.

Unequal Behaviour between Hydrolysable Functions of Nirmatrelvir under Stress Conditions: Structural and Theoretical Approaches in Support of Preformulation Studies.

Nirmatrelvir is an antiviral drug approved for the treatment of COVID-19. The available dosage form consists of tablets marketed under the brand name PAXLOVID®. Although knowledge of nirmatrelvir's intrinsic stability may be useful for any potential development of other pharmaceutical forms, no data regarding this matter is available to date. Preliminary forced degradation studies have shown that the molecule is stable under oxidative and photolytic conditions, while hydrolytic conditions, both acidic and basic, have proven deleterious. Indeed, the molecule presents a priori several functions that can undergo hydrolysis, i.e., three amide moieties and a nitrile function. However, considering the degradation products formed under forced conditions and which were detected and identified by LC-UV-HRMSn, the hydrolysis process leading to their formation is selective since it involved only 2 of the 4 hydrolysable functions of the molecule. Ab initio studies based on density functional theory (DFT) have helped better understand these reactivity differences in aqueous media. Some hydrolyzable functions of nirmatrelvir differ from others in terms of electrostatic potential and Fukui functions, and this seems to correlate with the forced degradation outcomes.

MALDI-TOF Mass Spectrometric Detection of SARS-CoV-2 Using Cellulose Sulfate Ester Enrichment and Hot Acid Treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here we report a novel strategy for the rapid detection of SARS-CoV-2 based on an enrichment approach exploiting the affinity between the virus and cellulose sulfate ester functional groups, hot acid hydrolysis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Virus samples were enriched using cellulose sulfate ester microcolumns. Virus peptides were prepared using the hot acid aspartate-selective hydrolysis and characterized by MALDI-TOF MS. Collected spectra were processed with a peptide fingerprint algorithm, and searching parameters were optimized for the detection of SARS-CoV-2. These peptides provide high sequence coverage for nucleocapsid (N protein) and allow confident identification of SARS-CoV-2. Peptide markers contributing to the detection were rigorously identified using bottom-up proteomics. The approach demonstrated in this study holds the potential for developing a rapid assay for COVID-19 diagnosis and detecting virus variants from a variety of sources, such as sewage and nasal swabs.

A new lipase (Alip2) with high potential for enzymatic hydrolysis of the diester diethyladipate to the monoester monoethyladipate.

Several putative lipase genes from the genome of the yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) LS3 were overexpressed in the yeast itself and screened for the desymmetrization of the dicarboxylic acid diester diethyl adipate (DEA) into the monoester monoethyl adipate (MEA). MEA can serve as a monomeric spacer group for functional polymers used in medical chemistry and dental applications. The selected lipase Alip2-c6hp was intracellularly located. After overexpression of the corresponding gene, it was purified and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. In fed-batch cultivation with constructed yeast strain B. raffinosifermentans G1212/YRC102-Alip2-c6h for large scale production of the Alip2-c6hp biocatalyst enzyme activities up to 674 U L-1 were reached. Several tested diesters were hydrolyzed selectively to monoesters. Under optimized conditions, the purified enzyme Alip2p-c6h converted 96 % of the substrate DEA to MEA within 30 min incubation, whereby only 1.6 % of the unwanted side-product adipic acid (AA) was formed. At room temperature the dicarboxylic acid esters diethyl malonate (DEM), diethyl succinate (DES), dimethyl adipate (DMA) and dimethyl suberate (DMSub) were completely hydrolyzed to their corresponding monoesters. A high yield of 87 % and 25 % could also be achieved with the dioldiesters 1,4-diacetoxybutane (DAB) and diacetoxyhexane (DAH). In conclusion the potential of the lipase Alip2-c6hp expressed in B. raffinosifermentans is very promising for selective hydrolysis of DEA to MEA as well as for the production of other monoesters.

Microwave Assisted Selective Hydrolysis of Polyamides from Multicomponent Carpet Waste.

Selective hydrolysis of polyamide-6 (PA-6) and polyamide-66 (PA-66) from commercial multicomponent PA-6/PA-66/polypropylene (PP) carpet is demonstrated by a microwave-assisted acid catalyzed hydrothermal process, yielding monomeric products and solid polypropylene residue. First, an effective method is established to chemically recycle neat PA-6 and PA-66 granules using microwave irradiation. The optimized, hydrochloric acid (HCl) catalyzed process leads to selective production of monomers, 6-aminocaproic acid or adipic acid and hexamethylenediamine, after only 30 min. A piece of commercial carpet is then recycled using the same reaction conditions, but with the alteration of the reaction time from 1 to 6 h. The produced water-soluble products and the remaining solid residue are carefully characterized, proving that the polyamide-part of the carpet is selectively hydrolyzed into water-soluble monomers and the polypropylene-part remains as an unconverted solid that can be further used to produce recycled filaments containing the carpet residue and virgin polypropylene. The developed process opens the possibility to recycle multicomponent materials, such as carpets, through selective hydrolysis. It can also contribute to a circular economy, producing original monomers and materials ready for a new life-cycle.

Enzymatic enrichment of n-3 polyunsaturated fatty acid glycerides by selective hydrolysis.

Most natural oils are low in n-3 polyunsaturated fatty acids (n-3 PUFAs) content, which limits their application in health products. In this study, n-3 PUFAs in glyceride form were selectively enriched by lipase-mediated hydrolysis of n-3 PUFA-containing oils. First, commercial lipases were screened, and the lipase AY "Amano" 400SD from Candida cylindracea was the best choice in producing n-3 PUFA glycerides from tuna oil. Subsequently, the hydrolysis conditions were optimized. Under the optimal conditions, the highest n-3 PUFA content in the glyceride fraction was found to be 57.7% after enzymatic hydrolysis. Addition of Ca2+ to the system significantly shortened the reaction time from 10 to 4 h. When algal oil was used as substrate, total PUFA contents in the glyceride fraction were 89.9%. This study provides an efficient enzymatic process to produce n-3 PUFA-enriched glyceride concentrates and demonstrates that AY "Amano" 400SD can effectively discriminate against n-3 PUFAs during hydrolysis.

Highly regio- and enantio-selective hydrolysis of two racemic epoxides by GmEH3, a novel epoxide hydrolase from Glycine max.

A novel epoxide hydrolase from Glycine max, designated GmEH3, was excavated based on the computer-aided analysis. Then, gmeh3, a GmEH3-encoding gene, was cloned and successfully expressed in E. coli Rosetta(DE3). Among the ten investigated rac-epoxides, GmEH3 possessed the highest and best complementary regioselectivities (regioselectivity coefficients, αS = 93.7% and ßR = 97.2%) in the asymmetric hydrolysis of rac-m-chlorostyrene oxide (5a), and the highest enantioselectivity (enantiomeric ratio, E = 55.6) towards rac-phenyl glycidyl ether (7a). The catalytic efficiency (kcatS/KmS = 2.50 mM-1 s-1) of purified GmEH3 for (S)-5a was slightly higher than that (kcatR/KmR = 1.52 mM-1 s-1) for (R)-5a, whereas the kcat/Km (5.16 mM-1 s-1) for (S)-7a was much higher than that (0.09 mM-1 s-1) for (R)-7a. Using 200 mg/mL wet cells of E. coli/gmeh3 as the biocatalyst, the scale-up enantioconvergent hydrolysis of 150 mM rac-5a at 25 °C for 1.5 h afforded (R)-5b with 90.2% eep and 95.4% yieldp, while the kinetic resolution of 500 mM rac-7a for 2.5 h retained (R)-7a with over 99% ees and 43.2% yields. Furthermore, the sources of high regiocomplementarity of GmEH3 for (S)- and (R)-5a as well as high enantioselectivity towards rac-7a were analyzed via molecular docking (MD) simulation.

Application of cyclodextrinase in non-complexant production of γ-cyclodextrin.

The production of γ-cyclodextrin usually includes the utilization of organic complexants. However, the non-complexant production of γ-cyclodextrin is always being explored due to the defects of organic complexants. However, in non-complexant production, the separation of γ-cyclodextrin from α- and ß-cyclodextrin is still a challenge. Here, the selective hydrolysis ability of a cyclodextrinase designated PpCD (cyclodextrinase from Palaeococcus pacificus) on α-cyclodextrin, ß-cyclodextrin, and γ-cyclodextrin was proved. The kcat /Km values of PpCD for α-cyclodextrin and ß-cyclodextrin were roughly 12-fold and 5-fold higher than that of γ-cyclodextrin. It was proved that PpCD had selective hydrolysis ability and its γ-cyclodextrin purification performance was apparent on various simulated cyclodextrin mixtures with reported proportions derived from different CGTases. Besides, the hydrolysis temperature was optimized and it could be seen that 85°C was appropriate for the production of γ-cyclodextrin. In addition, the production of γ-cyclodextrin was achieved by using PpCD in the γ-CGTase reaction products.

Comparison of selective hydrolysis of α-lactalbumin by acid Protease A and Protease M as alternative to pepsin: potential for ß-lactoglobulin purification in whey proteins.

The experiments reported in this research paper examine the potential of digestion using acidic enzymes Protease A and Protease M to selectively hydrolyse α-lactalbumin (α-La) whilst leaving ß-lactoglobulin (ß-Lg) relatively intact. Both enzymes were compared with pepsin hydrolysis since its selectivity to different whey proteins is known. Analysis of the hydrolysis environment showed that the pH and temperature play a significant role in determining the best conditions for achievement of hydrolysis, irrespective of which enzyme was used. Whey protein isolate (WPI) was hydrolysed using pepsin, Acid Protease A and Protease M by randomized hydrolysis conditions. Reversed-phase high performance liquid chromatography was used to analyse residual proteins. Regarding enzyme selectivity under various milieu conditions, all three enzymes showed similarities in the reaction progress and their potential for ß-Lg isolation.

A novel semi-synthesis of spinetoram-J based on the selective hydrolysis of 5,6-dihydro spinosyn A.

Spinetoram is a novel kind of fermentation-derived insecticide with a broad range of action against lots of insect pests. Due to the broad pest spectrum, high insecticidal activity, low environmental impact, and low toxicity to non-target species, spinetoram has been widely applied in pest control and stored-grain protection. Spinetoram is a mixture of spinetoram-J (XDE-175-J, major component) and spinetoram-L (XDE-175-L). In this study, a novel selective hydrolysis of C9-glycosidic bond of 5,6-dihydro spinosyn A was developed. And a 3-step semi-synthesis of spinetoram-J based on the selective hydrolysis was designed and discussed. All compounds in the synthesis were characterized by 1H NMR, 13C NMR and mass spectrum. In addition, the improved semi-synthesis also provides an efficient way to synthesize the rhamnose replacement analogues of spinetoram-J which may be promising in the pest control and fungus control.

Lipase-catalyzed selective enrichment of omega-3 polyunsaturated fatty acids in acylglycerols of cod liver and linseed oils: Modeling the binding affinity of lipases and fatty acids.

Present study employed molecular modeling method to elucidate the binding affinity of lipases with fatty acids of different chain lengths; and investigated the effects of lipases positional and fatty acids specificity on omega-3 polyunsaturated fatty acids (ω-3 PUFAs) enrichment in cod liver and linseed oils. Among the lipases studied, molecular modeling showed the active sites of Candida rugosa lipase (CRL) had a low C-Docker interactive energy for saturated (SFA) and monounsaturated (MUFA) fatty acids which predicted CRL to have highest preferences to selectively hydrolyze resulting in efficient enrichment of ω-3 PUFAs. Verification experiments showed the SFA and MUFA in the acylglycerol fraction includes monoacylglcyerols (MAG), diacyglycerols (DAG), and triacylglycerols (TAG) of CRL-hydrolyzed cod liver oil decreased from the initial 25.21 to 16.88% and 45.25 to 32.17%, respectively. In addition, CRL-hydrolyzed cod liver oil demonstrated 88.36% of ω-3 PUFAs enrichment. The regio-distribution of fatty acids in CRL-hydrolyzed cod liver oil were not significantly different than that of cod liver oil indicating the ω-3 PUFAs enrichment was due to fatty acids selectivity and not positional selectivity of CRL.

Synthesis of trilobatin from naringin via prunin as the key intermediate: acidic hydrolysis of the α-rhamnosidic linkage in naringin under improved conditions.

Trilobatin [4'-(ß-D-glucopyranosyloxy)-2',4",6'-trihydroxydihydrochalcone] was synthesized from commercially available naringin in three steps with an overall yield of 30%. The key step was the acid-catalyzed site-selective hydrolysis of terminal α-rhamnopyranosidic linkage in neohesperidose involved in naringin under controlled conditions, by applying a high-pressure steam sterilizer.

Impact of the environmental conditions and substrate pre-treatment on whey protein hydrolysis: A review.

Proteins in solution are subject to myriad forces stemming from interactions with each other as well as with the solvent media. The role of the environmental conditions, namely pH, temperature, ionic strength remains under-estimated yet it impacts protein conformations and consequently its interaction with, and susceptibility to, the enzyme. Enzymes, being proteins are also amenable to the environmental conditions because they are either activated or denatured depending on the choice of the conditions. Furthermore, enzyme specificity is restricted to a narrow regime of optimal conditions while opportunities outside the optimum conditions remain untapped. In addition, the composition of protein substrate (whether mixed or single purified) have been underestimated in previous studies. In addition, protein pre-treatment methods like heat denaturation prior to hydrolysis is a complex phenomenon whose progression is influenced by the environmental conditions including the presence or absence of sugars like lactose, ionic strength, purity of the protein, and the molecular structure of the mixed proteins particularly presence of free thiol groups. In this review, we revisit protein hydrolysis with a focus on the impact of the hydrolysis environment and show that preference of peptide bonds and/or one protein over another during hydrolysis is driven by the environmental conditions. Likewise, heat-denaturing is a process which is dependent on not only the environment but the presence or absence of other proteins.

Binuclear copper(II) complexes discriminating epimeric glycosides and α- and ß-glycosidic bonds in aqueous solution.

Two chiral binuclear copper(II) complexes were synthesized and characterized for the first time as efficient chemoselective catalysts for the hydrolysis of aryl glycosides and disaccharides in aqueous solution at near neutral pH. Under these conditions, discrimination of epimeric aryl α-glycopyranosides was observed both by 29-fold different reaction rates and 3-fold different proficiency of the catalyst. Additionally, large differentiation of the nature of α- and ß- glycosidic bond in aryl glycosides as model compounds is apparent, but also noted in selected disaccharides. The influence of the chirality of the complexes and the role of the configuration of the carbohydrate upon interaction with the catalyst is discussed in detail. Lastly, a putative mechanism for the metal complex-catalyzed hydrolysis is derived from the experimental evidence pointing at deprotonation of the hydroxyl group at C-2 as a pre-requisite for glycoside hydrolysis.

Selective hydrolysis of hemicellulose from wheat straw by a nanoscale solid acid catalyst.

A nanoscale catalyst, solid acid SO4(2-)/Fe2O3 with both Lewis and Brønsted acidity was found to effectively hydrolyze hemicellulose while keeping cellulose and lignin inactive, and selective hydrolysis of hemicellulose from wheat straw by this catalyst was also confirmed. The factors that significantly affected hydrolysis process were investigated with response surface methodology, and the optimum conditions for time, temperature, and ratio of wheat straw to catalyst (w/w) were calculated to be 4.10h, 141.97°C, and 1.95:1, respectively. A maximum hemicellulose hydrolysis yield of 63.5% from wheat straw could be obtained under these conditions. In addition, the catalyst could be recycled six times with high activity remaining.

New atropisomeric amino alcohol ligands for enantioselective addition of diethylzinc to aldehydes.

Efficient synthesis of several new atropisomeric amino alcohols having 1-phenyl-1H-pyrrole skeleton are reported. Steric arrangements of the products were confirmed by a single-crystal X-ray measurement. The consequences of the size of the N-substituents on enantioinduction were examined by employing the enantioselective catalytic addition of diethylzinc to a series of substituted benzaldehydes (yields 91-97%, up to 85% enantiomeric excess). The special effect of the ortho methoxy group of the substrate on the enantioinduction is also interpreted.

Selective hydrolysis of flavonoid glycosides by Curvularia lunata.

Twelve flavonoid glycosides were involved in the biotransformation of the glycosyl moieties by Curvularia lunata 3.4381, and the products were analyzed by UPLC/PDA-Q-TOF-MS(E). Curvularia lunata displayed hydrolyzing activities on the terminal Rha or Glc units of some flavonoid glycosides. Terminal Rha with a 1 → 2 linkage of isorhamnetin-3-O-neohesperidoside and typhaneoside could be hydrolyzed by Curvularia lunata, but terminal Rha with a 1 → 6 linkage of rutin, typhaneoside, and quercetin-3-O-apiosyl-(1 → 2)-[rhamnosyl-(1 → 6)]-glucoside could not be hydrolyzed. Curvularia lunata could also hydrolyze the Glc of icariin, floramanoside B, and naringin. This is the first report of the hydrolysis of glycosyl units of flavonoid glycosides by Curvularia lunata. A new way to convert naringin to naringenin was found in this research.

Enzymatic production of trans fatty acid free fat from partially hydrogenated soybean oil (PHSO)--theory, strategy and practicability.

Development of an advanced process/production technology for healthful fats constitutes a major interest of plant oil refinery industry. In this work, a strategy to produce trans fatty acid (TFA) free (or low TFA) products from partially hydrogenated soybean oil by lipase-catalysed selective hydrolysis was proposed, where a physically founded mathematic model to delineate the multi-responses of the reaction as a function of selectivity factor was defined for the first time. The practicability of this strategy was assessed with commercial trans-selective Candida antarctica lipase A (CAL-A) as a model biocatalyst based on a parameter study and fitting to the model. CAL-A was found to have a selectivity factor 4.26 and to maximally remove 73.3% of total TFAs at 46.5% hydrolysis degree.