Isoflavone during protease hydrolysis of defatted soybean meal

The study examined the effects of proteases on the isoflavones during enzymatic hydrolysis of soybean flour. Protease itself did not affect the isoflavones during hydrolysis, whereas the applied conditions and contaminated β-glucosidase in the enzyme could greatly affect the content and composition of isoflavones. Soybean flour hydrolysis by ENZECO Alkaline Protease L-FG at high pH (10) resulted in complete loss of malonylglucosidic and acetylglucosidic conjugates in the hydrolysate. However, these conjugates such as 6″-O-malonylgenistin and 6″-O-malonyldaidzin remained as the principal compounds accounting for 66.2, 58.3 and 70.5% of the total isoflavones in the Protease M “Amano”, Alcalase 2.4L, and neutral enzyme ENZECO Neutral Protease-NBP-L hydrolysates, respectively, compared to that of 57.8% in the original soybean flour. The residue prepared by Protease M contained 10 times higher aglycones than that of soy flour, which was due to the contaminated β-glucosidase activity in the enzyme preparation. Our result showed that β-glucosidase contaminated in Protease M has a unique selectivity compared to that of the purified almond β-glucosidase. Results from the study indicated that hydrolysis of soybean flour may provide another alternative approach to enrich aglycone isoflavones in soybean-containing products.     

酶法制取骨照相明胶工艺的研究

酶法从牛骨中制取照相明胶的新工艺与传统的碱法相比具有生产周期短、好胶率高以及产品质量相近等特点。应用差热分析研究了胶原蛋白在骨头预处理过程中的变性情况;用颗粒分布仪研究了骨素的溶胀作用;用凝胶过滤色谱技术测定了明胶分子量的分布;应用响应曲面分析优化了酶解工艺条件即1g骨素粉加1u的蛋白酶于27℃,pH7.5左右下水解6h。并对该工艺所得明胶产品的理化性能和照相性能进行了测定,结果表明该产品可用作国标中惰(Ⅰ)型照相明胶。     

产中性蛋白酶菌株B4发酵条件的研究

对从土壤中分离纯化得到的一株芽孢杆菌B4进行了发酵条件的探索.通过单因素碳源、氮源以及正交试验确定了B4最佳培养基配方.实验结果表明不同的碳、氮源及其配比对产酶有极大的影响.最佳培养基配方为玉米粉2%、豆饼粉3%、麸皮3%、Na2HPO4·12H2O0.4%、KH2PO4 O.03%、ZnCl2O.1%、pH8.0,在最佳培养条件下,酶活由271.56 U/ml提高到2519.35 U/ml,提高了8倍多.同时还研究了金属离子、起始pH值、接种量对产酶的影响.     

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors**

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly “druggable” or relatively easy-to-drug target is the coronavirus Main Protease (3CL^pro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.     

Partial characterization of a chymotrypsin-like protease in the larger grain borer (Prostephanus truncatus (Horn)) in relation to activity of Hyptis suaveolens (L.) trypsin inhibitor

Hyptis suaveolens trypsin inhibitor (HSTI) inhibits, in vitro, most of the trypsin-like serine proteases present in the gut of Prostephanus truncatus (Horn), the larger grain borer. In order to measure its in vivo effectiveness, different doses of the purified HSTI were incorporated into artificial seeds to measure their effects on the growth of P. truncatus larvae and pupae. Results showed that this protease inhibitor had no significant effect in vivo. Consequently, we investigated, identified, and partially characterized an inhibitor-insensitive protease that could explain the growth of P. truncatus on diet containing HSTI. This protease had an apparent molecular mass of 31 kDa, an optimum pH of 7.0, and an activity range extending from pH 6.0 to 8.0, which would allow activity in the gut of P. truncatus. This enzyme could be one of the physiological mechanisms in P. truncatus that allow the species to avoid the adverse effects of trypsin-like protease inhibitors present in the normal diet.     

大豆蛋白酶解制寡肽过程中蛋白酶再利用的研究

研究了在水解大豆蛋白反应中采用吸附法回收再利用蛋白酶的可行性。结果表明：吸附法可有效回收Akalase碱性蛋白酶。首次大豆蛋白水解反应中黑曲霉酸性蛋白酶的加入量为6％g/g蛋白,Alcalase加入量为15眺蛋白。用大豆蛋白原料吸附回收蛋白酶进行二次大豆蛋白水解反应,酶的补加量为：酸性酶的加入量为5％(g／g),AlcalaseIsn入量为2．5μl/g。     

Effects of Kudoa spores,endogenous protease activity and frozen storage on cooked texture of minced Pacific hake (Merluccius productus)

The effects of Kudoa infection of Pacific hake (Merluccius productus) on endogenous protease activity and on cooked mince texture were investigated. Texture was significantly (p < 0.05) negatively correlated with spore counts as well as protease activity. Soft texture (maximum force <150 g) was observed in fish with 10⁴–10⁶Kudoa thyrsites spores g⁻¹ mince, compared to 10⁵–10⁸K. paniformis spores g⁻¹ mince, suggesting that especially for fish having lower infection levels, K. thyrsites may have a greater impact than K. paniformis on Pacific hake quality. Pre-incubation for 15 min at 52 °C prior to cooking resulted in softer texture in some samples due to endogenous proteolytic action. This pre-incubation effect was not consistently observed in fish held 6 months or longer at −25 °C or after freeze-thaw cycling, which may be explained by an opposing toughening effect attributed to protein denaturation and aggregation during prolonged or abusive frozen storage.     

Double liposomes: hypoglycemic effects of liposomal insulin on normal rats

The biopharmaceutical characteristics of double liposomes (DLs) containing insulin were examined, and the usefulness of DLs in combination with aprotinin is discussed. Encapsulation of insulin was influenced by lipid composition, and the highest efficiency was observed with positively charged liposomes. Insulin encapsulated in liposomes, especially in DLs, was protected from enzymatic proteolysis. A portion of insulin molecules was adsorbed on the surface of the membrane when liposomes were prepared using a lipid with a positive charge and was degraded by enzymes. Remarkable hypoglycemic effects were observed after intragastric administration of DLs containing insulin at a dose of 20 IU/kg to normal male Wistar rats. The highest mean relative efficacy to administration was obtained with insulin-loading DLs containing aprotinin as a protease inhibitor. These results suggest that DLs are applicable as an oral dosage form for peptide drugs such as insulin etc., especially in combination with protease inhibitors.     

Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates

Enzymatic hydrolysis of hemp protein isolate (HPI) by six proteases (alcalase, flavourzyme, neutrase, protamex, pepsin and trypsin) and antioxidant activities of the resulting hydrolysates, obtained for 2 and 4 h were investigated. The yield of trichloroacetic acid (TCA)-soluble peptides (Y_sp), protein composition and surface hydrophobicity (H_o) of the hydrolysates were evaluated. The results showed that the hydrolysates exhibited varying DPPH radical scavenging (with lowest IC₅₀, ∼2.3 mg/mL) and Fe²⁺⁺ chelating (with lowest IC₅₀ of 1.6–1.7 mg/mL) abilities and reducing power (with highest absorbance at 700 nm of 0.31–0.35), depending on their Y_sp and H_o values. The DPPH radical scavenging and Fe²⁺⁺ chelating abilities of the hydrolysates were positively correlated with their Y_sp or H_o values. The results suggest that enzymatic hydrolysis can be used as an effective technique to produce high value-added products of hemp proteins.     

Synthesis and Structure-Activity Relationships of N-(4-Benzamidino)-Oxazolidinones: Potent and Selective Inhibitors of Kallikrein-Related Peptidase 6

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) and 34 ((5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) have single-digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32 , effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.