Chymotrypsin Research Articles

Dual-Locked Chemiluminescent Probe Enables Precise Imaging and Timely Diagnosis of Colitis via Chymotrypsin/Vanin-1 Cascade Activation.

The development of precise diagnosis and the discovery of individualized drugs go together to provide effective therapy against inflammatory bowel disease (IBD). The exploitation of the unique imaging advantages of chemiluminescent probes represents a pivotal strategy for achieving this goal. Nevertheless, the dual-locked strategy, which is believed to enhance precision, is rarely employed in the design of chemiluminescent probes. A novel dual-locked chemiluminescent probe, BPan-CL, was designed based on IBD candidate biomarkers chymotrypsin (CHT) and vanin-1. BPan-CL exhibited specific reactivity and chemiluminescence response when subjected to simultaneous stimulation of CHT and vanin-1, with a signal-to-noise ratio superior to that of the fluorescent probe with the same dual-locked mode. In both live cell and IBD mice imaging, BPan-CL demonstrated superior sensitivity compared to its single-locked counterpart, Pan-CL. In contrast to Pan-CL, BPan-CL was able to more accurately identify IBD and healthy mice by in vivo imaging and allowed for early prediction of IBD using a noninvasive fecal test. BPan-CL has identified CHT and vanin-1 as valuable combinatorial biomarkers for accurate and early IBD diagnosis. This strategy has significant potential for use in biomedical imaging and future individualized therapies.

Artificial Zymogen Based on Protein-Polymer Hybrids.

This study explores the synthesis and application of artificial zymogens using protein-polymer hybrids to mimic the controlled enzyme activation observed in natural zymogens. Pro-trypsin (pro-TR) and pro-chymotrypsin (pro-CT) hybrids were engineered by modifying the surfaces of trypsin (TR) and chymotrypsin (CT) with cleavable peptide inhibitors utilizing surface-initiated atom transfer radical polymerization. These hybrids exhibited 70 and 90% reductions in catalytic efficiency for pro-TR and pro-CT, respectively, due to the inhibitory effect of the grafted peptide inhibitors. The activation of pro-TR by CT and pro-CT by TR resulted in 1.5- and 2.5-fold increases in enzymatic activity, respectively. Furthermore, the activated hybrids triggered an enzyme activation cascade, enabling amplification of activity through a dual pro-protease hybrid system. This study highlights the potential of artificial zymogens for therapeutic interventions and biodetection platforms by harnessing enzyme activation cascades for precise control of catalytic activity.

Expression of a novel NaD1 recombinant antimicrobial peptide enhances antifungal and insecticidal activities

This study aimed to increase the antifungal and insecticidal activities of NaD1, as an antimicrobial peptides (AMP), by improving its interaction with the fungal cell wall and chitin monomeric units in insect midguts. Hence, the chitin-binding domains (CBDs) of wheat germ agglutinin protein (WGA) were fused to either N- or C-terminus of NaD1 generating transgenic Nicotiana tabacum hairy roots (HRs). Molecular assessments confirmed the integration of NaD1 transgenes, their transcription and production of recombinant peptides in the HR lines. Total protein of (CBD)4-NaD1 and NaD1-(CBD)4 transgenic lines inhibited the growth of Pyricularia oryzae mycelium, suggesting that fusion of CBD to NaD1 can increase NaD1 half-life, leading to higher affinity toward cell wall chitin. Furthermore, feeding the third-instar larvae of Chilo suppressalis with both (CBD)4-NaD1 and NaD1-(CBD)4 extracts exhibited a higher mortality rate. Both NaD1-CBDs caused a significant decrease in trypsin (TRY) and chymotrypsin (CTR) activities in the larvae, while enhancing the activity of antioxidant enzymes CAT, POD, APX, and SOD. Therefore, feeding the larvae by total extract of NaD1-(CBD)4 and (CBD)4-NaD1 HR lines probably increased affinity to midgut chitin in C. suppressalis, enhancing insecticidal activities. Overall, the results indicate that recombinant peptides are effective in enhancing fungal and insect resistance.

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.

Chymotrypsin etched ultrasmall gold nanoclusters for dual response diagnosis and deeply penetrated chemodynamic therapy of pancreatic cancer

Pancreatic cancer (PC) is a highly invisible malignancy for which the early diagnosis is crucial to achieve the best treatment outcome. Abnormalities in serum chymotrypsin (ChT) are commonly associated with the development of PC, suggesting its potential role as a novel biomarker candidate. Herein, we report a ChT-responsive ultrasmall apo catalase-stabilized gold nanoclusters (AuCATapo NCs, core size ∼2 nm) with remarkable fluorescence and peroxidase (POD)-like activity. It can achieve "fluorescence" and "colorimetric" dual detection of ChT, with a wide detection range (0 – 100 μg/mL) and a low detection limit (0.029 μg/mL). Additionally, the weak acidity (pH 6.2 – 6.8) in the tumor microenvironment can meet the optimal catalytic capacity of deeply penetrated AuCATapo NCs, which generates cytotoxic reactive oxygen species to cause thorough tumor damage effects. The ChT-cleaved AuCATapo NCs may expose more catalytic active sites, resulting in higher tumor cytotoxicity, as demonstrated in both in vitro and in vivo models. Generally, these NCs probes can simultaneously realize accurate detection of ChT and efficient pancreatic tumor ablation via nanozyme based catalytic therapy, opening up a new idea of "diagnosis and treatment integration" for PC.

A non-peptide-based chymotrypsin-targeted long-wavelength emission fluorescent probe with large Stokes shift and its application in bioimaging

As a hydrolase, chymotrypsin (CHT) is involved in many physiological activities, and its abnormal activity is closely related to diabetes, pancreatic fibrosis, chronic pancreatitis and pancreatic cancer. In this work, an innovative long-wavelength emission fluorescent probe TCF-CHT was designed and synthesized for the high specificity detection of CHT, which utilized TCF-OH and a mimetic peptide substrate 4-bromobutyryl as chromogenic group and recognition group, respectively. TCF-CHT exhibited excellent selectivity and eye-catching sensitivity (8.91 ng/mL) towards CHT, “off-on” long-wavelength emission at 670 nm and large Stokes shift (140 nm). Furthermore, the successful fulfillment and perfect performance in imaging endogenous CHT in complex organisms (P815 cells, HepG2 cells, zebrafish and tumor-bearing mice) verified its potential as a powerful tool for the recognition of CHT in complicated biological environments.

The Photoperiod Significantly Influences the Growth Rate, Digestive Efficiency, Immune Response, and Antioxidant Activities in the Juvenile Scalloped Spiny Lobster (Panulirus homarus)

This study aimed to elucidate the effects of different photoperiods (0 L:24 D, 6 L:18 D, 12 L:12 D, 18 L:6 D, 24 L:0 D, “Light (L) and Dark (D)”) on the growth performance and physiological responses of the juvenile scalloped spiny lobster (Panulirus homarus). Over a period of 56 days, parameters such as growth rate, digestive enzyme, immune enzyme, and antioxidant enzyme were meticulously evaluated in 90 lobsters subjected to these varying light conditions. The present study found no significant differences in survival rate (SR), molting frequency (MF), and meat yield production (MYP) among the various photoperiod treatments (p > 0.05). Notably, the highest weight gain rate (WGR) and specific growth rate (SGR) were observed under a 12 L:12 D photoperiod. In the continuous dark phase (0 L:24 D), pepsin (PEP) activity remained high in gastric tissues, while trypsin (TRYP) and chymotrypsin (CHT) activities reached the highest in hepatopancreas tissues. The α-amylase (AMS) activity in the hepatopancreas was most elevated under 18 L:6 D, and the optimal lipase (LPS) activity was recorded under 12 L:12 D. The activity of acid phosphatase (ACP) in the hepatopancreas was highest in the absence of light (0 L:24 D), whereas the activities of alkaline phosphatase (AKP) and lysozyme (LZM) were most effective under the 12 L:12 D photoperiod. The total antioxidant capacity (T-AOC), along with catalase (CAT) and superoxide dismutase (SOD) activities of the hepatopancreas reached the highest at 12 L:12 D. The highest activity of glutathione peroxidase (GSH-Px) was seen under 18 L:6 D. The concentration of malondialdehyde (MDA), a marker of oxidative stress, was found to be highest under 12 L:12 D. Consequently, this specific photoperiod is essential for achieving optimal growth and maintaining appropriate physiological balance in the scalloped spiny lobster during aquaculture. These findings provide a foundational guideline for establishing the lighting environment in the farming of the juvenile scalloped spiny lobster.

Enhancement of functional properties, digestive properties, and in vitro digestion product physiological activity of extruded corn gluten meal by enzymatic modification.

Enzymatic modification is an effective means of improving the functional properties, digestive properties, and in vitro digestion product physiological activity of proteins, thus significantly expanding protein uses in various food applications. In this study, the addition of chymotrypsin (CT) at pH 9.0 and 11.0 was found to significantly improve the functional properties (solubility, foaming properties, water holding capacity, oil holding capacity, etc.) and digestive properties of extruded corn gluten meal (ECGM). Similar changes were observed when treating ECGM with glutaminase, protein glutaminase, and papain. These changes were likely due to the increase in number of carboxyl groups and the multiple effects of change in protein net charge and conformation caused by enzymatic deamidation. Of note, ECGM deamidated by CT showed the highest degree of deamidation, solubility, and gastrointestinal digestibility at pH 11.0, up to 44.92%, 43.75%, and 82.22%, respectively. In addition, CT-ECGM digestion product exhibited strong antioxidant activity and potential to promote alcohol metabolism in both a static digestion model and dynamic digestion model, even comparable to commercial corn peptides (CCP), while being inexpensive and of low bitterness compared to CCP. Meanwhile, the physiological activity enhanced as the molecular weight of digestion product decreased with the digested component having strongest activity. This study may promote the application of ECGM as a food component in the food industry or even as a substitute for CCP. © 2023 Society of Chemical Industry.

Cellulose acetate microwell plates for high-throughput colorimetric assays.

Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene (PS) microwell plates form a part of this waste. These plates are the backbone of high throughput colorimetric measurements in academic, research, and healthcare settings for detection/quantification of wide-ranging analytes including proteins, carbohydrates, nucleic acids, and enzyme activity. Polystyrene (PS) microwell plates serve as a platform for holding samples and reagents, where mixing initiates chemical reaction(s), and the ensuing color changes are quantified using a microplate reader. However, these plates are rarely reused or recycled, contributing to the staggering amounts of plastic waste generated in scientific laboratories. Here, we are reporting the fabrication of cellulose acetate (CA) microwell plates as a greener alternative to non-biodegradable PS plates and we demonstrate their application in colorimetric assays. These easy to fabricate, lighter weight, customizable, and environmentally friendly plates were fabricated in 96- and 384-well formats and made water impermeable through chemical treatment. The plates were tested in three different colorimetric analyses: (i) bicinchoninic acid assay (BCA) for protein quantification; (ii) chymotrypsin (CT) activity assay; and (iii) alkaline phosphatase (AP) activity assay. Color intensities were quantified using a freely available smartphone application, Spotxel® Reader (Sicasys Software GmbH). To benchmark the performance of this platform, the same assays were performed in commercial PS plates too and quantified using a UV/Vis microplate reader. The two systems yielded comparable linear correlation coefficients, LOD and LOQ values, thereby validating the CA plate-cell phone based analytical method. The CA microwell plates, coupled with smart phone optical data capture, provide greener, accessible, and scalable tools for all laboratory settings and are particularly well-suited for resource- and infrastructure-limited environments.

Formulation of tablets containing lyophilized powder to improve the stability of alpha-chymotrypsin

This study aimed to develop a stable lyophilized formulation and the tableting process of lyophilized powder to improve the stability of the enzyme alpha-chymotrypsin (ACT). The impacts of the bulking agent and stabilizer on the freeze-drying process and the stability of ACT were determined. The effects of the filler excipients and the compression force on the activity of ACT tablets containing lyophilized powder were also evaluated. The findings revealed that arginine HCl, which had a multipoint interaction with the surface of ACT, and the crystalline excipient mannitol stabilized the structure of the enzyme and the lyophilized cake. The dehydration process through freeze-drying effectively protected the active substance against denaturing factors such as heat, moisture, and mechanical force. The study also demonstrated that the use of elastic fillers such as compressuc and a minor compression force contributed to the enhancement of ACT tablet stability. In summary, the study successfully developed a formulation for tablets containing ACT lyophilized powder, initially overcoming the poor stability of this enzyme to environmental factors, showing the applicability of lyophilization and stabilizing excipients in enhancing the stability of biomolecules.

The effect of cold atmospheric plasma treatment on the aggregation and conformational change of pepsin and alpha-chymotrypsin under physiological pH condition

Pepsin and alpha-chymotrypsin (ACT) are two common proteolytic enzymes responsible for the quality degradation in seafood postmortem. Cold atmospheric plasma (CAP) is a new non-thermal technology for maintaining and improving food quality. The conformation and structural behavior of pepsin and ACT induced by potentially denaturing CAP were investigated through various spectrofluorometric assays. Circular dichroism (CD) spectra indicated that the secondary structure of pepsin and ACT were significantly changed (p < 0.05), and spectrofluorometric assays on surface hydrophobicity, fluorescence emission, and fluorescence quenching constants indicated remarkable but varied tertiary structure alterations after treatment. The activities of pepsin and ACT decreased by 95.2% and 92.1% after CAP treatment. Phase diagram plots indicated that structural and conformational dynamics of pepsin and ACT were all-or-none processes leading to inactivation, and strongly indicated protein unfolding upon CAP treatment.

New Insights on the Size-Dependent Inhibition of Enzymes by Gold Nanoparticles.

Particle size might affect the inhibition behaviors of gold nanoparticles (AuNPs) on enzyme activity by influencing the density of binding sites (ρ), the association constant (Ka), the steric hindrance of enzymes by AuNPs, the binding orientations of the enzyme on AuNPs, as well as the structural changes of enzymes. In previous studies, the effects of the above-mentioned factors, which could not be ignored in the applications of enzymatic electrochemistry, were often overshadowed by the effects of surface area. In order to study the size effect on the inhibition types and inhibitory ability of enzymes by AuNPs, we investigated the inhibition behaviors of chymotrypsin (ChT) by AuNPs with three different sizes (D1-AuNCs, D3-AuNPs, and D6-AuNPs) under the same surface area concentration. The results showed that both of the inhibition types and the inhibition ability varied with the particle size of AuNPs. D1-AuNCs inhibited ChT noncompetitively, while D3/D6-AuNPs inhibited ChT competitively. Contrary to the common sense, D6-AuNPs showed a weaker inhibitory ability than D3-AuNPs. By means of zeta potential, agarose gel electrophoresis, isothermal titration calorimetry, synchronous fluorescence spectroscopy, and circular dichroism, the mechanism of the weak inhibitory ability of D6-AuNPs was found to be the standing binding orientation caused by the small curvature. This work had certain guiding significance for the biosafety of AuNPs, the development of nanoinhibitors, as well as the applications of AuNPs in enzymatic electrochemistry.

Effects of dietary Clostridium butyricum on the growth performance and intestinal health of hybrid groupers (Epinephelus lanceolatus♂ × E. fuscoguttatus♀)

This trial was performed to investigate the effects of Clostridium butyricum on growth and intestinal health of hybrid groupers (Epinephelus lanceolatus♂ × E. fuscoguttatus♀). 300 hybrid groupers were fed five diets containing C. butyricum at 0, 1 × 107, 2 × 107, 3 × 107, 4 × 107 CFU/g for 8 weeks. The results revealed that dietary C. butyricum significantly increased weight gain rate (WGR), specific growth rate (SGR) and relative feed intake (RFI) (P < 0.05). Moderate C. butyricum significantly promoted lipase (LPS), chymotrypsin (CTS), alpha amylase (α-AMS), total antioxidant capacity (T-AOC) activities and up-regulated intestinal manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), interleukin10 (IL-10), transforming growth factorβ (TGF-β), zonula occludens protein-2 (ZO-2), zonula occludens protein-3 (ZO-3), Occludin and Claudin-3a mRNA levels, while significantly decreased malondialdehyde (MDA) content and down-regulated intestinal Kelch-like-ECH-associated protein 1 (Keap1) and interleukin 8 (IL-8) mRNA levels (P < 0.05). 1 × 107 CFU/g C. butyricum significantly augmented villus length, muscle thickness and goblet cells in intestine of hybrid groupers (P < 0.05). 1 × 107 CFU/g C. butyricum significantly increased richness and diversity of intestinal microbiota (P < 0.05) and abundance of beneficial bacteria. Meanwhile, 1 × 107 CFU/g C. butyricum decreased the risk of conditional pathogenic bacteria to invade the host.

Paper-based flow sensor for detection of chymotrypsin and its inhibitors via viscosity change of gelatin

Chymotrypsin (CHT) is an important proteolytic enzyme that can quickly decompose denatured proteins. Herein, we reported a new, simple, rapid method for the detection of CHT and its inhibitors by a paper-based flow sensor for the first time. The principle of the sensor is the interconversion of solidification and dissolution of gelatin. Generally, the 3D structure of gelatin can be enzymatically hydrolyzed by CHT releasing internal water, leading to a gradual increase in the water mark coverage area. Conversely, water cannot be released and the water mark coverage area is 0 in the absence of CHT, which is attributed to the water being trapped in the gelatin hydrogel. The limit of detection of CHT is determined to be 0.8 U/mL. In addition, the detection of CHT in milk, screening of CHT inhibitors and determination of Bowman-Birk inhibitors in soymilk were successfully achieved. Interestingly, the sensor only requires gelatin hydrogel, pH indicator strips, and PVC plates. In summary, a rapid, high-throughput, low-cost strategy for the detection of CHT and screening of CHT inhibitors was developed. Moreover, this method also allows the detection of CHT in non-transparent samples such as milk and soymilk. Paper-based flow sensors are highly promising for applications in food quality control, new medicine discovery and disease diagnosis, which also have a high potential for commercialization.

Arg236 in human chymotrypsin B2 (CTRB2) is a key determinant of high enzyme activity, trypsinogen degradation capacity, and protection against pancreatitis

Pancreatic chymotrypsins (CTRs) are digestive proteases that in humans include CTRB1, CTRB2, CTRC, and CTRL. The highly similar CTRB1 and CTRB2 are the products of gene duplication. A common inversion at the CTRB1-CTRB2 locus reverses the expression ratio of these isoforms in favor of CTRB2. Carriers of the inversion allele are protected against the inflammatory disorder pancreatitis presumably via their increased capacity for CTRB2-mediated degradation of harmful trypsinogen. To reveal the protective molecular determinants of CTRB2, we compared enzymatic properties of CTRB1, CTRB2, and bovine CTRA (bCTRA). By evolving substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) inhibitory loop variants against the chymotrypsins, we found that the substrate binding groove of the three enzymes had overlapping specificities. Based on the selected sequences, we produced eight SGPI-2 variants. Remarkably, CTRB2 and bCTRA bound these inhibitors with significantly higher affinity than CTRB1. Moreover, digestion of peptide substrates, beta casein, and human anionic trypsinogen unequivocally confirmed that CTRB2 is a generally better enzyme than CTRB1 while the potency of bCTRA lies between those of the human isoforms. Unexpectedly, mutation D236R alone converted CTRB1 to a CTRB2-like high activity protease. Modeling indicated that in CTRB1 Met210 partially obstructed the substrate binding groove, which was relieved by the D236R mutation. Taken together, we identify CTRB2 Arg236 as a key positive determinant, while CTRB1 Asp236 as a negative determinant for chymotrypsin activity. These findings strongly support the concept that in carriers of the CTRB1-CTRB2 inversion allele, the superior trypsinogen degradation capacity of CTRB2 protects against pancreatitis.

Replacement of fishmeal with plant protein in the diets of juvenile lumpfish (Cyclopterus lumpus, L. 1758): Effects on digestive enzymes and microscopic structure of the digestive tract

This study investigated the effects of plant protein concentrates on the activity of digestive enzymes and microscopic structure of the intestine of juvenile lumpfish. The experiment was carried out using triplicate groups of lumpfish of 7 g average initial body weight, to 40 g average final weight, after 54 days. Four experimental diets were used; a fishmeal (FM) based control diet and three test diets with the plant protein concentrates; soy protein concentrate (SPC) and pea protein concentrate (PPC) (1:1 ratio) replacing FM in proportions of 25% (PP25), 50% (PP50) and 75% (PP75), respectively. Nested ANOVA showed several significant treatment differences in histomorphometry. Overall, fish fed test diets exhibited several changes such as shorter mucosal fold height (MFH) in the anterior intestine (AI), increased number of goblet cells (GCs) and width of lamina propria (WLP) in distal intestine (DI), compared to control. Principal component analysis (PCA) on histological indices showed that all three treatment groups (i.e. PP25, PP50 and PP75) had significantly altered overall intestinal architecture, compared to the control group. The observed enteritis was negatively related with the condition factor (CF). Regarding enzyme activities fewer changes were observed across diets. Fish fed the PP50 diet exhibited an increased activity of leucine aminopeptidase (LAP) in the pyloric caeca and a decrease on chymotrypsin (CHY) in the mid intestine (MI) compared to the control. Nevertheless, the overall changes captured by PCA on enzymes were associated only with condition factor (CF) and not with the diets. Overall, histological evaluation confirmed that lumpfish intestinal morphometry was significantly altered by plant protein ingredients at 25%, 50% and 75% levels, but replacing of FM up to 50% did not affected the growth and the enzyme activities up to 75% inclusion level.

RNA-seq analysis of the differential response to low-temperature stress in two morphs of mud crabs (Scylla paramamosain)

The mud crab (Scylla paramamosain) is an economically important crab species that is distributed along the southern coast of China. Previously we reported that various morphological types of mud crabs differentially adapt to temperature changes. This study investigates the effects of temperature on morphs with one or two spines (Sp1 and Sp2, respectively) on the outer margin of the carpus of cheliped at a low temperature (8 °C) and a control temperature (20 °C). The hepatopancreas were the transcriptome source, and a total of 81,853 unigenes were obtained by sequencing, with an average length of 420 bp, and an N50 of 1460 bp. Of these, 22.33 % were known genes. Under low-temperature stress, there were 361 differentially expressed genes (DEGs) between the two morphs; 96 and 265 were up- and down-regulated genes, respectively. There were no DEGs between the morphs at 20 °C. Functional enrichment analysis revealed that the DEGs encoded abundant metallocarboxypeptidase activity, extracellular space, proteolysis, and sequence-specific DNA binding, and were further enriched in signal pathway components, including ubiquinone and other terpenoid-quinone biosynthesis, phenylalanine metabolism, MAPK signaling pathway, apoptosis, and other signaling pathways. The gene expressions of acid phosphatase (ACP), chymotrypsin (CHY), serine carboxypeptidase (SCP), and trypsin (TRY) under low-temperature stress were detected. The expressions of ACP, CHY, and SCP gradually decreased with time. The ACP, CHY, SCP, and TRY gene expression of the Sp1 morph was lower than that of the Sp2 morph; however, the ACP gene expression of the Sp1 morph was higher than that of the Sp2 morph at several time points. In conclusion, adaptability differences between the morphs to low-temperature stress were confirmed, which will enable the selection of cold-tolerant, high-quality varieties of mud crabs for breeding.

Hybrid Mucin-Vaterite Microspheres for Delivery of Proteolytic Enzyme Chymotrypsin.

While the enteral delivery of proteolytic enzymes is widely established for combating many diseases as an alternative to antibiotic treatment, their local delivery only emerges as administration route enabling sustained release in a controlled manner on site. The latest requires the development of drug delivery systems suitable for encapsulation and preservation of enzymatic proteolytic activity. This study proposes hybrid microspheres made of mucin and biodegradable porous crystals of calcium carbonate (CC) as the carriers for chymotrypsin (CTR) delivery. CTR is impregnated into CC and hybrid CC/mucin (CCM) microspheres by means of sorption without any chemical modification. The loading of the CC with mucin enhances CTR retention on hybrid microspheres (adsorption capacity of ≈8.7mg g-1 vs 4.7mg g-1 ), recharging crystal surface due to the presence of mucin and diminishing the average pore diameter of the crystals from 25 to 8nm. Mucin also retards recrystallization of vaterite into nonporous calcite improving stability of CCM microspheres upon storage. Proteolytic activity of CTR is preserved in both CC and CCM microspheres, being pH dependent. Temperature-induced inactivation of CTR significantly diminishes by CTR encapsulation into CC and CCM microspheres. Altogether, these findings indicate promises of hybrid mucin-vaterite microspheres for mucosal application of proteases.

Chymotrypsin is a molecular target of insect resistance of three corn varieties against the Asian corn borer, Ostrinia furnacalis.

The Asian corn borer, Ostrinia furnacalis, is a serious insect pest that can infest corn leaves and stems. Due to its internal feeding behavior, its larvae are not exposed to insecticides that are usually sprayed for pest control. To minimize crop damage caused by O. furnacalis, improving insect resistance trait of corn has been considered as an optimal control tactic. This study screened 27 corn varieties for their insect resistance trait and selected three varieties of Ilmichal (IM), P3394, and Kwangpyeongok (KP) that showed insect resistance trait. O. furnacalis larvae did not show any significant difference in preference between these three insect-resistant corn varieties and a control susceptible variety. However, these resistant varieties after ingestion significantly interfered with larval development of O. furnacalis. This suggests that the insect resistance trait is induced by antibiosis, but not by antixenosis. Indeed, larvae fed with these varieties suffered from low chymotrypsin (CHY) activities in the midgut juice. To determine the target CHY inhibited by resistant corn varieties, a total of nine CHY genes (Of-CHY1~Of-CHY9) were predicted from the transcriptome of O. furnacalis. Six genes (Of-CHY1~Of-CHY6) were expressed in all developmental stages and tissues. Especially, Of-CHY3 was highly expressed in the midgut of O. furnacalis larvae. RNA interference (RNAi) using double-stranded RNA (dsRNA) specific to Of-CHY3 (2 μg of dsRNA injected to each L5 larva) resulted in significant reduction of Of-CHY3 expression level at 24 h post-treatment. Feeding L3 larvae with this dsRNA also significantly suppressed the expression level of Of-CHY3 and reduced its enzyme activity at 24 h post-treatment. A recombinant Escherichia coli expressing dsRNA specific to Of-CHY3 was constructed using L4440 vector. Feeding such recombinant bacteria suppressed the expression level of Of-CHY3 and prevented larval development of O. furnacalis. These results suggest that the three resistant varieties can produce a resistance factor(s) to inhibit the CHY activity of O. furnacalis and suppress larval growth. This study suggests that CHY might be an inhibition target in O. furnacalis for breeding insect-resistant corns.

Multi spectroscopy and molecular modeling aspects related to drug interaction of aspirin with alpha chymotrypsin; structural change and protease activity

Examining the structure of enzymes versus drugs can help us understand the effects of a drug or its side effects. In this study, we investigated the effect of Aspirin (N-Acetyl salicylic acid or NAS) on alpha chymotrypsin (α-Chy). NAS is a non-steroidal drug, that dose anti-inflammatory action by inactivating cyclooxygenase enzymes. It is also prescribed in medicine to treat fever and inflammation caused by wounds. α-Chy is a powerful secretory protease from the pancreas. In the study of Uv–Vis, the effect of different concentrations of NAS on α-Chy, NAS was able to increase absorption intensity of α-Chy. This is due the effect of NAS on the 3D structure of the α-Chy. In the fluorescence emission spectrum study, NAS caused the α-Chy emission to be quenched. Also, the type of perusal was considered static. Calculation of thermodynamic parameters showed that this reaction was performed spontaneously. The type of bond between NAS and α-Chy was hydrophobic forces. The kinetic reaction comes from the way of α-Chy activity illustrated that NAS acts as an activator. Other techniques performed included investigating the thermal stability of the enzyme (TM) in the presence of ligand. The melting temperature of α-Chy decreased in the presence of NAS, TM, and the enzyme became more unstable. Also, in the secondary structure study for α-Chy, in the complex with NAS, the alpha-helix value increased, indicating that the enzyme was partially open, as shown by the simulation and Tm results. This information suggests that NAS can activate and destabilize this enzyme by entering the gastrointestinal tract.