Enzymatic Activity Of Chymotrypsin Research Articles

Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis (Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins (Cry2A, Cry1C, Cry1Aa, and Cry1Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid (EDTA), PMSF, tosyl phenylalanine chloromethyl ketone (TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). EDTA could significantly inhibit the degradation of Cry2A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1C and Cry1Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.

Characterization of chymotrypsin–ι-carrageenan complex in aqueous solution: A solubility and thermodynamical stability study

The aim of this study is to report the results of research work on the molecular mechanism of complex formation between chymotrypsin and a negatively charged natural strong polyelectrolyte, ι-carrageenan, using spectroscopy techniques. The carrageenan–chymotrypsin complex showed a maximal non-solubility at pH around 4.50 with a stoichiometric ratio between 8 and 33g of chymotrypsin per g of carrageenan. These values were depended on the enzyme concentration, pH and ionic strength medium. The insoluble complex was redissolved by modifying the pH and by a NaCl concentration around 0.2M in agreement with a coulombic mechanism of complex formation. The non-soluble complex formation showed biphasic kinetics. A fast step was carried out around 10s and a coulombic mechanism takes place, and a slower step of around 120s, where participate only Van der Waals forces. The enzymatic activity of chymotrypsin was maintained even in the presence of carrageenan (0.005%, w/v).

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored “thiophilic-interaction chromatography” (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the “free” form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn 2+ ions (10 nM to 50 μM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn 2+ (10 μM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn 2+ (5 μM and lower), the enzymatic activity was enhanced.

Degradation of insulin-like growth factors in small intestine of suckling rats

Insulin-like growth factors (IFGs), IGF-I and IGF-II, present in mammalian milk, play an important role during gastrointestinal tract development. In this study we identified and localized the activities of the common intestinal proteolytic enzymes and investigated their degradation effect on IGFs. Results indicated that the enzymatic activities of chymotrypsin, trypsin, and elastase progressed from the lowest in the duodenum, to the highest in the midjejunum, and declined in the ileum. Chymotrypsin exhibited the greatest IGFs degradation activities in neonatal intestinal lumen followed by elastase. These data furnish a potential strategic design to supplement IGFs into milk formulas.

Increased Chymotrypsin Activity in AOT/Bile Salt Reversed Micelles

Enzymatic activity of chymotrypsin in AOT reversed micelles is facilitated by the addition of a bile salt cosurfactant, sodium taurocholate (NaTC). NaTC diversifies the interfacial properties of the reversed micelles and increases their water capacity, resulting in a more favorable environment for enzymatic catalysis. The reaction velocity for the hydrolysis of the substrateN-glutaryl-l-phenylalaninep-nitroanilide (N-GPNA) by chymotrypsin more than doubles when NaTC is added to AOT reversed micelles in heptane. The enzymatic reaction obeys Michaelis–Menten kinetics in AOT/heptane reversed micelles over the range of NaTC concentrations studied and within a concentration range of 0.05–0.30 mMN-GPNA. NaTC causes changes in the enzyme turnover number,kcat, the Michaelis constant,KM, and the catalytic efficiency of the enzyme,kcat/KM, that are generally consistent with increased enzymatic activity. Similar effects are seen in dodecane, suggesting that exchange of reactants and products among aqueous pools is not a rate-limiting factor in this system.

Novel preparation method for surfactant-coated enzymes using W/O emulsion

A novel preparation method for surfactant-coated enzymes has been developed using a W/O emulsion. The enzymatic activity of chymotrypsin in isooctane significantly increased with the coating of surfactants. The surfactant-coated chymotrypsin showed a high enzymatic activity for amidation, although powdered chymotrypsin did not show the activity. Further, the coated enzyme showed a remarkably high storage stability.

Correction - Enzymatic Activity of Chymotrypsin and Its Poly(Ethylene Glycol) Conjugates Toward Low and High Molecular Weight Substrates

Correction - Enzymatic Activity of Chymotrypsin and Its Poly(Ethylene Glycol) Conjugates Toward Low and High Molecular Weight Substrates