Enhanced biocatalysis mechanism under microwave irradiation in isoquercitrin production revealed by circular dichroism and surface plasmon resonance spectroscopy.

An efficient and rapid process for isoquercitrin production by hesperidinase-catalyzed hydrolysis of rutin was successfully developed under microwave irradiation detecting the affinity by circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy. A maximum isoquercitrin yield of 91.5±2.7% was obtained in 10min with the conditions of 10g/L hesperidinase, 2g/L rutin, 30°C and microwave power density 88.9W/L. Enzymatic reaction rate and Vm/Km in the microwave reactor were 6.34-fold higher than in a continuous flow microreactor and 1.24-fold higher than in a biphasic system. CD and SPR analysis results also showed that hesperidinase has a better selectivity and affinity (3.3-fold than in a batch reactor) to generate isoquercitrin under microwave irradiation. Microwave irradiation greatly improved the reaction efficiency and productivity, leading to a more positive economical assessment. The binding affinity indicates the presence of strong multivalent interactions between rutin and hesperidinase under microwave irradiation.

[The effect of magnetic fields on the activity of proteinases and glycosidases in the intestine of the crucian carp Carassius carassius].

It has been demonstrated by the example of the crucian carp (Carassius carassius) that a 1-hour stay of fish in a combined magnetic field with resonance parameters for calcium ions decreases the proteolytic and amylolytic activities of their intestinal enzymes. It has been found that a 1-hour exposure to a combined magnetic field with resonance parameters for potassium ions has almost no effect on the activity of proteinases, but it decreases the amylolytic activity. It has been noted that the activity of proteases and glycosidases is lower under hypomagnetic conditions. Upon the inversion of the vertical component of the geomagnetic field, the proteolytic activity of the intestinal mucosa in C. carassius decreases, while the amylolytic activity becomes higher compared to the control. Possible effects of magnetic fields on the activity of digestive hydrolases in fish are discussed.

[The effects of geomagnetic storms on proteinase and glycosidase activities in fish intestinal mucosa].

It has been demonstrated that the glycosidase activity of cyprinoid fishes (carp and crucian carp) exposed to a geomagnetic storm for up to 20 h considerably decreases; however, the proteinase activity is weakly altered (a statistically significant decrease in the enzyme activity has been observed only in fasting fish). An in vitro study of the effects of individual half hour intervals of the geomagnetic storm that correspond to the main and recovery phases on the same enzyme activities demonstrates the opposite trend. Independently of the experimental conditions, geomagnetic storms have been shown to influence the enzyme system of fasting fish negatively.

[The effect of urea, gamma- and UV-irradiation on physico-chemical characteristics of native and immobilized inulinase]. / Vozdeistvie mocheviny, gamma- i UV--izluchenii na fiziko-khimicheskie svoistva svobodnoi i immobilizovannoi inulazy.

The immobilization of inulinase by ionexchange AB-26 and AB-17-2P has been made by adsorbtion and glutaraldehyde methods. The effect of UV-radiation, carbamide and gamma-rays on the stability of native and immobilized enzyme has been investigated. The stability of inulinase in relation to denaturation agents has been shown to increase with the immobilization of ionexchange. The character of binding with the matrix affects greatly the stability of immobilized enzyme to physical factors.

Structural changes in a cryo-cooled protein crystal owing to radiation damage.

The high intensity of third-generation X-ray sources, along with the development of cryo-cooling of protein crystals at temperatures around 100 K, have made it possible to extend the diffraction limit of crystals and to reduce their size. However, even with cryo-cooled crystals, radiation damage becomes a limiting factor. So far, the radiation damage has manifested itself in the form of a loss of overall diffracted intensity and an increase in the temperature factor. The structure of a protein (myrosinase) after exposure to different doses of X-rays in the region of 20 x 10(15) photons mm(-2) has been studied. The changes in the structure owing to radiation damage were analysed using Fourier difference maps and occupancy refinement for the first time. Damage was obvious in the form of breakage of disulfide bonds, decarboxylation of aspartate and glutamate residues, a loss of hydroxyl groups from tyrosine and of the methylthio group of methionine. The susceptibility to radiation damage of individual groups of the same kind varies within the protein. The quality of the model resulting from structure determination might be compromised owing to the presence of radiolysis in the crystal after an excessive radiation dose. Radiation-induced structural changes may interfere with the interpretation of ligand-binding studies or MAD data. The experiments reported here suggest that there is an intrinsic limit to the amount of data which can be extracted from a sample of a given size.

The damaging effect of UV rays below 320 nm on the rabbit anterior eye segment. II. Enzyme histochemical changes and plasmin activity after prolonged irradiation.

Prolonged irradiation of the rabbit eyes with UVB rays (312 nm) caused serious enzymatic disturbances in the cornea and lens and the development of an inflammatory reaction in the whole anterior eye segment, particularly in the cornea. In the corneal stroma many inflammatory cells with high activities of acid glycosidases and lysosomal proteases were present. This was accompanied with significantly elevated plasmin activity in the tear fluid (1.6 IU/ml). Plasmin appeared also in the aqueous humour (0.8 IU/ml). For the treatment of these changes catalase (1 mg/1 ml saline), aprotinin (100 micrograms/1 ml saline) and catalase-aprotinin mixture (1:1) were applied on the eye surface during irradiation. The catalase-aprotinin mixture was most efficient and decreased plasmin activity in the tear fluid and diminished disturbances of the anterior eye segment. Obviously both, active oxygen species and elevated plasmin activity in the tear fluid contribute to the damage of the anterior eye segment and development of intracorneal inflammation after irradiation of the eye with UVB rays.

The radiation-induced inactivation of external yeast invertase in dilute aqueous solution.

The inactivation of external yeast invertase by irradiation in dilute aqueous solution has been investigated. The contributions of the individual radical species from water radiolysis to inactivation and amino acid degradation were estimated from the results of experiments in which solutions were saturated with nitrogen, nitrous oxide or oxygen, and on addition of hydroxyl radical scavengers. Under conditions where inactivation by hydroxyl radicals predominates, the rate of inactivation increased with increasing dose, indicating that in the initial stages of the radiolysis the mannose-rich oligosaccharide chains of the glycoprotein protect the polypeptide chain from radical attack. Amino acid analysis of the irradiated external invertase showed that there was significant destruction of tyrosine, phenylalanine, methionine and histidine residues. Destruction of methionine and histidine residues may be responsible for the free radical-induced inactivation of this enzyme.

Radiation inactivation of the glycoprotein, invertase.

The radiation target size for invertase activity has been determined for the Saccharomyces cerevisiae glycoprotein which contains 50% carbohydrate. Identical inactivation curves were observed for the native enzyme as well as samples depleted of carbohydrate by incubation with Endo-beta-N-acetylglucosaminidase H. The functional unit of 120,000 daltons was unaltered by the per cent of oligosaccharide cleaved by the enzyme, or by the presence or absence of the released sugars. The irradiated samples showed no change in hexose content even after radiation exposures which grossly destroyed enzymatic activity. Reducing sugars appeared in the irradiated samples, indicating radiation damage to the oligosaccharides. These results unequivocally identify the enzymatically functional portion of the invertase molecule as the polypeptide homodimer, independent of the covalently-bound carbohydrates, and indicate that transfer of radiation energy from protein to oligosaccharide or vice versa is inefficient.