Autolysis control and structural changes of purified ficin from Iranian fig latex with synthetic inhibitors.

The fig's ficin is a cysteine endoproteolytic enzyme, which plays fundamental roles in many plant physiological processes, and has many applications in different industries such as pharmaceutical and food. In this work, we report the inhibition and activation of autolysis and structural changes associated with reaction of ficin with iodoacetamide and tetrathionate using high-performance liquid chromatography (HPLC), ultra filtration membrane, and dynamic light scattering (DLS) methods. The ficin structural changes were also determined using UV-absorption, circular dichroism (CD), fluorescence spectroscopy, and differential scanning calorimetry (DSC) techniques. These techniques demonstrated that iodoacetamide completely inhibited ficin autolysis, which was irreversible. However, tetrathionate partially and reversibility inhibited its autolysis. The ficin structural changes with two synthetic inhibitors were associated with secondary structural changes related to decreased alpha-helix and increased beta sheet and random coil conformations, contributing to its aggregation.

Influence of wheat-based diets supplemented with xylanase, bile acid and antibiotics on performance, digestive tract measurements and gut morphology of broilers compared with a maize-based diet.

1. The aim of the present study was to compare wheat-based diets supplemented with arabinoxylanase, bile acid and virginiamycin with a maize-based diet in their effects on the performance of broilers. 2. The diets were fed to 5 groups of broilers. Group A received a maize-based diet (control), and groups B, C, D and E were fed on wheat-based diets. Diets C, D and E were supplemented with arabinoxylanase (150 mg/kg), bile acid (500 mg/kg) and virginiamycin (200 mg/kg), respectively. 3. A total of 450 1-d-old Ross 385 chicks were randomly divided into 5 groups, each with three pens as replicates (30 birds per pen). The experimental diets were given for 18 d. On d 18, three chickens from each group were slaughtered by cervical dislocation and the liver weight, and duodenal, jejunal and ileal lengths were measured for length and width of the villi and the height of crypts. 4. Arabinoxylanase, bile acid and virginiamycin supplementation significantly increased feed intake and daily gain but had no significant effect on feed conversion ratio. The liver weight was significantly greater in virginiamycin supplemented chicks as compared with those on the diet containing bile acid. 5. Virginiamycin inclusion increased full and empty caeca weight significantly compared with groups A and D. Group A had the lightest caeca. Significant effects of diets were observed on duodenal, ileal and intestinal length, but jejunal length was not affected by diet. The heights of duodenal and ileal, but not jejunal, villi were significantly affected by the treatments. The lengths of villi in groups B and E were significantly higher than other groups. The mean height of ileal villi in group A was significantly higher than in groups receiving the wheat-based diets. The mean width of duodenal and ileal villi was significantly affected by the ration. The height of crypts in group B was significantly higher than in group E. 6. It could be concluded that supplementation of the wheat-based broiler ration with virginiamycin had significant positive effects on gastrointestinal tract characteristics for absorption. Supplementation with bile acid reduced the weight of liver and caeca and the length of duodenum, jejunum, ileum and in small intestine and the height and width of villi.

Chemical modification of bacterial alpha-amylases: changes in tertiary structures and the effect of additional calcium.

A comparative study was performed on the effect of calcium on native and chemically modified forms of mesophilic and thermophilic alpha-amylases. Circular dichroism (CD) and irreversible thermoinactivation studies were carried out in the absence and presence of 10 mM calcium. From the CD experiments, changes in the tertiary structure of these enzymes, brought about by modification, were concluded. Furthermore, these changes were found to be influenced by the presence of calcium. Sorbitol was very effective in affording protection against irreversible thermoinactivation of native and modified forms of the enzymes, both in the absence and presence of calcium. Results are discussed in terms of the usefulness of this new approach involving a combination of medium and chemical modification for protein stabilization and enhancement of catalytic potential.

Proteolysis of mesophilic and thermophilic alpha-amylases: a comparative study.

A comparative study was performed on limited and extensive proteolysis of mesophilic (from Bacillus amyloliquefaciens [BAA]) and thermophilic (from Bacillus licheniformis [BLA]) alpha-amylases using trypsin. As expected, the thermophilic enzyme showed greater resistance to digestion by the protease. While the catalytic potential of BLA was enhanced on proteolysis, that of BAA was diminished owing to this process. Combined with greater catalytic activity, a lower thermal stability was observed for BLA on proteolytic treatment. For both enzymes, the extent of proteolytic cleavage was reduced in the presence of various stabilizing agents. The digestion patterns are explained in terms of available information in the literature on the structure of these proteins, especially in relation to segmental mobility.

Chemical modification of lysine residues in Bacillus alpha-amylases: effect on activity and stability.

Chemical modification of lysine residues in two bacterial alpha-amylases, a mesophilic enzyme from Bacillus amyloliquefaciens (BAA) and a thermophilic enzyme from Bacillus licheniformis (BLA) was carried out using citraconic anhydride. 13 +/- 1 residues in BAA and 10 +/- 1 residues in BLA were found modified under defined experimental conditions. Modification brought about dramatic enhancement of thermal stability of BAA and catalytic activity of BLA. Such alterations were found dependent on the temperature and pH. Results obtained on Tm, the extent of deamidation, changes in the circular dichroism (CD) spectra and kinetic parameters before and after modification are discussed in terms of their contributions to the mechanism of irreversible thermoinactivation and activity enhancement.

Comparative studies on a mesophilic and a thermophilic alpha-amylase.

A comparative study was performed on thermal stability of mesophilic and thermophilic alpha-amylases, and the effects of various denaturing agents, organic solvents, and stabilizers were investigated. As expected, the thermophilic enzyme showed higher resistance toward denaturation in water as its natural medium, but such a difference could not be detected in nonaqueous environments. Furthermore, stability of these molecules was improved by including various stabilizing agents. Of the compounds tested, sorbitol provided the highest degree of protection, which was found to be owing to its effect on increasing Tm and its ability in totally preventing deamidation of amino acid residues in the protein molecules.