New differences between isoenzymes A and B from Candida rugosa lipase

Water adsorption isotherms of pure lipases A and B from Candida rugosa are different and can be used to distinguish between the isoenzymes. The maximum esterification yield (50%, 20h) can be achieved at initial 0.9<1.0. Lipase B is more stereoselective (49% yield, 98% enantiomeri excess) than lipase A (47% yield, 72% enantiomerci excess) but both isoenzymes mainly esterify the (S) 2(4-isobutylphenyl)propionic acid (Ibuprofen).     

Improved stability of the lipase from Candida rugosa in different purification degrees by chemical modification

Summary Semipurified lipase of Candida rugosa and pure isoforms (lipase A and lipase B) have been chemically modified using two methodologies based on polyethyleneglycol (PEG). The activation of PEG with p-NO₂-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG in the enzymatic activity of the lipase. The chemical modification increases the stability of pure lipases in isooctane at 50 °C.     

Improvement of activity and stability of chloroperoxidase by chemical modification

Background  

Enzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water. These limit the industrial applications of enzymes.     

Improved thermal stability and activity in the cold-adapted lipase B from Candida antarctica following chemical modification with oxidized polysaccharides

In order to improve the thermal stability (t_1/2) and activity of lipase B from cold-adapted Candida antarctica (CALB), amino groups of the enzyme were chemically linked to a range of oxidized polysaccharides using a range of reducing agents. By chemically modifying CALB using 0.1% dextran (250 kDa) at pH 8.6 for 10 days using borane–pyridine complex as reducing agent, increased thermal stability (t_1/2, 168 min at 70°C) and activity (65% higher specific activity) was achieved compared to the unmodified enzyme (t_1/2, 18 min at 70°C). Improvements in thermostability were generally better with high molecular weight polymers such as dextran (40 and 250 kDa) or ficoll (70 and 400 kDa) in comparison to low molecular weight inulin (5 kDa). The shape of the polymer also appeared to be important with elongated, elipsoidal-shaped dextran providing better thermostabilization than spherical-shaped ficoll. Borane–pyridine complex was found to be a good, non-toxic reducing agent for improving thermostability, compared with sodium borohydride and sodium cyanoborohydride. An interesting finding was that, in all cases, specific activity of the modified enzymes increased with a concomitant increase in thermostability. This response defies the general principle of a trade-off between activity and stability, and demonstrates that chemical modification provides new avenues for improving the thermal stability of enzymes from psychrophiles without sacrificing their activity.     

Rapid purification of two lipase isoenzymes from Candida rugosa

Summary We describe a two-step method for the purification of two lipases (lipases A and B) from C. rugosa. The purification procedure includes Phenyl-Sepharose and Sephacryl HR 100 chromatographies. The enzymes obtained were pure according to criteria of specific activity and neutral sugar content.     

Effect of chemical modification on thermal stability of horseradish peroxidase]

Soluble preparations of horse radish peroxidase are obtained by means of its amino groups modification with glutaric aldehyde, maleic anhydride and inert proteins including albumin. The enzyme activity is found to decrease under the modification with glutaric aldehyde and to be unchanged at all other cases. Thermal stability of the enzyme preparations obtained is studied within the temperature range from 56 to 80 degrees C. Thermostability of glutaric aldehyde-modified peroxidase is approximately 2.5-fold decreased at 56 degrees C. Thermostability of other preparations exceeds the stability of native peroxidase in 25--90 times at 56 degrees C. Thermodynamic parameters of activation for the process of irreversible thermoinactivation of native and modified enzyme are calculated. A strong compensation effect between activation enthalpy and entropy values is observed, which were changed in 1.5--2 times, while the free activation energy is changed by 2--3 kcal/mol only. Possible mechanism of the change of the enzyme thermal stability under its chemical modification is discussed.     

Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes

The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.     

The effect of modification of lactate and malate dehydrogenases on their stability and activity

The inactivation of lactate and malate dehydrogenases (LDH and MDH) modified by progesterone in the water-dimethylformamide (DMF) medium is described by the first-order equation up to large conversion degrees. The MDH modification is accompanied by the increase of its stability by 7-14%, while LDH modification leads to the enzyme stability decrease by 67%. The enzymes catalytic activities are changed simultaneously. The main factors of the stability and activity changes are the DMF influence upon the quaternary structure of the proteins at the modification and a hydrofobization of the external and internal protein sites by progesterone.     

Effect of chemical modification of cellulose on the activity of a cellulase from Aspergillus niger

Five chemically modified forms of cellulose were prepared, characterized, and tested as substrates for a homogeneous glucanohydrolase from A. niger. The relative order of reactivity at pH 4.0 was DEAE = PEI > benzyl DEAE > cellulose > P > CM.The following abbreviations are used throughout the article: (RBB) Remazol brilliant blue R; (DEAE) diethylamino ethyl; (PEI) polethyleneimine; (CM) carboxymenthyl; (P) phospho; (DS) degree of RBB dye substitution of cellulose, in mol dye/100 glucose. This indicates that positively charged cellulose substrates are more susceptible to hydrolysis by the cellulase. This observation strengthens an earlier proposal that caroxyl groups on the enzyme are involved in substrate binding and catalytic action. Chemical modification is suggested as a method to increase the rate of enzymatic hydrolysis of cellulose, a process now in the commercial development stage.     

Thermodynamics of carbonic anhydrase catalysis. A comparison between human isoenzymes B and C

The CO2 hydration and HCO3- dehydration activities of human red cell carbonic anhydrase isozymes B and C (HCAB and HCAC) have been studied as a function of temperature from 0 degrees to 37 degrees C. The Arrhenius plots of ln kcat versus 1/T are linear for both isozymes in both hydration and dehydration reactions, indicating that the rate-determining steps remain unchanged over this temperature range. The 37 degrees C hydration kcat, at pH 7.5, is 13 X 10(5) s-1 for isozyme C and 0.71 X 10(5) s-1 for isozyme B. Km, for hydration, is 10 mM for C and 5 mM for B, and invariant with temperature. The uncatalyzed reactions are significantly affected by temperature, 30- to 40-fold rate enhancements being observed from 0 degrees to 37 degrees C. The enzyme-catalyzed processes are much less sensitive to temperature, the rate enhancements being 2- to 3-fold for HCAB and 5- to 6-fold for HCAC in this temperature range. These observations are consistent with a significant lowering of the free energy of activation by both isozymes. This effect is greater for C accounting for its higher catalytic power. The enthalpy of activation, at pH 7.5 and 8.2, in the rate-limiting step is considerably less for the B enzyme compared to C. This is, however, more than offset by a large negative entropy of activation in the case of HCAB. This observation indicates either a mechanistic difference in the rate-limiting events or a difference in the structural organizations of the active sites of the two isozymes, or both.     

Acid glycosaminoglycans and their chemical modification

The modem state of chemical modification of hyaluronic acid, chondroitin sulfates, and heparin is considered, and the possible application of modified glycosaminoglycans as potential drugs is discussed.     

Acid glycosaminoglycans and their chemical modification

The modern state of chemical modification of hyaluronic acid, chondroitin sulfates, and heparin is considered, and the possible application of modified glycosaminoglycans as potential drugs is discussed.     

Influence of chemical modification on enzyme inactivation kinetics and stability

Enzymes are placed in different categories depending on the effect of chemical modification on their inactivation kinetics and residual activity. This is done using a series-type mechanism involving degraded but stable enzyme states. The major distinction in the three basic categories is the effect of modification on residual activity. Each category is further sub-divided depending on the effect of modification on the values of the deactivation rate constants. The classification provides for a framework for comparison of a wide variety of enzyme deactivation data. Structure-function relations are provided wherever possible.     

Effect of chemical modification on struture and activity of glucoamylase fromAspergillus candidus andRhizopus species

The histidine, tyrosine, tryPtoPhan and carboxyl grouPs in the enzyme glucoamylase fromAsPergillus Candidus andRhizoPus sPecies were modified using grouP sPecific reagents. Treatment of the enzyme with diethylPyrocarbonate resulted in the modification of 0.3 and 1 histidine residues with only a slight loss in activity (10% and 35%) of glucoamylase fromAsPergillus candidus andRhizoPus sPecies resPectively. Modification of tyrosine either by N-acetylimidazole or [I¹²⁵]-leads to a Partial loss of activity. Under denaturing conditions, maltose did not helP in Protecting the enzyme against tyrosine modification or inactivation. Treatment with 2-Hydroxy-5-nitro benzyl bromide in the Presence of urea, Photooxidation at PH 9.0, N-bromosuccinamide at PH 4.8 resulted in a comPlete loss of activity. However, the results of exPeriments in the Presence of maltose and at PH 4.8 Photooxidation and N-bromosuccinamide treatment suggested the Presence of two tryPtoPhan residues at the active site. There was a comPlete loss of enzyme activity when 10 and 28 carboxyl grouPs fromAsPergillus candidus andRhizoPus, resPectively were modified. Modification in the Presence of substrate maltose, showed at least two carboxyl grouPs were Present at the active site of enzyme and that only one active center seems to be involved in breaking ally 3 tyPes of α-glucosidic linkages namely α-1, 4, α-1, 6 and α-l, 3.     

The actions of low-molecular fragments of substrate and their analogs on the activity of isoenzymes of phospholipase C from Clostridium perfringens]

The interaction of the lecithin molecule fragments and their analogues with phospholipase C Cl. perfringens was studied by gel-diffusion in agarose-lecithin gels. It was found intense inhibition of phospholipase C activity in the presence of cathionic compounds; this phenomenon shows the existence of anionic centre in the active site of enzyme. The esteric centre is probably hydrophobic nature and is not capable to bind the negatively charged groups. However, phosphoserine, phosphothreonine, gamma-aminobutyric, aspartic and glutamic acids can interact with an additional cathionic centre, whose location in phospholipase C differs from that in pancreatic phospholipase A2.     

Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes

Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6',7'-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 microM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 microM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56+/-0.12 microM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56+/-0.72 microM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 microM and 13.86 microM, respectively.