DENATURATION OF NATIVE AND DEGLYCOSYLATED  -GALACTOSIDASES FROM PENICILLIUM CANESCENS BY GUANIDINE HYDROCHLORIDE

The aim of this work was the study of native and deglycosylated  -galactosidases from Penicillium canescens under denaturing conditions caused by guanidine hydrochloride. Calculation of kinetics and constants of enzymes inactivation was carried out on using experimental kinetic curves of enzyme denaturation. We observed significant differences in the kinetics of inactivation of native and deglycosylated  -galactosidases from P. canescens caused by guanidine hydrochloride. Native enzyme was stable within the selected range of guanidine hydrochloride concentrations (from 0.1 to 3.0 M), retaining no less than 50% of the initial enzyme activity for 3 days. Deglycosylated enzyme preparations were less stable and they lost their activity within 5–30 minutes, when they were treated with guanidine hydrochloride in concentrations above 1 M. Dissociation rate constant of native and deglycosylated forms of the enzyme differed by 10 to 100 folds. It was shown that subunit interactions play a major role in the process of inactivation of the enzyme, and the carbohydrate component is essential for stabilizing of subunit bonds and maintaining conformational stability of the enzyme under denaturing conditions of chemical agents.

Investigation of the mechanism of inactivation and reactivation of enzymes is very interesting from a theoretical and practical point of view. The processes of folding and unfolding of the molecule, and hence the catalytic properties of the enzyme are largely dependent on the conditions of the reaction medium. The interest in these processes is also associated with the appearance of work concerning genetic engineering, gene cloning, folding and the acquisition of the catalytically active conformation of recombinant and modified proteins.
It is known that a carbohydrate component of extracellular enzymes may have a significant effect on their stability and activity [1,2].The amount of protein glycosylation sites of the molecule may vary substantially depending on the way of producer growing. So, the study concerning the stability of various forms of enzymes that were obtained as a result of modifications of the carbohydrate component of the protein molecule can determine the role of carbohydrate in maintaining of active conformation and quaternary structure of the protein. One of the ways of such modification is the growing of enzyme producers in the presence of inhibitors of glycosylation: tunicamycin, 2-deoxy-D-glucose, -factor, and others [3][4][5][6].
We have previously obtained -galactosidase (EC 3.2.1.22) from Penicillium canescens and shown that this enzyme is a glycoprotein with a molecular weight of 400 kDa, in which the carbohydrate content is 16% (m / m) [7]. The carbohydrate component includes mannose, D-glucosamine, arabinose and rhamnose.
The aim of this work was to explore how the inhibition of N-and O-glycosylation affects the stability of -galactosidase from P. canescens under condition of chemical denaturation, to investigate the kinetics of enzyme denaturation using guanidine hydrochloride and find opportunities for renaturation of enzymes with different content of carbohydrate component.
The aim of this work was the study of native and deglycosylated -galactosidases from Penicillium canescens under denaturing conditions caused by guanidine hydrochloride. Calculation of kinetics and constants of enzymes inactivation was carried out on using experimental kinetic curves of enzyme denaturation. We observed significant differences in the kinetics of inactivation of native and deglycosylated -galactosidases from P. canescens caused by guanidine hydrochloride. Native enzyme was stable within the selected range of guanidine hydrochloride concentrations (from 0.1 to 3.0 M), retaining no less than 50% of the initial enzyme activity for 3 days. Deglycosylated enzyme preparations were less stable and they lost their activity within 5-30 minutes, when they were treated with guanidine hydrochloride in concentrations above 1 M. Dissociation rate constant of native and deglycosylated forms of the enzyme differed by 10 to 100 folds. It was shown that subunit interactions play a major role in the process of inactivation of the enzyme, and the carbohydrate component is essential for stabilizing of subunit bonds and maintaining conformational stability of the enzyme under denaturing conditions of chemical agents.

Materials and Methods
It was used native and modified preparations of -galactisidase from Penicillium canescen that were purified from culture liquid of the producer by precipitation with 90% ammonium sulfate. The preparations were purified to homogeneity by gelfiltration on TSK Toyoperl HW-55 and Sepharose 6B, аnd using ionexchange chromatography on DEAE-Toyoperl 650М [7]. Treatment of enzymes was carried out with guanidine hydrochloride (AppliChem GmbH, Germany).
Effect of inhibitors on the secretion of -galactosidases was studied in this manner. Micromycetes culture was pregrown for 48 hours then mycelium was separated by centrifugation and transferred to medium of standard composition containing the desired inhibitor.The cultures were grown in the presence of 100 and 150 μg/ml of 2-deoxy-D-glucose or 15 μg/ml of tunicamycin. Then the cultures were grown for 3 days. -Galactosidase activity was determined by the rate of degradation of p-nitrophenyl substrate.
The degree of substrate hydrolysis was defined by the colorimetric method as it was shown in [8]. One unit of activity corresponded the amount of enzyme that catalyzes the hydrolysis of 1 μM substrate for 1 min under standard test conditions (37 C, pH 5.2).
Inactivation of the enzyme with guanidine hydrochloride was carried out by the following procedure. To a solution containing 1.5 U/ml of -galactosidase in 0.1 M phosphate-citrate buffer (PCB, pH 5.2) we added guanidine hydrochloride in different concentrations (from 0.1 to 4 M) and at the enzyme activity was registered at regular intervals.
To perform reactivation the solutions of enzyme treated with guanidine hydrochloride were dialyzed against 0.1 M PCB, pH 5.2. Changes in the activity of the native and modified enzyme were recorded over several days.
The study of the kinetics of inactivation and calculation of correspondent constants were performed according to [9].
All experiments were carried out at least in 3-5 repetitions. Statistical processing was performed by standard methods with the definition of Student's t test at the 5% significance level.

Results and Discussion
We used three -galactosidase preparations from Penicillium canescens: the native preparation isolated from the culture medium under standard conditions and two deglycosylated samples obtained by cultivation of the producer in the presence of tunicamycin and 2-deoxy-D-glucose.
The total carbohydrate content was reduced from 16.5% (native enzyme) to 11.7% (N-modified) and 8.2% (O-modified). After appropriate purification, we obtained enzymes that were different by quantitative content of neutral monosaccharides (Fig. 1).
Our further investigations concerned the role of carbohydrate component in supporting of active conformation of the enzyme under influence of guanidine hydrochloride and the kinetics of the protein denaturation. There are data about denaturation of different proteins in the presence of guanidine hydrochloride or urea [10]. This process can take place in one irreversible stage or include some reversible. After removing of the denaturant the spontaneous recovery of activity sometimes takes place. The investigated preparations of the enzyme in 0.1 M PCB (pH 5.2) possessed 100% of activity under the room temperature for 7 days. We chose these conditions as initial to carry out denaturation of -galactosidase from P. canescens by guanidine hydrochloride. When guanidine hydrochloride was added to different preparations of -galactosidase the gradual decrease of enzyme activity was observed (Fig. 2). However, there were significant differences in kinetics of native and deglycosylated preparations. First of all, the degree of denaturating action of guanidine hydrochloride depended on enzyme preparation. The native enzyme was more resistant to different concentrations of guanidine hydrochloride (from 0.1 to 3.0 M) keeping no less than 50% of the initial activity for 3 days. Deglycosylated preparations were less resistant to high concentrations of the denaturant. These preparations lost brashly their activity at denaturant concentration more than 1M over 5-30 min. At lower concentrations the native preparation decreased its activity over the first 150 min, then we observed the increase of activity followed by the decrease and then the rate was not high. Probably, in this case there was a change of some conformation states of the enzyme when it was switching to a less stable form. The decrease of activity was 20-30%, so we can suggest that partial unfolding of the molecule does not touch the active center of the enzyme.
There are data concerning the presence of lag-periods during thermal inactivation of different proteins in cases when the intermediate form of the enzyme appears, whose activity is comparable to the initial one [11].
The analysis of inactivation curves of -galactosidase from P. canescens under influence of guanidine hydrochloride at 20 С let us suggest that at all used concentrations of guanidine hydrochloride the dependence activity on the time can be described by two exhibitors. These kinetics curves are similar  [12]. It proves the oligomeric structure of this enzyme. So, dissociative character of the enzyme denaturation takes place in case of guanidine hydrochloride: dissociation of tetramer into less active monomers with their following irreversible inactivation.
Inactivation of deglycosylated -galacto sidases from P. canescens takes place at the higher rate. To achieve the total loss of activity it needed from 5 to 120 min d e p e n d i n g o n t h e p r e p a r a t i o n a n d guanidine hydrochloride concentration. When the denaturant concentration was more than 1M the inactivation of these enzymes was observed over first 5 min of incubation.
Our attempts to carry out reactivation of modified enzymes had no effect. Reactivation was only observed for native -galactosidase in case of removing of guanidine hydrochloride over first 3 hours, to the point of fracture (Fig. 3). It proves the presence of monomers or dimers that are capable of reversible denaturation. At the later stages the observed denaturation was irreversible. Partially deglycosylated enzymes were not capable of renaturation.