Héctor L. Ramírez

Stabilization of invertase by modification of sugar chains with chitosan

Chitosan was linked to invertase by covalent conjugation to periodate-activated carbohydrate moieties of the enzyme. The thermostability of modified enzyme was enhanced by about 10 °C. The half-life at 65 °C was increased from 5 min to 5 h. The enzyme stability was enhanced by 20% at pH below 3.0. The half-life of denaturation by 6 M urea was increased by 2 h.

Stabilization of α-amylase by chemical modification with carboxymethylcellulose

Carboxymethylcellulose activated by periodate oxidation was covalently linked to porcine pancreatic α-amylase (EC 3.2.1.1). The specific activity of the conjugate prepared was 54% of the native enzyme. The carbohydrate content was estimated to be 62% by weight as a result of the modification of 67% of the amino groups from the protein. In comparison with the native enzyme, the thermostability and pH stability were improved for α-amylase by this modification. The conjugate was also more resistant to the action of denaturant agents such as urea and sodium dodecylsulfate. We conclude that modification of enzymes by the anionic polysaccharide carboxymethylcellulose might be a useful method for improving enzyme stability under various denaturing conditions.

Structure/Function Relationships of Several Biopolymers as Related to Invertase Stability in Dehydrated Systems

Structure/function relationships of different biopolymers (alginate, dextran, or beta-cyclodextrin) were analyzed as single excipients or combined with trehalose in relation to their efficiency as enzyme stabilizers in freeze-dried formulations and compared to trehalose. Particularly, a novel synthesized polymer beta-cyclodextrin-branched alginate (beta-CD-A) was employed as excipient. During freeze-drying, the polymers or their mixtures did not confer better protection to invertase compared to trehalose. Beta-CD-A (with or without trehalose), beta-cyclodextrin (beta-CD), or dextran with trehalose were the best protective agents during thermal treatment, while beta-CD and alginate showed a negative effect on invertase activity preservation. The beta-CD linked alginate combined the physical stability provided by alginate with the stabilization of hydrophobic regions of the enzyme provided by cyclodextrin. Beta-CD-A was effective even at conditions at which trehalose lost its protective effect. A relatively simple covalent combination of two biopolymers significantly affected their functionalities and, consequently, their interactions with proteins, modifying enzyme stability patterns.

Modification of α-amylase by sodium alginate

Sodium alginate, activated by periodate oxidation, was covalently linked to porcine pancreatic α-amylase via reductive alkylation with NaBH4. The enzyme-polymer conjugate, purified by gel filtration on Fractogel EMD BioSEC (S), retained about 50% of the native specific amylolytic activity. The sugar content was estimated to be 712 mol of monosaccharides per mol of enzyme protein. An average of 11 amino groups out of 21 groups from α-amylase were modified with the polysaccharide. The functional stability was improved for α-amylase after conjugation with sodium alginate. In comparison with the native enzyme, the thermostability of α-amylase was increased by this modification. In addition, the stability in the range of pH 5.0–11.0 was improved for the modified enzyme. The conjugate was also more resistant to denaturation by 0.3% sodium dodecylsulphate, retaining about 10% of its initial activity after 120 min of incubation. The formation of stabilizing salt bridges in the protein surface of the α-amylase-polysaccharide complex was confirmed by FT-IR spectrometry. Attending to the results obtained, we conclude that the covalent attachment of the anionic polysac-charide sodium alginate to the enzymes might be a useful and non-expensive method for improving the stabilization of these biocatalysts under various denaturing conditions.

Invertase Stabilization by Chemical Modification of Sugar Chains with Carboxymethylcellulose

Invertase from Saccharomyces cerevisiae was activated by periodate treatment and further reacted with ethylenediamine/sodium borohydride. Carboxymethylcellulose was then attached to ethylenediamine-modified invertase using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as coupling agent. The modified enzyme contained 3.5 mol of polysaccharide per mol of holoenzyme, and retained about 56% of the initial invertase activity. The thermostability of invertase increased from 64 to 70° C, the thermal inactivation at different temperatures ranging from 60 to 70° C was markedly reduced for polymer-modified enzyme. An increase of 9.1 kJ mol−1 in activation free energy of inactivation was determined for invertase after modification. Functional stability was increased for carboxymethylcellulose-invertase complex in the range of pH between 2.0 and 12.0. The conjugate was also more resistant to denaturation by 6 M urea solution.

Partial purification and properties of cyclodextrin glycosiltransferase (CGTase) from alkalophilic Bacillus species

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.9) is an unique enzyme capable of converting starch and related substrates into cyclodextrins (CDs). In this paper, we report an one step gel purification method of CGTase from Bacillus sp. and later enzyme characterization. The Bacillus sp. strain was isolated from a Colocacia esculenta rizospheric soil sample and the CGTase production was carried out in alkaline medium (pH=10). The CGTase purification from the culture supernatant was performed by gel filtration. The enzyme was purified in one step with a recovery of 87.3% activity and 40-fold purification for specific enzymatic activity of 2.24 U/mg. Optimal activity was observed at pH 5.0 in citrate-phosphate buffer, and the enzyme retained almost 100 % of its activity between pH 5.5 and 10 after incubation for 1 h at 4°C. The enzyme exhibited maximum activity at 55°C and showed a T50% of 70°C. The ratio of α:β:γ CD formed by the enzyme was 0.74:1:0.61 for soluble starch and 0.29:1:0.85 for cocoyam starch.

Solubilization and Stabilization of Sodium Dicloxacillin by Cyclodextrin Inclusion

The aim of this work is to analyze the effect of cyclodextrin (CD) complexation on the solubilization and stabilization of sodium dicloxacillin in acid aqueous solutions. The effect of four cyclodextrins alpha-, beta-, gamma- and hydroxypropyl-beta-CD was studied. Phase solubility diagrams obtained are AL or BS type, depending on the cyclodextrin used and on the pH of the solution. The highest stability constants of the inclusion complexes are obtained with gamma-CD at pH 1 and 2 and HPbeta-CD at pH 3. The structure of the inclusion complex in solution is characterized by nuclear magnetic resonance (1H-NMR). This study suggests that the 7-oxo-4-thia-1-azabicyclo group is located in the CD cavity. Nevertheless, molecular modelling calculations predict two different orientations of dicloxacillin in the gamma-CD cavity in vacuum and in aqueous solution. In vacuum, the results predict the inclusion of the dichlorophenyl ring of dicloxacillin instead of 7-oxo-4-thia-1-azabicyclo group into the gamma-CD cavity. However, the results are different in aqueous solution and this conformation is confirmed by the NMR study. The effect of gamma-CD and HPbeta-CD in the stability of the drug in solution was studied. The degradation of sodium dicloxacillin in solution follows a pseudo-first-order kinetics and the cyclodextrin do not change this fact. Both cyclodextrins increase the stability of the drug but the efficacy is higher with gamma-CD.

Impact of supramolecular interactions of dextran-β-cyclodextrin polymers on invertase activity in freeze-dried systems.

β‐Cyclodextrin (β‐CD)‐grafted dextrans with spacer arms of different length were employed to evaluate the impact of supramolecular interactions on invertase activity. The modified dextrans were used as single additives or combined with trehalose in freeze‐dried formulations containing invertase. Enzyme activity conservation was analyzed after freeze‐drying and thermal treatment. The change of glass transition temperature (Tg) was also evaluated and related to effective interactions. Outstanding differences on enzyme stability were mainly related to the effect of the spacer arm length on polymer–enzyme interactions, since both the degree of substitution and the molecular weight were similar for the two polymers. This change of effective interactions was also manifested in the pronounced reduction of Tg values, and were related to the chemical modification of the backbone during oxidation, and to the attachment of the β‐CD units with spacer arms of different length on dextran.