Ipsita Roy

Freeze‐drying of proteins: some emerging concerns

Freeze‐drying (lyophilization) removes water from a frozen sample by sublimation and desorption. It can be viewed as a three‐step process consisting of freezing, primary drying and secondary drying. While cryoprotectants can protect the protein from denaturation during early stages, lyoprotectants are needed to prevent protein inactivation during drying. The structural changes as a result of freeze‐drying have been investigated, especially by FTIR (Fourier‐transform IR) spectroscopy. In general, drying results in a decrease of α‐helix and random structure and an increase in β‐sheet structure. In the case of basic fibroblast growth factor and γ‐interferon, enhanced FTIR showed large conformational changes and aggregation during freeze‐drying, which could be prevented by using sucrose as a lyoprotectant. It is now well established that structural changes during freeze‐drying are responsible for low activity of freeze‐dried powders in nearly anhydrous media. Strategies such as salt activation can give ‘activated’ enzyme powders, e.g. salt‐activated thermolysin‐catalysed regioselective acylation of taxol to give a more soluble derivative for therapeutic use. In the presence of moisture, freeze‐dried proteins can undergo disulphide interchange and other reactions which lead to inactivation. Such molecular changes during storage have been described for human insulin, tetanus toxoid and interleukin‐2. Some successful preventive strategies in these cases have also been mentioned as illustrations. Finally, it is emphasized that freeze‐drying is not an innocuous process and needs to be understood and used carefully.

Lactose hydrolysis by Lactozym™ immobilized on cellulose beads in batch and fluidized bed modes

LactozymTM is a commercially available preparation of b-galactosida se from Kluyveromyces fragilis. It has valid generally recognized as safe (GRAS) status for whey hydrolysis and production of low lactose milk. Immobilized b-galactosidase from K. fragilis has been less studied. In this work, LactozymTM was immobilized on cellulose beads via epichlorohydrin coupling chemistry. The optimized preparation was characterized in terms of its kinetic parameters. The fluidized bed hydrolyzed whey lactose ( > 90% conversion) in 5 h as compared to 48 h taken by the same enzyme in continuous batch mode. The immobilized enzyme could be reused three times without any change in the performance of the fluidized bed reactor. The fluidized bed could also hydrolyze milk lactose up to 60% within 5 h. The above data show that this enzyme from a GRAS status source can also be used to develop a process for lactose hydrolysis for whey utilization as well as production of low lactose milk.

Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads.

Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As “proof of concept”, soybean trypsin inhibitor was purified 18‐fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL‐1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity.

Non-thermal effects of microwaves on protease-catalyzed esterification and transesterification

and 50-methylthioadenosine phosphorylase) were from a thermophile Sulfolobus solfataricus. It was found that the inactivation (due to microwaves) kinetics did not depend upon protein concentration. The significant inactivation of enzymes due to exposure to radiation was in agreement with conformational changes revealed by fluorescence emission spectra and far ultraviolet CD spectra. More recently, the same group has looked at inactivation of alcohol dehydrogenase from the same source and confirmed that a nonthermal effect was responsible for enzyme inactivation.12 In the present work, we have chosen two proteases, subtilisin and a-chymotrypsin, and investigated the effect of microwaves alone at fixed temperatures on the rates of esterification13 and transesterification14 reactions catalyzed by these enzymes.

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

ABSTRACT The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.

Exploiting unusual affinity of usual polysaccharides for separation of enzymes on fluidized beds

Two polysaccharides, alginate and chitosan, showed unusual affinity and bound alpha-amylase (from various sources) and Aspergillus niger cellulase, respectively. The beads prepared from these polymers were successfully used for the purification of the respective enzymes by fluidized bed affinity chromatography. alpha-amylase from wheat germ could be purified by 58-fold with about 90% recovery of activity. Aspergillus niger cellulase, on the other hand, was purified by 30-fold with 80% recovery of enzyme activity. Both purified preparations show single band on SDS-PAGE.

Purification of lysozyme from other hen's-egg-white proteins using metal-affinity precipitation.

It was found that the presence of 5 mM Cu2+ caused precipitation of protein present in hens egg white to a large extent. About 85% of lysozyme activity remained in the supernatant and the enzyme was purified by approx. 13‐fold. A further gel‐filtration step on Sephadex G‐75 resulted in an overall yield of 80% for the enzyme with 655‐fold purification, and showed a single band on SDS/PAGE.

Comparison of batch, packed bed and expanded bed purification of A. niger cellulase using cellulose beads

Rigid macroporous cross-linked cellulose beads were prepared and used as a useful affinity medium for purification of A. niger cellulase from commercial preparation, in batch; packed bed and expanded bed modes. The beads bound 99% activity in both packed bed and expanded bed modes and upto 91% activity could be recovered by washing the adsorbent with 1 M phosphate buffer, pH 7.0. While batch adsorption and elution gave only 4-fold purification, packed bed operation gave 14-fold purification and expanded bed, the highest, 36-fold purification.

A smart bioconjugate of alginate and pectinase with unusual biological activity toward chitosan

The commercial preparation of pectinase (Pectinex Ultra SP‐L) was conjugated to alginate by noncovalent interactions by employing 1% alginate during the conjugation protocol. The optimum “immobilization efficiency” was 0.76. The pH optimum and the thermal stability of the enzyme remained unchanged upon conjugation with alginate. The soluble bioconjugate showed a 3‐fold increase in Vmax/Km as compared to the free enzyme when the smart biocatalyst was used for chitosan hydrolysis. Time course hydrolysis of chitosan thus showed higher conversion of chitosan into reducing oligosaccharides/sugars. The smart bioconjugate could be reused five times without any detectable loss of chitosanase activity.

Accelerating Enzymatic Hydrolysis of Chitin by Microwave Pretreatment

Response surface analysis was used to determine optimum conditions [2% (w/v) chitin, 57.5 °C, 38 min] for microwave irradiation of chitin to improve its enzymatic hydrolysis. Vmax/Km of cabbage chitinase toward untreated and microwave‐irradiated chitin was found to be 21.1 and 31.7 nmol h‐1 mg‐2 mL, respectively. Similar improvement was observed in the case of pectinase in its unusual catalytic activity of chitin degradation. It was found that a greater extent of chitin hydrolysis by chitinase was possible after the substrate chitin was irradiated with microwaves.