Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N2 adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. 27Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N2 adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. Km was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.

Acid activated montmorillonite: an efficient immobilization support for improving reusability, storage stability and operational stability of enzymes

Three enzymes, α-amylase, glucoamylase and invertase, were immobilized on acid activated montmorillonite K 10 via two independent techniques, adsorption and covalent binding. The immobilized enzymes were characterized by XRD, N2 adsorption measurements and 27Al MAS-NMR spectroscopy. The XRD patterns showed that all enzymes were intercalated into the clay inter-layer space. The entire protein backbone was situated at the periphery of the clay matrix. Intercalation occurred through the side chains of the amino acid residues. A decrease in surface area and pore volume upon immobilization supported this observation. The extent of intercalation was greater for the covalently bound systems. NMR data showed that tetrahedral Al species were involved during enzyme adsorption whereas octahedral Al was involved during covalent binding. The immobilized enzymes demonstrated enhanced storage stability. While the free enzymes lost all activity within a period of 10 days, the immobilized forms retained appreciable activity even after 30 days of storage. Reusability also improved upon immobilization. Here again, covalently bound enzymes exhibited better characteristics than their adsorbed counterparts. The immobilized enzymes could be successfully used continuously in the packed bed reactor for about 96 hours without much loss in activity. Immobilized glucoamylase demonstrated the best results.

Characterization of laccase from Streptomyces psammoticus: an enzyme for eco-friendly treatment of environmental pollutants

The present study has identified an actinomycete culture (S. psammoticus) which was capable of producing all the three major ligninolytic enzymes. The study revealed that least explored mangrove regions are potential sources for the isolation of actinomycetes with novel characteristics. The laccase production by the strain in SmF and SSF was found to be much higher than the reported values. The growth of the organism was favoured by alkaline pH and salinity of the medium. The enzyme also exhibited novel characteristics such as activity and stability at alkaline pH and salt tolerance. These two characters are quite significant from the industrial point of view making the enzyme an ideal candidate for industrial applications. Many of the application studies to date are focused on enzymes from fungal sources. However, the fungal laccases, which are mostly acidic in nature, could not be used universally for all application purposes especially, for the treatment of effluents from different industries, largely due to the alkaline nature of the effluents. Under such situations the enzymes from organisms like S. psammoticus with wide pH range could play a better role than the fungal counterparts. In the present study, the ability of the isolated strain and laccase in the degradation of dyes and phenolic compounds was successfully proved. The reusability of the immobilized enzyme system made the entire treatment process inexpensive. Thus it can be concluded from the present study that the laccase from this organism could be hopefully employed for the eco-friendly treatment of dye or phenol containing industrial effluents from various sources.

Enzyme reactions as index of freshness of Fish and Shellfish

An attempt has been made in this study to screen some fish muscle enzymes to assess their potential worth in testing the degree of freshness of fish. A problem with routine enzyme activity determinations is the complexity of the method of enzyme assay. Hence, in the present study as far as possible simple assay techniques were adopted. Several species were screened to assess the possibility of employing this procedure on a large scale. It is hoped that findings of this study will lead to the development of meaningful criteria in testing the freshness of fish. This thesis has been divided into five chapters