Immobilization of cells of filamentous fungi - producer pectolytic enzymes

Abstract

One of the most effective approaches to the enzymes’ preparation and stabilization, as well as increase in their economic attraction is immobilization using insoluble carriers. Such systems allow create high concentration of cells in the reactor volume. They increase the process efficiency and accumulation rate of extracellular pectinases in the culture medium and have possibility of technically simple separation from reagents, that allows use them repeatedly and obtain a product uncontaminated by the enzyme. Besides, the enzyme immobilization on solid carriers allows significantly improve mechanical properties of enzymatic agents. Also enzymatic systems, based on immobilized cells, have increased resistance to negative factors’ influence (pH, temperature, culture medium components, etc.) and long-term conservation of metabolic activity at their multiple use in the biotechnological processes in the absence of noticeable “laking” of cells. A method of the cell immobilization into a cryogel of polyvinyl alcohol has been technologically studied and approved to produce a high-productive immobilized biocatalyst on the basis of filamentous fungus cells Aspergillus awamori 56-2-53-85-375, able in the immobilized state to secrete extracellular pectinases during long culture period. This carrier is a macroporous visco-elastic gel material, obtained in a result of cryogenic treatment, i.e. after freezing – frozen conditioning – melting of the polymer’s water solutions. It has a high strength, marked porosity, biocompatibility and stability in the biological environment. Prepared in a such manner highly active immobilized biocatalyst on the basis of polyvinyl alcohol cryogel is characterized by long and stable biosynthesis of the pectolytic enzyme complex without loss of technological mode. The polyvinyl alcohol does not exert negative influence on the immobilized cells’ pectolytic enzyme biosynthesis, on the contrary, increases their vitality and enzymic activity due to the porous structure of this carrier matrix (pore section 0.5-1.2 micron), which provides a light diffusion in the culture medium components and metabolism products. Besides, the cryogel of the polyvinyl alcohol is a visco-elastic unbreakable material, which is almost non-abrasive wear, has soft operational characteristics during long culture period (up to 600 hours) and able to take any form of granules, appropriate for various reactors with various operating modes. An even mycelium germination by the whole volume of the polyvinyl alcohol cryogel granules has been established. Increase in the mycelium packing density inside the granules reduced their moisture from 90% to 80%. Increase in the heterogeneous biocatalyst granules sizes and mass due to the high rate of the mycelium growth has also been noted. Summarily accumulative pectolytic activity level of the developed immobilized biocatalyst exceeded in 3.5 times the level of A. awamori 56-2-53-85-375 strain, obtained in a result of many-staged selection used without immobilization. Also, the immobilized cells of А. awamori 56-2-53-85-375 strain have higher general pectinase and polygalacturonase activity, that is favorable for a red wine technology. Thus, there is no need to clean and activate the obtained pectolytic enzymes, they can be used in a native view in the wine industry.