MICROBIAL PRODUCTION AND CHARACTERIZATION OF CHITIN DEACETYLASE BY PENICILLIUM OXALICUM

Abstract

The present investigation was undertaken to isolate a potent microorganism with chitin deacetylase production ability and to develop and economize the process by employing agrohorticultural residual resources. The fungal strain secreting notably higher levels of extracellular chitin deacetylase was isolated from the residual materials of the sea food processing industry. The isolated strain SA-1 was able to produce notable levels of chitin deacetylase and identified as Penicillium oxalicum ITCC 6965 by Indian Agricultural Research Institute,New Delhi. To achieve an improvement in the enzyme production ability, the selected strain was subjected to genetic manipulation using single-stage and mixed mutagenesis. Mutagenesis by microwave irradiation and ethidium bromide had led to the development of strain SAEM-51 with ability for considerably higher levels of extracellular chitin deacetylase along with lower levels of intracellular enzyme production. Mutant strain had-2.0 fold improvement in the kinetic variables mainly Qp, Qs, Yp/x , Yp/s , qP, qs over the parental strain. Mutagenized strain P. oxalicum SAEM-51 had discrete morphological features. The mycelia of mutant SAEM-51 were elongated, flattened and smooth as compared to wild type SA-1 which were irregular, rough surfaced and coiled. Similarly, spores from the wild type were crescent shaped while those from mutant strain were oval and irregular. Analysis of enzyme activity by P. oxalicum SAEM-51 using chitin, colloidal chitin and glucose revealed that chitin comparatively results into lower levels of enzyme activity. Highest CDA levels were attained using glucose as a carbon source under submerged condition. Nutritional and cultural parameters for enzyme production were derived further using response surface methodology under submerged condition. A 6.0 fold increase in enzyme levels was attained under derived conditions. Further in an attempt to see if the process can be made economical and further increase in the production can be obtained, the level of enzyme production was evaluated using solid-state fermentation. Among various solid supports used for i SSF process, mustard oil cake, was found to be the most ideal for fungal colonization and enzyme production. Derivation of fermentation parameters viz. amount of substrate, moisture level and inoculum level using response surface methodology had resulted in 10.9 fold enhancement in enzyme production levels. To develop an industrially compatible enzyme preparation, CDA from culture supernatants was purified using ultrafiltration, cation and anion exchange chromatography and characterized for its biochemical and molecular properties. Thermodynamic parameters had depicted the notable stability of CDA at higher temperatures (upto 70 °C). Analysis of kinetic constants had enumerated that enzyme had effectively deacetylated chitin and its oligomers having degree of polymerization more than four. Metal ions i.e. Cu+2, Co+2, Fe+2 and Cd+2 were observed as inducers of enzyme activity. Developed enzyme preparation was not inhibited with acetate (upto 70 mM concentration), an end product of the enzyme reaction. Enzyme was observed to possess the characteristic secondary structure consisting of both a and P helices at its optimal pH. Significant homology of the enzyme when compared with the existing sequences in the database was observed with CDAs from bacterial, fungal and yeast strains. Deacetylation potential of the developed enzyme preparation was evaluated for bioconversion of chitin to chitosan in a two stage chemical and enzymatic deacetylation process. Variations in morphology, crystallinity and thermal properties of the deacetylated chitinous substrates were evaluated by scanning electron microscopy, X-ray diffraction, thermogravimetric analysis and differential scanning calorimetry. Degree of deacetylation of the substrates before and following chemical and enzymatic treatments was determined using FTIR and elemental analysis. The pretreatment of the substrate led to the decrease in crystallinity and formation of amorphous chitinous substrates to facilitate enzyme reaction. Among the various substrates analyzed, superfine chitin appeared to be most suitable substrate for enzyme action which had resulted into the chitosan with 79.52 %of the deacetylation. Further, improvement in deacetylation was attained by deriving the reaction variables i.e. substrate amount and enzyme li dose through central composite design. This had led into a further 10 % improvement in deacetylation with the formation of chitosan with 90%of the deacetylation.