Production and Characterization of Enzymes of Commercial Importance

Abstract

In contrast to most of the world already done on enzyme inductions in the bacterial cells, this work was undertaken with a view to elucidate certain aspect of lipase induction mechanisms in the fungal cells. The various organism involved in the study was Rhizopus arrhizus, M.racemosus, M.fragillis and M. Pusillus. After a general study on lipase production on the shark flask culture by the various species, indepth investigations were undertaken on M.hiemalis. The rizopus species were growth in the medium containing glycerides mainly of eleic acid, erucic acid, saturated fatty acids of 8-16 carbon and butric acid. In the case of R.arrhizus and R.japonicus there was a decrease in the in the extracellular lipase activity when the glucose was replaced by the different trightycerides, but the enzyme activity increased in the mycelia. In the cares of R.oryzae both the exacelluer and mycelial lipase activities increased in some extent, when grown in the presence of triglycerides instead of glucose. Tribtyrin was not utilized well for the growth of these species yet the enzyme produced in the presence of glucose and other triglycerides hyderolysed it at the highest rate and the order of activity was followed by those against the glycerines of coconut oil, oleic acid and eruic acid in decreasing order. Oleic and erucic acid glycerides affected greater induction of lipase in all the Rhizopus species as compared to coconut oil. Olic acid erotic acid glycerines in general caused greater inhabitation of the enzyme as well, as compared to the glycerines of coconut oil. The lipase produced in the presence of particular triglycerides neither show preferential activity against the same triglycides nor the enzyme was inhibited pereferantly by those. In case of the various Mucor species it was observed that when glucose was placed by olive, coconut and mustard oils in the growth medium there was increase in lipase activities nothy in the mycelia and the both in all these case. Olive oil was founded to cause greater lipase induction was compared to coconut and mustard oils. The enzume produce by these species was not specifically active against the triglycerides in the presence of which it was produced. Irresepective of the triglycides used as a carbon source the lapse produced hydrolysed coconut oil at a greater rate and this activity was followed by those against olive and mustard oils. Optimum ph at which maximum hydrolysis took place was determined to be 7.0 when all these species were produced in the medium containing glucose as the carbon sources. When M.hiemelias was grown in the medium containing olive oil instead of glucose the optimum ph for lipase activity remained the same. M.hiemalis being the best producer of lipase among the various Mucor species was studied in detail. Initial ph of the fermentation medium at which M.hiemalis produced maximum lipase was 4.0. Addition of ca to the medium did not increase lipase production. Optimum ph for hydrolysis for both the mycelial and the extracellular lipase was founded to br 7.0. The fungus produced significant amount of lipase in the presence of glucose. However, the lipase activity increased markedly when olive oil was added to the medium at the begging of fermentation. Addition of olive oil at a later stage did not induce the enzyme as much. Studies with wised mycelia shoed that greater amount of lipase was released in the presence of olive oil. Olive oil and mustard oil hydrolytic products inhibited the lipase induced by a particular type of triglycides did not specifically hydrolyses the same triglyceride nor was it inhibitated specifically. The mycelial growth, extracellular and cell-bound lipase production And lipid formation within the cell of M.hiemalis were observed to be associated with glucose utilization. Most of the glucose was used toward the end of the growth phase when the mycelial showed maximal lipase activity as well as maximal lipid content maximal broth lipase activity was observed after the cell lusis had started. The fraction indentified in the mycelial lipase. The proportion between the neutral and the polar lipids was only 0.06 at 44 hours, increasing gradually to 1.51 at 164 hours of fermentation. Effect of adding triglycides on the growth of macro hiemalis and production of lipase and the lipids was studied as well. A concentration of 1% added triglycides was best for the mycelial as wells the birth lipase production. The added triglycerides were utilized through phase most of the were consumed. The mycelial lipase activity was maximum at this stage while the maximal broth lipase activity was achieved after the cell lyses had started. The lipids produced per gram mycelial were high initially reducing gradually later. With increase in growth the maximum mycelial lipids per 100 grams culture medium was obtained after 96 hours. The various frations deleted in the mycelial lipid extracts were sterol esters, triglycerides, free fatty acids , triglycerides , sterols, ,onoglycerides , phosphatidyl ethanolamine , phosphatidyl chorine and small amounts of unknown lipids. In contests to muvelial lipid composition of the glucose medium, the proportion between the total neutral and the total polar lipids remained nearly constant throughout the growth. Purification of the M.hiemalis extracellular lipase when produced in the presence of triglycerides showed the presence of two lipases, which, however, need to be further characterised. Apart from providing useful information on lipase induction in fungal cells, it has been possible to affect manifold increase in the lipase production during these studies.