Production and Characterization of Enzymes of Commercial Importance

Abstract

In contrast to most of the work already done on enzyme induction in the bacterial cells, this work was undertaken with a view to elucidate certain aspects of lipase induction mechanism in the fungal cells. The various organisms involved in these studies were Rhizopus arrhizus, R. japonicus, R. oryzae, Mucor hiemalis, M. phillipovi, M. circinelloides, M. racemous, M. fragilis and M. pusillus. After a general study on lipase production in shake of flask cultures by the various species, in-depth investigations were undertaken on M. Hiemalis. The Rhizopus species were grown in the medium containing glycerides mainly of the oleic acid (glycerol trioleate and olive oil), erucic acid (mustard oil), saturated fatty acids of 8-16 carbon (coconut oil) and butyric acid (tributyrin). In the case of R. Arrhizus and R. Japonicus there was a decrease in the extracellular lipase activity when glucose was replaced by the different triglycerides, but the enzyme activity increased in the mycelia. In the case of R. oryzae both the extracellular and the mycelial lipase activities increased to some extent, when grow in the presence of triglycerides instead of glucose. Tributyrin was not utilized well for the growth of these species yet the enzyme produced in the presence of the glucose and other triglycerides hydrolyzed it at the highest rate and the order of activity was followed by those against the glycerides of coconut oil, oleic acid and erucic acid in decreasing order. Oleic and erucic acid glycerides affected greater induction of lipase in all the Rhizopus species as compared o coconut oil. Oleic and erucic acid glycerides in general caused greater inhibition of the enzyme as well, as compared to the glycerides of coconut oil. The lipase produced in the presence of particular triglycerides neither show preferential activity against the same triglycerides nor the enzyme was inhibited preferentially by these. In case of various Mucor species it was observed that when glucose was replaced by olive, coconut and mustard oil in the growth medium there was increase in lipase activities both in the mycelia and the broth in all cases. Olive oil was found to cause greater lipase induction as compared to coconut and mustard oils. The enzyme produced by these species was not specifically active against the triglycerides in the presence of which it is produced. Irrespective of the triglycerides used as carbon source the lipase produced hydrolyzed 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 source. When M. Hiemalis 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 of hydrolysis for both mycelial and the extracellular lipases was found to be 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 beginning of fermentation. Addition of olive oil at a later stage did not induce enzyme as much. Studies with washed mycelia showed that greater amount of lipase was released in the presence of olive oil. Olive oil and mustard oil hydrolytic products inhibited the lipase more than those of coconut oil. The lipase induced by a particular type of triglycerides did not specifically hydrolyse the same triglyceride nor it inhibited specifically. The mycelial growth, extracellular and cell-bound lipase production and lipid formation within cells of M. hiemalis were observed to be associated with glucose utilization. Most of the glucose was used towards the end of the growth phase when the mycelia showed maximal lipase activity (17U/g dry mycelia) as well as maximal lipid content (13.4 mg/g dry mycelia). Maximal broth lipase activity (76 U/100ml) was observed after the cell lysis had started. The fractions identified in the mycelial lipid extracts were sterol esters, triglycerides, fatty acids, diglycerides, sterols monoglycerides, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine and an unknown polar lipid. 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 triglycerides on the growth of Mucor Hiemalis and production of lipase and the mycelial lipids was studied as well. A concentration of 1% added triglycerides was best for the mycelial as well as the broth lipase production. The added triglycerides were utilized through formation of free fatty acids and towards the end of the growth phase most of these were consumed. The mycelial lipase activity was maximum (66 U/g mycelia) at this stage while the maximal broth lipase activity (216 U/g mycelia) was achieved after the cell lysis had started. The lipids produced per gram mycelia were high initially (260 mg/g after 48 hours), reducing gradually later. With increase in growth the maximum mycelial lipids per 100ml culture medium was obtained after 96 hours (296 mg/100ml). the various fractions detected in the mycelial lipid extracts were sterol esters, triglycerides, free fatty acids, diglycerides, sterols, monoglycerides, phosphatidyl choline and small amounts of unknown lipids. In contrast to the mycelial lipid composition of the glucose medium, the proportion between the total neutral and 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 characterized. 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.