Isolation and Molecular Characterization of Pectinase from Penicillium chrysogenum and Aspergillus niger Grown on some Agro wastes in Lagos Nigeria.
A Thesis Submitted to the School of Postgraduate Studies, University of Lagos.
Waste generated by several agricultural activities has many alternative uses. Unfortunately, much of it is burnt in the open fields, resulting in environmental pollution. In view of the economic and ecological importance of agro wastes, microfungi were isolated from decaying agro wastes samples and screened for pectinolytic activity using a plate assay method containing pectin alone. The effects of carbon, nitrogenous sources and pH on pectinase production by the strains with maximum pectinolytic activities were investigated. Hyper- production of pectinase by these strains using ultra-violet irradiation (UV) as mutagen was also investigated. The selected mutants of Penicillium chrysogenum and Aspergillus niger were characterized using catabolite repression. The genetic variability between the wild and mutant strains was investigated using Random Amplified Polymorphic DNA (RAPD) primers. The polygalacturonase (Pga) produced by the mutant strains were partially purified and the enzyme properties were investigated. Five fungi strains were isolated they are: Aspergillus niger, Aspergillus clavetus, Penicillium chrysogenum, Fusarium species and Trichoderma harzianum. Pectinase production by these isolates showed that the highest pectinase activity index was observed in Aspergillus niger (A.n) followed by Penicillium chrysogenum (P.c). The growth profile of the A.n and P.c in various agro wastes studied showed that these fungi were able to grow in both simple sugar (metabolites) and various agro wastes. The production of pectinase by A.n and P.c in various agro wastes studied showed that wheat bran medium supported pectinase production better than other agro wastes used with a yield of 350.48 IU/ mg protein and 450 IU/ mg protein respectively with maximum yield at 48 h. The inclusion of various nitrogenous salts revealed that urea best supported the production of pectinase (195.75 IU/ mg protein) while polygalacturonase (Pga) production by the addition of urea (33.52 IU/ mg protein) was highly reduced. When P.c was used as the fermenting organism, pectinase production was supported best by peptone 177.97 IU/ mg protein but its polygalacturonase activity was highly reduced (40.35 IU/ mg Protein). Pectinase and polygalacturonase optimization by P.c was best achieved when urea was used as nitrogen source, 124.26 IU/ mg protein for pectinase and 93.00 IU/ mg protein for polygalacturonase after 72 h post fermentation. The stimulation of polygalacturonase and pectinase in A.n using various agro wastes, revealed that sugar cane (73.40 IU/mg protein) and wheat bran (181.43 IU/mg protein) best sustained polygalacturonase and pectinase production respectively and when P.c was induced for both polygalacturonase and pectinase production, wheat bran best supported the production of both polygalacturonase and pectinase. The outcome of pH effect on pectinase production showed an optimal pH of 3.5 and 5.5 for A.n and P.c respectively. Three 2 deoxy-D- glucose (2DG) UV mutants of Aspergillus niger and five 2DG UV mutants of P. chrysogenum were isolated post exposure of the strains to UV irradiation. The comparison of the pectinase production by the suspected mutants and wild strains revealed that; 2 DG UV M2 mutant of Aspergillus niger was selected as the best mutant with enzyme activities of 465% and 230% in solid state fermentation (SSF) and submerged fermentation (SmF) respectively, using wheat bran as the sole carbon source. P. chrysogenum 2DG-UV W1 gave a percentage enzyme activity of 218% in solid state fermentation, and 2DG-UV W2 gave a better pectinase yield (135%) in submerged fermentation over 2DG-UV W1 (122.58%). The glucose showed no catabolite repression (CR) on pectinase production abilities of both the 2DG UV M2 and 2DG UV W1. However, sucrose repressed pectinase production in both the wild and the 2DG UV W1 mutant strain of P.chrysogenum, while 2DG UV M2 of A. niger was able to produce pectinase. The results of RAPD-PCR on possible polymorphisms showed that there was considerable genetic variation between the wild and mutant strains of A. niger and P.chrysogenum using OPA 02, OPC 02, OPE 02 and OPA 04. A purification fold of 7.94 and 4.7 were achieved for Pga of 2 DG UV M2 (A.niger) and 2 DG UV W1 (P.chrysogenum) respectively. The molecular weight of Pga of 2 DG UV M2 (A.niger) and 2 DG UV W1 (P.chrysogenum) were 105 and 95 kDa respectively. An optimal temperature of 40oC was recorded for the Pga of both mutant strains, and the Pga retained 60% of its activity at 80oC and an optimal pH of 4.5 and 5.0 for 2 DG UV M2 (A.niger) and 2 DG UV W1 (P.chrysogenum) respectively. The Vmax of 156.25 IU/ mg protein was obtained for both Pga of 2 DG UV M2 (A.niger) and 2 DG UV W1 (P.chrysogenum) and Km of 1.27 and 2.6 g/l were obtained for 2 DG UV M2 (A.niger) and 2 DG UV W1 (P.chrysogenum) respectively. It was also established that Mg2+ activated the Pga of both 2 DG UV M2 (A.niger) and 2 DG UV W1 (P.chrysogenum). This study showed that the natural process of biodegradation could be harnessed for the biotransformation of agro waste materials into a valuable resource. There is a good prospect for agro wastes as the sole carbon source for pectinase production and the use of UV irradiation in hyper-pectinase induction in wild mutants.