5533
Gehad Hassan El-Sayed Mohammed Badran
Biochemical Studies On The Production Of Polygalacturonase From Locally Isolated Streptomycetes
Streptomyces sp, Polygalacturonase , Numerical taxonomy , 16S rRNA sequencing , Streptomyces sp. GHB5 , Optimum conditions , Nutritional requirements ,Purification , Immobilization , Application
Biotechnological products like enzymes are most significant products for human needs in large scale part of environmental, industrial and food technology especially the one that obtained from microbial origin. The present study began with isolation process, where twenty five cultivated soil samples were collected from different soil samples of Egypt, 526 streptomycetes isolates were recovered and screened for polygalacturonase production. By primary and secondary screening of active Streptomyces sp., five isolates (2, 5, 8, 72 and 103) give the best results (≥15 U/mg) specific enzyme activity. By screening using different agro-industrial waste products as lemon peel, orange peel, rice husk and wheat bran, isolate 5 give maximum activity (21.5 U/mg) with lemon peel nitrate medium. Based on morphological, cultural, physiological, biochemical and chemo-taxonomical characteristics, the numerical identification was made by using UPGMA program, confirmed that the five active strains of Streptomyces sp. were assigned to four hierarchical clusters: first cluster consists of two strains and one strain present in the 2nd, 3rd and 4th clusters. Streptomyces sp. GHB5, was the most active Streptomyces sp. for maximum polygalacturonase production identified by 16S rRNA sequencing. It was provided a Gene Bank accession number LC405930. The optimum culture conditions for the maximal enzyme productivity were studied. The best obtained conditions were achieved using broth medium with working volume of 20 % (v/v), containing 1.5% (w/v) lemon peel and 0.28% (w/v) potassium nitrate with initial medium pH of 7.5, when incubated at 30°C and 180 rpm for 6 days with an inoculum size of 4% (v/v) of 5 days inoculum age. Maximum polygalacturonase production was achieved at g/l: lemon peel, 15; KNO3, 2.8; (C/N ratio= 10.58) and K2HPO4, 1.0; CaCO3, 3; MgSO4, 0.5; NaCL, 0.5 and FeSO4.7H2O, 0.01. The enzyme was purified by 40 % ethanol precipitation followed by gel filtration chromatography using Sephedex G-100 column, with about 28.40 % yield and 11.29 fold purification. The optimum pH and temperature for the purified polygalacturonase activity was 9 and 45 oC, respectively. The enzyme was stable within the pH range of 7.0 to 10 and temperature up to 50 oC. The polygalacturonase showed specific activity towards different pectins tested. The polygalacturonase exhibited Km and Vmax values of 4.35 mg/ml and 80.65 µmol/min/mg respectively. Different metal ions at 1 mM, showed different effect on enzyme activity, where Cu2+, Ag+, Cd2+ and Hg2+ were found to increase the Polygalacturonase activity. On the other hand K+, Ca2+, Mg2+ inhibit enzyme activity, while Zn2+ Mn2+ Ba2+ ,Co2+ ,Pb2+, Ni2+ and Li+ were partially inhibited the enzyme activity. The purified enzyme was stable for more than 24 week at 4 oC. The purified enzyme has a molecular weight of 35 KDa. Laboratory produced polygalacturonase from Streptomyces sp. GHB5 used for clarification of orange juice, apple juice and hydrolysis of agriculture waste corn cub, wheat brane, orange peel, lemon peel and banana fiber. The immobilized polygalacturonase produced by Streptomyces sp. GHB5 was reused via four catalytic cycles.
2018
Ph.d
Helwan
Science