Production, Purification and Characterization of Polygalacturonase from Bacillus licheniformis SHG10

Document Type : Original Article

Authors

1 Department of Biochemistry, Faculty of Science, Alexandria University

2 City of Scientific Researches and Technological Applications, Borg El Arab, Alexandria, Egypt

3 Department of Biotechnology, Institute of Graduate studies and Research, Alexandria University

Abstract

 




 Background: Microorganisms are the best source of pectinolytic enzymes as they allow an economical technology with low resource consumption. Objectives: The present study was carried out to isolate, purify and characterize polygalacturonase (PGase) form Bacillus licheniformis SHG10. Methods: The concentrated dialysed cell free extract was loaded on prepacked DEAE-Sepharose Fast-Flow ion exchange chromatography. The active PGase fractions were concentrated and loaded again on sephacryl Fast-Flow High Resolution (FF S-100 HR) chromatography. Molecular weight was determined using slab-gel SDS-Polyacrylamid gel electrophoresis and Characterization of the purified enzyme was performed. Results: The results showed that the enzyme was purified with a purification fold (33.34) and yield (41.73%) with specific activity (8.67 μ moles/min/mg protein). It has a molecular mass of about 68.0 kDa. While, on SDS-PAGE electrophoresis, the purified PGase appeared as single band with molecular mass of about 34.0 kDa, suggesting that the purified PGase is a dimer protein. The purified enzyme exhibited maximal activity at a temperature of 45oC and pH 8.5. The maximum velocity of the enzyme in presence of citrus pectin and pectate as substrates were 10.98 and 14.7 μ mol galacturonate/min/mg protein, respectively. The Michaelis constant (Km) values were 0.085 and 0.039 mM, respectively, indicating that the purified PGase has higher affinity to pectate (non-methylated pectic substance) than citrus pectin as substrate. 

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