Molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a Bacillus isolate and properties of its recombinant enzyme |
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| Institution: | 1. Tochigi Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, 321-3497 Tochigi, Japan;2. Japan Marine Science and Technology Center, 2-15 Natsushima, Yokosuka 237-0061, Japan;1. College of Horticulture and Forestry Sciences, Huazhong Agricultural University and Key Laboratory of Horticultural Plant Biology, Ministry of Education, Wuhan, 430070, PR China;2. Department of Horticulture, College of Agriculture, University of Sargodha, Pakistan;1. VIP Department, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, No. 33 Ba-Da-Chu Road, Shi Jing Shan District, Beijing 100144, PR China;2. 4th Department, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, No. 33 Ba-Da-Chu Road, Shi Jing Shan District, Beijing 100144, PR China;1. Department of Plant Pathology, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India;2. Department of Veterinary Microbiology, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India;3. Department of Microbiology, CSK-Himachal Pradesh Agricultural University, Palampur, 176062, India;1. Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao, 266003, China;2. Department of Microbiology, University of Central Punjab, Lahore, Pakistan;3. Department of Microbiology, Hazara University, Mansehra, Pakistan |
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| Abstract: | An exopolygalacturonase (exo-PGase; EC 3.2.1.82) was found in the culture broth of a Bacillus isolate. The gene encoding the exo-PGase, pehK, was cloned by polymerase chain reaction using mixed primers designed from N-terminal and internal amino acid (aa) sequences of the enzyme (PehK). The determined nucleotide (nt) sequence of pehK revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103 810 Da). The recombinant enzyme was purified to homogeneity from a culture broth of Bacillus subtilis harboring a pehK-containing plasmid. It had a molecular mass of 105 kDa and a pI value of 5.0. The maximum activity was observed at pH 8 and 55°C in Tris–HCl buffer. The degradation products from polygalacturonic or oligogalacturonic acids were digalacturonic acid, like the exo-PGases, PehX of Erwinia chrysanthemi and PehB of Ralstonia solanacearum. The deduced aa sequence of PehK exhibited moderate homology to those of PehX and PehB with approx. 30% identity for both. High homology was observed in a suitably aligned internal region of the three enzymes (65% identity), and some of the conserved aa residues appeared to form the catalytic core of the enzymes. |
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