Gene cloning and characterization of the cyclodextrin glycosyltrasferase gene from thermotolerant Paenibacillus sp. RB01 and Paenibacillus sp. T16

Abstract

Genes encoding cyclodextrin glycosyltransferase (CGTase), from the thermotorelant Paenibacillus sp. RB01 and Paenibacillus sp. T16 isolated from the hot spring area in Ratchaburi and Tak provinces were cloned into Escherichia coli. Genomic DNA was extracted and used as the template for CGTase gene amplification using a pair of designed primers that amplified a whole CGTase gene including its promoter. The PCR product was ligated to pGEM®-T vector and then transformed into E. coli JM109. The transformants of Paenibacillus sp. RB01 and Paenihacillus sp. T16 were pRB and pT, respectively. Their nucleotide sequences from pRB and pT consisted of 2194 and 2139 bp open reading frame with 732 and 713 deduced amino acid residues, respectively. The deduced amino acid sequences of pRB and pT showed 97% and 99% identity with the CGTase of Paenibacillus sp. All, respectively. The recombinants required one-third culture time of wild types and a neutral pH for culture medium to produce compatible amount of CGTase. The recombinant and wild type CGTases were purified and characterized in parallel. Both enzymes showed almost similar biochemical characteristics in terms of molecular weight, optimum pH and temperature. There were some significant differences in pH, temperature stability and kinetic parameters. The recombinant enzymes were more stable at higher temperature and low pH. Molecular weight of GCTase from pRB and pT were estimated to be 65 and 77 kDa by SDS-PAGE and pl of 5.85. The optimum pH and temperature for dextrinizing activities of CGTases from RB01 and pRB were 5-6, 60°C and 5-9, 50-70°C. The optimum pH and temperature for cyclization activity of CGTases from RB and pRB were 6.5, 60-70°C and 6.5, 50-60°C. The optimum pH and temperature for dextrinizing activity of CGTases from T16 and PT were 5-9, 60°C and 5-9, 40-70°C. The optimum pH and temperature for cyclization activity of CGTases from T16 and PT were 6.5, 60-70°C and 6.5, 50-70°C. The enzymes were stable in a wide pH range of 6-10 and temperature at 40-60°C within 30 minutes. The enzymes were stable at pH 7-9 and temperature of 45-55°C within 60 min. The enzymes were specific for substrates with α-1,4 glycosidic bonds, with minimum of 3 glucose units. The turnover numbers (k[subscript cat]) of the CGTases from both RB01 and pRB with G- α-CD were higher than those of β-CD and its derivatives. The turnover numbers (k[subscript cat]) of the CGTases from T16 and pT with natural CD (α-CD and γ -CD) were higher than those of β-CD and its derivatives.. The efficiency (k[subscript cat]/ K[subscript m]) of CGTases from both RB01 and PRB were highest with HP-B-CD and the efficiency (k[subscript cat]/ K[subscript m]) of CGTases from T16 and pT were highest with β-CD. The dominant end-products obtained were β-CDs. CGTases catalyzed the conversion of starch to CDs with a ratio of α-: β-: γ-CD of 0.57 : 1 : 0.13 for RB01 and 0.21 : 1 : 0.05 for pRB and 0.95 : 1 : 1.57 for T16 and 0.25 : 1: 0.51 for pT, respectively.