Biochemical and molecular characterization of some cyclodextrin glycosyl transferase enzyme producing bacteria and actinomycetes
Samar Zaim Rashid Al-Sharawi;
Abstract
In the present study, eleven CGTase-producing isolates were obtained from different isolation sources. These isolates were screened for CGTase production and only four isolates were selected.
The most potent isolates AB2, TB1, TA1 and TA4 were selected and identified by biochemical methods and partial 16S rDNA sequence as Bacillus hunanensis, Bacillus caldotenax, Thermoactinomyces vulgaris and Laceyella sacchari, respectively.
CGTase gene isolation from actinobactria was difficult as actinobacteria have high GC ratio. While, CGTase gene from alkalophilic bacteria AB2 was amplified by PCR primer pair DF1 and DR1 and identified as cyclodextrin glucotransferase (cgt) gene. The AB2 CGTase revealed partial protein sequence alignment revealed the presence of 3 conserved regions (I, II and III) out of the 4 conserved regions that have been identified in the α-amylase family. CGTase from alkalophilic isolate AB2 and thermophilic isolate TB1 sequence using degenerate primer pair S209 and S211 were identified as maltose ABC transporter substrate-binding protein (MalE).
The nutritional and physical conditions for CGTase production were optimized. The best carbon source was soluble starch at concentration 15 g/l and the best nitrogen source was peptone and yeast extract (1:1) (w:w) at concentration 30 g/l for AB2, 20 g/l for TB1 and TA4 and 15 g/l for TA1. The best sodium chloride concentration was 25 g/l for alkaliphilic isolate AB2 and 10 g/l for thermophilic isolates TB1, TA1 and TA4. Sodium carbonate concentration was optimized for the alkaliphilic isolate only and the best concentration was 7.5 g/l. The optimum incubation temperature for alkaliphilic isolate AB2 was 37°C, for thermophilic bacteria TB1
was 70°C and for thermophilic actinomycetes TA1 and TA4 was 50°C. The best harvesting time for CGTase was after 48 h and the best agitation speed was 180 r.p.m.
Production of CGTase in large scale was carried out usin
The most potent isolates AB2, TB1, TA1 and TA4 were selected and identified by biochemical methods and partial 16S rDNA sequence as Bacillus hunanensis, Bacillus caldotenax, Thermoactinomyces vulgaris and Laceyella sacchari, respectively.
CGTase gene isolation from actinobactria was difficult as actinobacteria have high GC ratio. While, CGTase gene from alkalophilic bacteria AB2 was amplified by PCR primer pair DF1 and DR1 and identified as cyclodextrin glucotransferase (cgt) gene. The AB2 CGTase revealed partial protein sequence alignment revealed the presence of 3 conserved regions (I, II and III) out of the 4 conserved regions that have been identified in the α-amylase family. CGTase from alkalophilic isolate AB2 and thermophilic isolate TB1 sequence using degenerate primer pair S209 and S211 were identified as maltose ABC transporter substrate-binding protein (MalE).
The nutritional and physical conditions for CGTase production were optimized. The best carbon source was soluble starch at concentration 15 g/l and the best nitrogen source was peptone and yeast extract (1:1) (w:w) at concentration 30 g/l for AB2, 20 g/l for TB1 and TA4 and 15 g/l for TA1. The best sodium chloride concentration was 25 g/l for alkaliphilic isolate AB2 and 10 g/l for thermophilic isolates TB1, TA1 and TA4. Sodium carbonate concentration was optimized for the alkaliphilic isolate only and the best concentration was 7.5 g/l. The optimum incubation temperature for alkaliphilic isolate AB2 was 37°C, for thermophilic bacteria TB1
was 70°C and for thermophilic actinomycetes TA1 and TA4 was 50°C. The best harvesting time for CGTase was after 48 h and the best agitation speed was 180 r.p.m.
Production of CGTase in large scale was carried out usin
Other data
| Title | Biochemical and molecular characterization of some cyclodextrin glycosyl transferase enzyme producing bacteria and actinomycetes | Authors | Samar Zaim Rashid Al-Sharawi | Issue Date | 2018 |
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