An extracellular glucoamylase produced by endophytic fungus EF6 |
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Authors: | P Tangngamsakul A Karnchanatat P Sihanonth P Sangvanich |
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Institution: | 1.Biotechnology Programme, Faculty of Science,Chulalongkorn University,Bangkok,Thailand;2.Research Institute of Biotechnology and Genetic Engineering,Chulalongkorn University,Bangkok,Thailand;3.Department of Microbiology, Faculty of Science,Chulalongkorn University,Bangkok,Thailand;4.Research Centre for Bioorganic Chemistry, Department of Chemistry, Faculty of Science,Chulalongkorn University,Bangkok,Thailand |
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Abstract: | A strain of endophytic fungus EF6 isolated from Thai medicinal plants was found to produce higher levels of extracellular
glucoamylase. This strain produced glucoamylase of culture filtrate when grown on 1% soluble starch. The enzyme was purified
and characterized. Purification steps involved (NH4)2SO4 precipitation, anion exchange, and gel filtration chromatography. Final purification fold was 14.49 and the yield obtained
was 9.15%. The enzyme is monomeric with a molecular mass of 62.2 kDa as estimated by SDS-PAGE, and with a molecular mass of
62.031 kDa estimated by MALDI-TOF spectrometry. The temperature for maximum activity was 60°C. After 30 min for incubation,
glucoamylase was found to be stable lower than 50°C. The activity decrease rapidly when residual activity was retained about
45% at 55°C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0–7.0 at 50°C. The activity
of glucoamylase was stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg2+. Various types of starch were test, soluble starch proved to be the best substrate for digestion process. The enzyme catalyzes
the hydrolysis of soluble starch and maltose as the substrate, the enzyme had K
m values of 2.63, and 1.88 mg/ml and V
max, values of 1.25, and 2.54 U/min/mg protein, and V
max/K
m values of 0.48 and 1.35, respectively. The internal amino acid sequences of endophytic fungus EF6 glucoamylase; RALAN HKQVV
DSFRS have similarity to the sequence of the glucoamylase purified form Thermomyces lanuginosus. From all results indicated that this enzyme is a glucoamylase (1,4-α-D-glucan glucanohydrolase). |
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