Purification of extracellular amylolytic enzymes from the thermophilic fungus <i>Thermomyces lanuginosus</i>

Jensen, Bo; Olsen, John Elmerdahl; Allermann, K.

doi:10.1139/m88-041

“…The glucoamylase from the thermophilic fungus Thermomyces lanuginosus is a candidate in this respect. Glucoamylase from this thermophile has been described by Taylor et al (1978) and Basaveswara Rag et al (1979Rag et al ( , 1981, Jensen et al (1988) showed that several extracellular amylolytic activities viz. alpha-amylase, glucoamyOffprint requests to: I. Haasum lase and alpha-glucosidase were excreted when T. lanuginosus was cultivated in agar-free YpSs medium (Emerson 1941).…”

Section: Introductionmentioning

confidence: 86%

“…1457, The Royal Verterinary and Agricultural University, Copenhagen, Denmark). Conidiated cultures were produced as previously described (Jensen et al 1988) and maintained on YpSs agar (Emerson 1941) (Vogel 1964). In some of the experiments starch was replaced by maltose (5885, Sigma, St. Louis, Mo., USA) and yeast extract by one of eight nitrogen sources in concentrations equivalent to the amount of nitrogen in yeast extract.…”

Section: Methodsmentioning

confidence: 99%

“…One unit of activity was defined as the amount of protein which liberated 1 Ixmol glucose/min under the specified conditions. With crude enzyme extracts this method will not be specific for the content of glucoamylase in the fermentation broths as the presence of alpha-amylase and alpha-glucosidase interferes (Yamamoto 1988;Jensen et al 1988).…”

Section: Methodsmentioning

confidence: 99%

“…Purification by DEAE was selected to remove interfering alpha-amylase and alpha-glucosidase from the glucoamylase preparation (Jensen et al 1988). The crude culture filtrate from a 96-h-old fermentation was concentrated by ultraflltration on a membrane (GR 61 PP, De Danske sukkerfabrikker, DDS RO-Division, Nakskov, Denmark) with a cut-off at 20 kDa.…”

Section: Deae Ion-exchan~te Chromato[lraphymentioning

confidence: 99%

“…A simultaneous shift in buffer to 50 mM imidazole buffer, pH 7.0, containing 5 mM NaN3 was performed. Samples were applied to the column and chromatographed as previously decribed (Jensen et al 1988). Fractions with glucoamylase activity were pooled and dialysed against the 50 mM imidazole buffer, pH 7.0, and rechromatographed using a linear gradient of NaCI (70-140 mM) in a total volume of 500 ml.…”

Section: Deae Ion-exchan~te Chromato[lraphymentioning

confidence: 99%

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Growth and glucoamylase production by the thermophilic fungusThermomyces lanuginosus in a synthetic medium

Haasum

¹

,

Eriksen

²

,

Jensen

³

et al. 1991

Appl Microbiol Biotechnol

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The production of glucogenic amylase from the thermophilic fungus Thermomyces lanuginosus was studied in shake flasks and laboratory fermentors. As conidia were not able to germinate in media without yeast extract, pregerminated conidia were applied as inoculum. By this procedure it was possible to use different NH~-salts as the sole source of nitrogen for growth and amylase formation in a synthetic medium. In pH-controlled fermentors a fourfold increase in the extracellular glucogenic amylase activity was obtained with (NHn)H2PO4 as the nitrogen source as compared with yeast extract. However, by fractionation of these activities, comparable yields of partially purified glucoamylases were obtained. The glucoamylase preparation from fermentations with either of the nitrogen sources had a temperature optimum at 70°C and showed similar thermal stability. By incubation without substrate at 60 ° C, 90% of the activity was still present after 5 h. At 70 ° C, 50% of the activity was retained after 30 min incubatiom

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Thermostableα-Amylase and Glucoamylase fromThermomyces lanuginosus F1

Odibo

¹

,

Ulbrich‐Hofmann

²

2001

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An α-amylase and a glucoamylase produced by Thermomyces lanuginosus F 1 were separated by ionexchange chromatography on Q-Sepharose fast flow. The enzymes were further purified to electrophoretic homogeneity by chromatography on Sephadex G-100 and Phenyl-Sepharose CL-4B. The molecular weights and isoelectric points of the enzymes were 55,000 Da and pH i 4.0 for α-amylase and 70,000 Da and pH i 4.0 for glucoamylase, respectively. The optimum pH and temperatures for the enzymes were found to be 5.0 and 60°C for α-amylase, and 6.0 and 70°C for glucoamylase, respectively. Both enzymes were maximally stable at pH 4.0 and retained over 80% of their activity between pH 5.0 and 6.0 for 24 h. After incubation at 90°C (1 h), the α-amylase and glucoamylase retained only 6% and 16% of their activity, respectively. The enzymes readily hydrolyzed soluble starch, amylose, amylopectin and glycogen but hydrolyzed pullulan very slowly. Glucoamylase and α-amylase had highest affinity for soluble starch with K M values of 0.80 mg/ml and 0.67 mg/ml, respectively. The α-amylase hydrolyzed raw starch granules with a predominant production of glucose and maltose. The activities of α-amylase and glucoamylase increased in the presence of Mn 2+ , Co 2+ , Ca 2+ , Zn 2+ and Fe 2+ , but were inhibited by guanidine-HCl, urea and disodium EDTA. Both enzymes possess pH and thermal stability characteristics that may be of technological significance.

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Thermostable -Amylase and Glucoamylase from Thermomyces lanuginosus F1

Odibo

¹

,

Ulbrich‐Hofmann

²

2001

View full text Add to dashboard Cite

An α-amylase and a glucoamylase produced by Thermomyces lanuginosus F 1 were separated by ionexchange chromatography on Q-Sepharose fast flow. The enzymes were further purified to electrophoretic homogeneity by chromatography on Sephadex G-100 and Phenyl-Sepharose CL-4B. The molecular weights and isoelectric points of the enzymes were 55,000 Da and pH i 4.0 for α-amylase and 70,000 Da and pH i 4.0 for glucoamylase, respectively. The optimum pH and temperatures for the enzymes were found to be 5.0 and 60°C for α-amylase, and 6.0 and 70°C for glucoamylase, respectively. Both enzymes were maximally stable at pH 4.0 and retained over 80% of their activity between pH 5.0 and 6.0 for 24 h. After incubation at 90°C (1 h), the α-amylase and glucoamylase retained only 6% and 16% of their activity, respectively. The enzymes readily hydrolyzed soluble starch, amylose, amylopectin and glycogen but hydrolyzed pullulan very slowly. Glucoamylase and α-amylase had highest affinity for soluble starch with K M values of 0.80 mg/ml and 0.67 mg/ml, respectively. The α-amylase hydrolyzed raw starch granules with a predominant production of glucose and maltose. The activities of α-amylase and glucoamylase increased in the presence of Mn 2+ , Co 2+ , Ca 2+ , Zn 2+ and Fe 2+ , but were inhibited by guanidine-HCl, urea and disodium EDTA. Both enzymes possess pH and thermal stability characteristics that may be of technological significance.

show abstract

Purification of extracellular amylolytic enzymes from the thermophilic fungus Thermomyces lanuginosus

Cited by 40 publications

References 5 publications

Growth and glucoamylase production by the thermophilic fungusThermomyces lanuginosus in a synthetic medium

Growth and glucoamylase production by the thermophilic fungusThermomyces lanuginosus in a synthetic medium

Thermostableα-Amylase and Glucoamylase fromThermomyces lanuginosus F1

Thermostable -Amylase and Glucoamylase from Thermomyces lanuginosus F1

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