Purification and functional properties of a novel glucoamylase activated by manganese and lead produced by Aspergillus japonicus.

Microbial amylases are used to produce ethanol, glucose and can be applied in textiles products, detergents and other industries. This study aimed to determine the best carbon source concentration to induce the amylase production by A. japonicus, and its purification and biochemical characterization. For that, this fungus was cultivated in Khanna medium, pH 5.5, for 4 days, at 25°C, in static condition, supplemented with potato starch and maltose in different concentrations. The fungal crude enzymatic extract was purified in a unique elution in DEAE-cellulose column and the molecular mass was determined as 72kDa. The optimum temperature and pH was 65°C and 5.0, respectively. Amylase remained 75% of its activity after one hour at 50°C and was stable in the pH range 3.0-7.0. The analysis of the end-products by thin layer chromatography showed only glucose formation, which characterizes the purified enzyme as a glucoamylase. Amylopectin was the best substrate for the enzyme assay and Mn+2 and Pb+2 were good glucoamylase activators. This activation, in addition to the biochemical characteristics are important results for future biotechnological applications of this glucoamylase in the recycling and deinking process by the paper industries.

An extracellular glucoamylase produced by endophytic fungus EF6.

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 degrees C. After 30 min for incubation, glucoamylase was found to be stable lower than 50 degrees C. The activity decrease rapidly when residual activity was retained about 45% at 55 degrees 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 degrees 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 Km values of 2.63, and 1.88 mg/ml and Vmax, values of 1.25, and 2.54 U/min/mg protein, and Vmax/Km 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-alpha-D-glucan glucanohydrolase).

Amylolytic hydrolysis of native starch granules affected by granule surface area.

Initial stage of hydrolysis of native starch granules with various amylolytic enzymes, alpha-amylase from Bacillus subtilis, glucoamylase I (GA-I) and II (GA-II) from Aspergillus niger, and beta-amylase from sweet potato showed that the reaction was apparently affected by a specific surface area of the starch granules. The ratios of the reciprocal of initial velocity of each amylolytic hydrolysis for native potato and maize starch to that for rice with the amylolytic enzymes were nearly equivalent to the ratio of surface area per mass of the 2 starch granules to that of rice, that is, 6.94 and 2.25, respectively. Thus, the reciprocal of initial velocity of each enzymatic hydrolysis as expressed in a Lineweaver-Burk plot was a linear function of the reciprocal of surface area for each starch granule. As a result, it is concluded that amylolytic hydrolysis of native starch granules is governed by the specific surface area, not by the mass concentration, of each granule.

Purification, identification and molecular cloning of glycoside hydrolase family 15 glucoamylase from the brown-rot basidiomycete Fomitopsis palustris.

The brown-rot basidiomycete Fomitopsis palustris produces a major extracellular enzyme of 72 kDa when the fungus is incubated in cellulose culture with 0.2% cellobiose. This protein was purified by column chromatography, and the amino acid sequences of its proteolytic fragments were analyzed. The N-terminal amino acid sequence of one of the fragments showed high identity with fungal glycoside hydrolase family 15 glucoamylases. As its kinetic efficiency increased in proportion to the degree of polymerization of the substrate, the protein was identified as a glucoamylase. A cDNA encoding the glucoamylase (gla) was cloned by reverse transcriptase PCR.

Degradation of raw starch granules by alpha-amylase purified from culture of Aspergillus awamori KT-11.

Raw-starch-digesting alpha-amylase (Amyl III) was purified to an electrophoretically pure state from the extract of a koji culture of Aspergillus awamori KT-11 using wheat bran in the medium. The purified Amyl III digested not only soluble starch but also raw corn starch. The major products from the raw starch using Amyl III were maltotriose and maltose, although a small amount of glucose was produced. Amyl III acted on all raw starch granules that it has been tested on. However, it was considered that the action mode of the Amyl III on starch granules was different from that of glucoamylase judging from the observation of granules under a scanning electron microscope before and after enzyme reaction, and also from the reaction products. Glucoamylase (GA I) was also isolated and it was purified to an electrophoretically pure state from the extract. It was found that the electron micrographic features of the granules after treatment with the enzymes were quite different. A synergistic effect of Amyl III and GA I was observed for the digestion of raw starch granules.

Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins.

The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.

Studies on glucoamylase produced from Aspergillus awamori (NRRL-3112) and their effect on saccharification of potato starch.

Efficiency of an enzymatic starch saccharification process depends not only on the activity of the glucoamylase but also its purity. About 96.82% unwanted proteins present in 2-day old culture broth of A. awamori NRRL-3112 (grown in MYGP medium) were separated by precipitation with ammonium sulphate which was followed by dialysis. More than 80% activity of the glucoamylase was recovered. Three protein fractions (A, B, C) were identified using gel permeation chromatography. Fractions A and B showed comparatively higher glucoamylase activity than fraction-C. Moreover, fraction-B showed no product inhibition. The optimum temperature and pH of the purified enzyme (fraction-B) were 60 degrees C and 4.0 respectively. The saccharification efficiency of potato pulp was more in case of using purified glucoamylase (fraction-B) as compared to that of crude enzyme.