Glucoamylases: Microbial Sources, Industrial Applications and Molecular Biology ? A Review (original) (raw)
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Glucoamylases: structural and biotechnological aspects
Applied Microbiology and Biotechnology, 2011
Glucoamylases, one of the main types of enzymes involved in starch hydrolysis, are exo-acting enzymes that release consecutive glucose units from the non-reducing ends of starch molecules. Glucoamylases are microbial enzymes, present in bacteria, archaea, and fungi but not in plants and animals. Structurally, they are classified in family 15 of glycoside hydrolases and characterised by the invariable presence of a catalytic domain with (α/α)6-fold, often bound to a non-catalytic domain of diverse origin and function. Fungal glucoamylases are biotechnologically very important as they are used industrially in large amounts and have been extensively studied during the past 30 years. Prokaryotic glucoamylases are of biotechnological relevance for being generally thermophilic enzymes, active at elevated temperatures.
Microbial Glucoamylases: Biochemical and Biotechnological Features
Starch - Stärke, 1989
gel which became firm on ageing. In addition they generally have poor retention of gel structure on thawing after freezing. The dextrinised starches produced uniform brownish solutions which did not gel on ageing at room temperature. The highest acetyl substituted starch on the other hand formed paste which was clearer than that of unmodified starch with a better freezethaw stability and reduced syneresis. Lesser acetyl substituted starches also gave pastes of improved clarity but exhibited a poor freeze-thaw stability. The unmodified starch gave a clear, translucent, uniform and non-coloured gel, on ageing, however, the product retrograded, which has been reported by Chan and Toledo [16].
Cultural conditions for production of glucoamylase fromLactobacillus amylovorusATCC 33621
Journal of Applied Bacteriology, 1995
Lactobacillus amylovorus ATCC 33621 is an actively amylolytic bacterial strain which produces a cell-bound glucoamylase (EC 3.2.1.3). Conditions of growth and glucoamylase production were investigated using dextrose-free de Man-Rogosa-Sharpe (MRS) medium in a 1.5 1 fermenter, with varying dextrin concentration (0*1-1*5% (w/v)), pH (4.5-6.5) and temperature (25-55°C). Cell extracts were prepared by subjecting cells to treatment with a French Pressure cell in order to release intracellular proteins. Glucoamylase activity was then assayed. The effects of pH (4. k 9.0), temperature (15-8SOC) and substrate (dextrin and starch, 0-2Yo w/v) concentration on crude enzyme activity were investigated. Optimal growth was obtained in MRS medium containing 1% (w/v) dextrin, at pH 5.5 and 37°C. Glucoamylase production was maximal at the late logarithmic phase of growth, during 16-18 h. Crude enzyme had a pH optimum of 6-0 and temperature optimum of 60°C. With starch as the substrate, maximal activity was obtained at a concentration of 1*5O/o (w/v). The effects of ions and inhibitors on glucoamylase activity were also investigated. Enzyme activity was not significantly influenced by Ca2+ and EDTA at 1 mmol I-' concentration ; however Pb2+ and Co2+ were found to inhibit the activity at concentrations of 1 mmol I-'. The crude enzyme was found to be thermolabile when glucoamylase activity decreased after about 10 min exposure at 60°C. This property can be exploited in the brewing of low calorie beers where only mild pasteurization treatments are used to inactivate enzymes. The elimination of residual enzyme effect would prevent further maltodextrin degradation and sweetening during long-term storage, thus helping to stabilize the flavour of beer.
Characterization of two forms of glucoamylase from aspergillus niger
Carlsberg Research Communications, 1982
Aspergillus niger glucoamylases GI and GII (E.C. 3.2.1.3) were isolated from a commercial enzyme preparation by ammonium sulfate precipitation followed by DEAE-cellulose ion exchange chromatography. Both enzymes consist of a single glycosylated polypeptide chain. The molecular weights of GI and GII were determined by sedimentation equilibrium ultracentrifugation to 52,000 and 46,000, respectively, and by molecular sieving to 65,000 and 55,000. The amino acid compositions of GI and GII were very similar. Furthermore, the N-terminal amino acid sequence of the intact GI and GII as well as of their cyanogen fragments were identical, suggesting great homology in the primary structure of the two forms. In addition the digests of GI and GII produced respectively by Armillaria mellea protease, Staphylococcus aureus V8 protease, and submaxilla~ protease were analyzed by high pressure gel permeation chromatography. The elution profiles were also consistent with GI and GII having similar polypeptide chains. However, digestion with carboxypeptidase Y showed different C-terminal residues of the two forms. 1. INTRODUCTION Glucoamylase (l,4-aD -glucan glucohydrolase, E.C. 3.2.1.3) catalyzes the release of Dglucose from the non-reducing end of starch, glycogen and gluco-oligosaccharides. Although the ct-i,6-glucosidic linkages are cleaved less readily than the a-l,4-glucosidic linkages (17, 28, 38), the debranching capacity of glucoamylases is sufficient to make them important in the industrial production of glucose from starch, a Abbreviations: GI and GII denote two forms of glucoamylase.
2017
Improvements in the industrial process involved in starch degradation have led to the search for thermostable, salt-loving amylolytic enzymes especially glucoamylase, an enzyme that can completely hydrolyze starch to glucose. In this research, glucoamylase was optimally produced in liquid culture from Aspergillus fumigatus and purified to homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and gel filtration, giving a yield of 8.19% with 20-fold purification. The 50 kDa glucoamylase, molecular weight estimated by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was highly stable over a wide pH range in the acidic region and also thermostable; retaining about 70% of initial activity after 60 min of incubation at 60ºC. The purified enzyme was active on amylose, dextran and different starches with K m and V max values of 1.59 mg/mL and 14.33 U/ mg respectively, when raw cassava starch was used as substrate. Remarkably, this glucoamylase poss...
African Journal of Biotechnology, 2010
Glucoamylase (GA) from Aspergillus flavus HBF34 strain was partially purified 120 folds using starch affinity chromatography. Two isoenzymes (GA1 and GA2) were identified by polyacrylamide gel electrophoresis (PAGE) zymography. Sodium dodecyl sulfate (SDS)-PAGE analysis revealed that one of the enzymes consist of one subunit and the other, two subunits. The optimum pH of the purified GA was 6.0 and the optimum temperature was 60°C. GA was found to be stable at temperatures up to 50°C and at a pH range between 3.0 and 9.0. Km and Vmax values of the enzymes were determined using soluble potato starch, glycogen, amylopectin and amylose as substrates and calculated to be 0.046, 0.075, 0.1 and 0.125 mg/ml and 769, 1250, 3333 and 2500 U/mg protein, respectively. While GA was activated by Mn 2+ , Ca 2+ , Co 2+ and Ba 2+ , it was inhibited by Hg 2+ , Fe 3+ , Al 3+ , Zn 2+ and Cu 2+. The activity of GA was found to be tolerant up to 5 M NaCl concentration. N-bromosuccinimide (NBS) and phenylmethanesulfonylfluoride (PMSF) inhibited the enzyme, suggesting the involvement of tryptophan and serine residues in the catalytic process. Raw corn starch adsorption of GA was found to be 93%. Thin-layer chromatography (TLC) results showed that amylase was in fact a glucoamylase.
Characterization of glucoamylase from Lactobacillus amylovorus ATCC 33621
Biotechnology Letters, 1996
An intracellular glucoamylase, purified from Lactobacillus amylovorus, reacted selectively with polysaccharides. Kinetic studies indicated low affinity for maltose and maltotriose (K, 58 g/ml and 178 g/ml) and higher affinity for starch and dextrin (K, 0.01 g/ml and 0.02 g/ml). Glucoamylase was inhibited almost 50% by 10 mM glucose. Cu2' and Pb2+ inhibited glucoamylase at 1 .O mM but EDTA and other metal chelators had no effect on the enzyme activity. Acarbose and Tris inhibited the enzyme by 84% and 98%, respectively at 1 mM, while iodoacetate and p-chloromecuribenzoic acid inhibited activity by 98% and 78%, respectively at 10 mM. The purified enzyme was thermolabile at temperatures greater than 55'C and thus has potential for application in the brewing industry.
Properties of a purified thermostable glucoamylase from Aspergillus niveus
Journal of Industrial Microbiology & Biotechnology, 2009
A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40°C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0–5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0–9.5. The temperature optimum was 65°C and retained 100% activity after 240 min at 60°C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K m was calculated as 0.32 mg ml−1. Circular dichroism spectroscopy estimated a secondary structure content of 33% α-helix, 17% β-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.
Purification and properties of two forms of glucoamylase fromAspergillus niger
Folia Microbiologica, 1996
A. niger produced c~-glucosidase, a-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylascs, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5-9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65 ~ respectively, and were stable for 1 h at temperatures of up to 60 ~ The kinetic parameters K m and Vshowed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GAl.