title: Properties of a purified thermostable glucoamylase from Aspergillus niveus.
abstract: A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40 degrees 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 degrees C and retained 100% activity after 240 min at 60 degrees 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% alpha-helix, 17% beta-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.
authors.affiliation: Departamento de Biologia, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Monte Alegre, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Tony Marcio
authors.initials: TM
authors.lastname: da Silva
authors.forename: Alexandre
authors.initials: A
authors.lastname: Maller
authors.forename: André Ricardo de Lima
authors.initials: AR
authors.lastname: Damásio
authors.forename: Michele
authors.initials: M
authors.lastname: Michelin
authors.forename: Richard John
authors.initials: RJ
authors.lastname: Ward
authors.forename: Izaura Yoshico
authors.initials: IY
authors.lastname: Hirata
authors.forename: João Atilio
authors.initials: JA
authors.lastname: Jorge
authors.forename: Héctor Francisco
authors.initials: HF
authors.lastname: Terenzi
authors.forename: Maria Lourdes T M
authors.initials: ML
authors.lastname: de Polizeli
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000378:metabolism; D002845:Chromatography; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight
chemical_list: D005087:Glucan 1,4-alpha-Glucosidase
references.citation: J Microbiol. 2005 Dec;43(6):561-8
references.pmid: 16410774
references.citation: Biosci Biotechnol Biochem. 2003 Feb;67(2):334-40
references.pmid: 12728995
references.citation: Crit Rev Biochem Mol Biol. 1989;24(4):329-418
references.pmid: 2548811
references.citation: J Agric Food Chem. 2008 Jan 9;56(1):126-31
references.pmid: 18072737
references.citation: Appl Microbiol Biotechnol. 2003 May;61(4):323-8
references.pmid: 12743761
references.citation: Appl Microbiol Biotechnol. 2001 Oct;57(1-2):146-52
references.pmid: 11693912
references.citation: J Mol Biol. 1994 May 13;238(4):575-91
references.pmid: 8176747
references.citation: Trends Biotechnol. 1997 Mar;15(3):97-101
references.pmid: 9080715
references.citation: Comput Appl Biosci. 1993 Apr;9(2):197-9
references.pmid: 8481823
references.citation: J Biol Chem. 1951 Nov;193(1):265-75
references.pmid: 14907713
references.citation: Appl Environ Microbiol. 1995 Dec;61(12):4258-62
references.pmid: 16535181
references.citation: Biotechnol Appl Biochem. 2000 Apr;31(2):135-52
references.pmid: 10744959
references.citation: J Ind Microbiol Biotechnol. 2008 Jan;35(1):17-25
references.pmid: 17938981
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Appl Microbiol Biotechnol. 1999 May;51(5):673-5
references.pmid: 10390823
references.citation: Biochim Biophys Acta. 2005 Jun 15;1750(1):61-8
references.pmid: 15886078
references.citation: Ann N Y Acad Sci. 1964 Dec 28;121:404-27
references.pmid: 14240539
references.citation: J Mol Biol. 1996 Jun 28;259(5):970-87
references.pmid: 8683599
references.citation: Biotechnol Adv. 2006 Jan-Feb;24(1):80-5
references.pmid: 16091302
references.citation: Biotechnol Bioeng. 1989 Aug 20;34(5):681-8
references.pmid: 18588152
references.citation: Extremophiles. 2005 Feb;9(1):85-9
references.pmid: 15378403
references.citation: Extremophiles. 2007 Nov;11(6):781-8
references.pmid: 17619813
references.citation: World J Microbiol Biotechnol. 1995 Mar;11(2):242-3
references.pmid: 24414515
references.citation: Protein Eng. 1993 Jun;6(4):383-90
references.pmid: 8332596