Penicillium chrysogenum glucose oxidase - a study on its antifungal effects (original) (raw)

Enhancement of glucose oxidase production by Penicillium variabile P16

Enzyme and Microbial Technology, 1999

Effects of the polysaccharides alginate and locust bean gum, and oligosaccharides oligomannuronate OM ( ) ( ) and oligoguluronate OG , on glucose oxidase GOD production by Penicillium variabile P16 were studied. Small increases were obser¨ed when the cultures were supplemented with OG and OM blocks with an a¨erage ( ) y1 degree of polymerization DP of approximately ten. With 200 mg l OM blocks addition at 0 h, the increase ( reached 32.1% compared with the control; howe¨er, regardless of the time of addition, large increases up to ) y 1 approximately 70% in GOD production were obtained with 100 and, particularly, 200 mg l of alginate-de-( ) ri¨ed oligosaccharides OG andr or OM blocks with a DP of approximately se¨en. No significant influence was obser¨ed on mycelial biomass. ᮊ 1999 Else¨ier Science Inc. All rights reser¨ed.

Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

Brazilian Archives of Biology and Technology, 2013

This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%), DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P.

ARTICLE Characterization of the Distribution of Glucose Oxidase in Penicillium sp. CBS 120262 and Aspergillus niger NRRL-3 Cultures and Its Effect on Integrated Product Recovery

Glucose oxidase (GO) is an important industrial enzyme typically purified from Penicillium and Aspergillus sp. As GO distribution within the cultures influences process design for maximal product recovery, distribution of GO activity in Penicillium sp. CBS 120262 and Aspergillus niger NRRL-3, during mid-exponential and stationary phases, is compared. On progression from mid-exponential to stationary phase, the percentage GO activity in the cytoplasm decreased 1.6-and 1.3-fold in Penicillium sp. and A. niger respectively. In Penicillium sp., a concomitant 1.8-and 1.9-fold decrease in the percentage GO activity in the cell envelope and slime mucilage respectively, translated into a 2.0-fold increase in the extracellular fluid. In A. niger, decreasing cytoplasmic GO activity was accompanied by 1.3fold increases in the cell envelope and slime mucilage, with a 1.3-fold decrease in the extracellular fluid. Similar trends were observed in specific GO activities. As final GO activity recovered is governed by the purification program, recovery from the extracellular fluid plus cell extract or from the extracellular fluid only were compared through simulating processes of varying complexity. A critical yield for each purification stage was identified above which recovery from the extracellular fluid plus cell extract exceeded that from extracellular fluid alone. These results highlight the influence of microorganism, harvest time and efficiency of downstream process on GO activity delivered. In the systems studied, Penicillium sp. is the organism of choice and should be harvested during stationary phase. The purification process chosen should be informed by both enzyme distribution and individual purification stages yields. Biotechnol. Bioeng. 2008;99: 910-918.

Application of high-performance chromatographic and electrophoretic methods to the purification and characterization of glucose oxidase and catalase from penicillium chrysogenum

Journal of Chromatography A, 1987

The high resolving power of the preparative and analytical high-performance chromatographic and electrophoretic methods recently developed in this laboratory for the separation of biopolymers has been demonstrated by the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum. Crude glucose oxidase was purified to homogeneity in one step by high-performance hydrophobic-interaction chromatography (HIC) on a pentylagarose column. Crude catalase was purified by a combination of HIC and high-performance anion-exchange chromatography on 3-diethylamino-2-hydroxypropylagarose. The homogeneity of the enzymes was monitored by high-performance electrophoresis and free zone electrophoresis. The pl values of these two enzymes determined by isoelectric focusing in the high-performance electrophoresis apparatus were 4.2 and 6.5, respectively. Their molecular weights were determined by high-performance molecular sieve chromatography on an agarose column. Glucose oxidase has a molecular weight of 175 000 and probably consists of two identical subunits, as sodium dodecyl sulphate polyacrylamide gel electrophoresis gave a molecular weight of around 72000. The molecular weight of catalase, which is probably composed of non-identical subunits, as indicated by sodium dodecyl sulphate electrophoresis, is around 320 000. Some other characteristics of these two enzymes were also investigated, e.g., electrophoretic mobility, pH stability and optimum pH.

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

Analytical Biochemistry, 2004

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of D D-glucose at carbon 1 into D D-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide (2 H 2 O). The intensity of the D D-glucono-1,5-lactone band maximum at 1212 cm À1 due to CAO stretching vibration was measured as a function of time to study the kinetics of D D-glucose oxidation. The extinction coefficient e of D D-glucono-1,5-lactone was determined to be 1.28 mM À1 cm À1. The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V max , K m , k cat , and k cat /K m) determined by Lineweaver-Burk plot were 433.78 ± 59.87 U mg À1 protein, 10.07 ± 1.75 mM, 1095.07 ± 151.19 s À1 , and 108.74 s À1 mM À1 , respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36 ± 42.83 U mg À1 protein, 6.47 ± 0.85 mM, 1187.77 ± 108.16 s À1 , and 183.58 s À1 mM À1 for V max , K m , k cat , and k cat /K m , respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense

Applied Microbiology and Biotechnology, 1997

Glucose oxidase from Penicillium amagasakiense was puri®ed to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel ®ltration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not aect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buer concentration and was enhanced by certain additives, particularly 1 M (NH 4) 2 SO 4 , which stabilised glucose oxidase 100-to 300-fold at 50°C and pH 7±8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60°C and pH 6. In sodium acetate buer, changes in pH (4±6) aected the anity for glucose but had no eect on the V max of the reaction. In contrast, in TRIS buer, pH 8, a 10-fold decrease in V max and a 2-fold decrease in K m were observed.

Glucose oxidase from Penicillium amagasakiense . Primary structure and comparison with other glucose-methanol-choline (GMC) oxidoreductases

European Journal of Biochemistry, 1998

The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage. The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N-glycosylation sites, of which at least five were confirmed to be glycosylated. Glucose oxidase from P. amagasakiense shows a high degree of identity (66%) and 79% similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose-methanol-choline (GMC) oxidoreductase family. The tertiary structures of glucose oxidase from A. niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family. The general topology of the GMC oxidoreductases is conserved, with the exception of the presence of an active site lid in cholesterol oxidase and the insertion of additional structural elements in the substrate-binding domain of alcohol oxidase. The overall structure can be divided into five distinct sequence regions: FADbinding domain, extended FAD-binding domain, flavin attachment loop and intermediate region, FAD covering lid, and substrate-binding domain. The FAD-binding and the extended FAD-binding domains are composed of several separate sequence regions. The other three regions each comprise a single contiguous sequence. Four major consensus patterns have been identified, including the nucleotide-binding consensus sequence close to their N-termini. The functions of the two motifs recently selected by the Genetics Computer Group, Madison, Wisconsin, as additional signature patterns of the GMC oxidoreductases are discussed. The other consensus patterns belong to either the FAD-binding or the extended FAD-binding domain. In addition, the roles of conserved residues are discussed wherever possible.

Expression of Penicillium variabile P16 glucose oxidase gene in Pichia pastoris and characterization of the recombinant enzyme

Enzyme and Microbial Technology, 2006

Glucose oxidase (GOX) is a glycoprotein that finds wide application in food industry and clinical analysis. The gene encoding the GOX from Penicillium variabile P16 was expressed in Pichia pastoris X 33 using the methanol inducible AOX1 promoter. Among 11 transformants resistant toward high zeocin concentrations, six Mut + strains were screened in shaken flasks and the strain X33 c9, producing 0.33 U ml −1 of heterologous GOX after 11 days of fermentation, was selected. Recombinant GOX (ca. 50 U ml −1 ) was produced in a 3-l fermenter under not optimized conditions, recovered and purified in order to characterize and to compare it with the native one. The GOX from P. pastoris had a molecular weight of 82 kDa. Comparison of carbohydrate moieties showed a slight over-glycosylation of the GOX from Pichia over the native enzyme (17 and 14%, respectively). pH behavior of the recombinant enzyme, in terms of both activity and stability, was similar to that of the native one; on the other hand, a certain difference was observed in optimal temperature for activity and in thermal stability. P. pastoris appears to be a good expression system for GOX production.