Extracellular Glucose Oxidase of Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1 using ultrafiltration membranes was developed. Two samples of the enzyme with a specific activity of 914–956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16°C was 2.74 × 10^–6 s^–1. This constant decreased significantly as the pH of the medium increased (4.0–10.0). The temperature optimum for glucose oxidase–catalyzed -D-glucose oxidation was in the range 30–65°C. At temperatures below 30°C, the activation energy for -D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase–catalyzed -D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Selection of Penicillium funiculosum Strains with High Glucose Oxidase Activity

Metabolic inhibitors, riboflavin, and end products of glucose oxidation were shown to be effective for the selection ofPenicillium funiculosum mutant strains with a high glucose oxidase activity. The incidence of positive mutations was highest in clones resistant to sodium azide, riboflavin, and -D-glucono--lactone. Enzyme activity in Penicillium funiculosum mutants was studied in submerged culture. The level of glucose oxidase synthesis in seven cultures was 24–56% higher than that in the parent strain of Penicillium funiculosum NMM95.132.     

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A ¹¹⁶KSKRKKKNKK¹²⁵ and B ¹⁷⁵KKATKKESKKQTK¹⁸⁷ reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein–protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.     

RNA binding is more critical to the suppression of silencing function of Cucumber mosaic virus 2b protein than nuclear localization

Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios ~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.     

The cellular immune response in different forms of diphtheria in adults

In 109 adult diphtheria patients with different forms of the disease (toxic, subtoxic, localized) and carriers, the latter including 6 persons having had the localized form of diphtheria, 12 different subpopulations of T and B lymphocytes and neutrophils were studied by the methods of rosette formation with sheep, mouse and bovine red blood cells. The study showed that, starting from week 2 of the disease, an increase in the content of the subpopulations under study was registered in all forms of diphtheria; the disease, as well as in patients with the localized form of the disease accompanied by complications and in long-term carriers. In all forms of diphtheria a decrease in the content of neutrophil rosette-forming cells was shown. The marker tests for prognostication of the complicated course of the disease, viz. the elevated content of M-rosette-forming cell and T gamma-rosette-forming cells during the first week of then disease, were established.     

Immobilization of extracellular glucose oxidase from Penicillium funiculosum 46.1 on gels of aluminum or zinc hydroxides

Different methods of immobilization of extracellular glucose oxidase (GO) from Penicillium funiculosum 46.1 on gels of aluminum or zinc hydroxides have been compared. GO from the culture liquid filtrate (CLF) associated with Zn(OH)₂ but not Al(OH)₃ gels. Preparation of samples of immobilized GO does not require isolation of the enzyme (CLF may be used). GO immobilized on Zn(OH)₂ gels from CLF was 1.6 times more efficient in catalyzing D-glucose oxidation than the enzyme contained in the original culture liquid. Crosslinking of gel-adsorbed CLF proteins affected the properties of GO adversely and to a considerable extent. Various means of polymerization and immobilization of GO isolated from CLF have been studied. Optimum results were obtained when GO polymeric products were pre-synthesized in solution, followed by adsorption to Al(OH)₃, but not Zn(OH)₂ gels. The catalytic efficiency of GO immobilized on a Zn(OH)₂ gel was significantly lower than that of the enzyme associated with Al(OH)₃.     

Immobilizalion of extracellular glucose oxidase from penicillium funiculosum 46.1 on gels of aluminum or zinc hydroxides

Different methods of immobilization of extracellular glucose oxidase (GO) from Penicillium funiculosum 46.1 on gels of aluminum or zinc hydroxides have been compared. GO from the culture liquid filtrate (CLF) associated with Zn(OH)2 but not Al(OH)3 gels. Preparation of samples of immobilized GO does not require isolation of the enzyme (CLF may be used). GO immobilized on Zn(OH)2 gels from CLF was 1.6 times more efficient in catalyzing D-glucose oxidation than the enzyme contained in the original culture liquid. Crosslinking of gel-adsorbed CLF proteins affected the properties of GO adversely and to a considerable extent. Various means of polymerization and immobilization of GO isolated from CLF have been studied. Optimum results were obtained when GO polymeric products were pre-synthesized in solution, followed by adsorption to Al(OH)3 but not Zn(OH)2 gels. The catalytic efficiency of GO immobilized on a Zn(OH)2 gel was significantly lower than that of the enzyme associated with Al(OH)3.     

Cooxidation of phenol and 4-aminoantipyrin, catalyzed by polymers and copolymers of horseradish root peroxidase and Penicillium funiculosum 46.1 glucose oxidase

Polymers and copolymers of horseradish root peroxidase (HRP) and Penicillium funiculosum 46.1 glucose oxidase (GO) have been synthesized and their catalytic properties have been characterized (free and immobilized forms of each enzyme were studied). The cooxidation reaction of phenol and 4-aminoantipyrin (4-AAP), performed in an aqueous medium in the presence of equimolar amounts of GO and HRP, was characterized by effective K(M) and k(cat) of 0.58 mM and 20.9 s(-1) (for phenol), and 14.6 mM and 18.4 s(-1) (glucose), respectively. The catalytic efficiency of polymerization products (PPs) of GO (GO-PPs) depended on the extent of their aggregation. The combinations GO + HRP-PP and HRP + GO-PP, as well as the copolymer HRP*-GO-PP, proved promising as reagents for enzyme-based analytical systems. When adsorbed on aluminum hydroxide gels, GO-PPs exhibited higher catalytic activity than the non-polymeric enzyme. Maximum retention of GO-PP activity on the inorganic carrier was observed in the case of GO-PP copolymers with an activated HRP. Polymerization of HRP in the presence of a zinc hydroxide gel, paralleled by HRP-PP immobilization onto the gel, increased both the activity of the enzyme and its operational stability.