Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus.

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.     

Stability of minute virus of mice against temperature and sodium hydroxide.

Treatment with steam and/or dilute NaOH are commonly used techniques to disinfect manufacturing vessels and tools in the pharmaceutical industry. The aim of this procedure is sanitisation and inactivation of microbiological and viral contaminants. Here we describe the inactivation of the mouse parvovirus Minute Virus of Mice (MVM) under these conditions. Parvoviruses are known to be resistant to physico-chemical treatment and one representative of this family, the human parvovirus B19, is a potential contaminant of blood plasma. We show inactivation kinetics for MVM treated with wet-heat (70, 80, 90 degrees C) and with 0.01-1 M NaOH solutions (pH >/=11.9). Robust inactivation was only achieved at 90 degrees C for at least 10 min and in NaOH solutions of pH >/=12.8 (0.1 M NaOH). It was observed, that aggregation of viruses might protect viral particles from inactivation by NaOH. Therefore, appropriate sample preparation of spiking material is important for accurate simulation of the naturally occurring situation. The observed stability at pH 11.8 exceeds the previously reported upper limit of pH 9. Inactivation was due to disintegration of the viral capsid as assessed by accessibility of viral DNA for endonucleases.     

Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution

With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.     

Identification of the major positional isomer of pegylated interferon alpha-2b

Interferons display a wide range of antiviral, antiproliferative, and immunomodulatory activities on a variety of cell types and have been used to treat many diseases including hairy-cell leukemia and hepatitis B and C and have also been applied to other therapeutic areas. To improve the pharmacological properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with average molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated proteins with differing sites of PEG attachment. To identify the major positional isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were conducted on a subtilisin-digested N-acetylated peptide of the major positional isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-34, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a novel interferon-polymer attachment site as a histidine-linked interferon conjugate. We show that the major component of PEG-IFN is pegylated in the imidazole side chain of histidine-34. Chemical shift data suggest that pegylation occurs mainly at the N(delta)(1) position in the imidazole side chain of this residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the current experimental conditions at pH 6.5 with an electrophilic derivative of PEG, succinimidyl carbonate PEG. The reversibility of the histidine modification was examined. The PEG-imidazole adduct in the intact protein, PEG-IFN(His-34), is labile but much more stable than in the peptide, PEG-IFN(His-34)dig. Apparently, the tertiary structure of the intact protein protects the His(34)-imidazole ring from depegylation.     

TAM selection of Drosophila somatic cell hybrids.

The selection of MDR3, an adenine-salvage-deficient variant of the Kc line, is described. It is resistant to methylpurine and to diaminopurine and is TAM (thymidine, adenine, methotrexate) sensitive. Two wild-type (TAM-resistant) cell lines, Schneider's line 3 (S3) and Dübendorfer's line 1 (D1), due to their different nutritional requirements, are unable to proliferate in medium ZH1% used for line MDR3. This allowed the selection of hybrids between MDR3 and either D1 or S3 in TAM cloning medium after treatment with polyethyleneglycol. Hybrids were identified by the isoenzyme pattern of NADP-dependent isocitrate dehydrogenase.