A. P. G. M. Van Loon

The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila.

Phytases catalyse the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. In this study genes encoding novel phytases from two different filamentous fungi, Aspergillus terreus strain 9A-1 and Myceliophthora thermophila were isolated. The encoded PhyA phytase proteins show 60% (A. terreus) and 48% (M. thermophila) identity, respectively, to the PhyA of Aspergillus niger and have 21-29% identity compared to other histidine acid phosphatases. All three PhyA proteins, in contrast to the A. niger pH 2.5-optimum acid phosphatase, prefer phytic acid as substrate and show enzyme activity at a broad range of acidic pH values. Based on their enzyme characteristics and protein sequence homology, the phytases form a novel subclass of the histidine acid phosphatase family.

An expression system matures : A highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha

An efficient process was developed for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups, previously reported as repressive substrates, were used as main carbon sources during fermentation. Glucose was even the most productive substrate for high-level production of phytases. Compared with the process using glycerol, the standard carbon source used for this process until now, the use of glucose led to a reduction of more than 80% in the raw materials costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g/L) were obtained in the medium, with phytase representing over 97% of the total accumulated protein. These levels greatly exceed those reported so far for any yeast-based expression system. Very efficient downstream processing procedures were developed with product recovery yields over 90%. Both the fermentation and downstream processing were successfully tested in pilot scale up to 2000 L. As a result, H. polymorpha can be used as a highly competitive system for low-cost phytase production.

Maintenance of NF-kappa B activity is dependent on protein synthesis and the continuous presence of external stimuli.

The activation of NF-kappa B-like activities (called NF-kappa B) by tumor necrosis factor alpha (TNF alpha) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) were compared. High levels of NF-kappa B activity were found 2 to 4 min after TNF alpha addition to human HL60 cells and lasted for at least 3 h, although the half-life of active NF-kappa B was less than 30 min. Inactive NF-kappa B, however, was relatively stable. NF-kappa B activation by TNF alpha was initially cycloheximide insensitive, but maintenance of NF-kappa B activity required ongoing protein synthesis and continuous stimulation by TNF alpha. Thus, the cells did not remain in an activated state without stimulation. In HL60 cells, NF-kappa B induction by PMA required 30 to 45 min and was completely dependent on de novo protein synthesis, while PMA (and interleukin-1) induced NF-kappa B activity rapidly in mouse 70Z/3 cells via a protein synthesis-independent mechanism. The NF-kappa B-like activities obtained under each condition behaved identically in methylation interference and native proteolytic fingerprinting assays. The NF-kappa B-like factors induced are thus all very similar or identical. We suggest that cell-specific differences in the protein kinase C-dependent activation of NF-kappa B may exist and that TNF alpha and PMA may induce expression of the gene(s) encoding NF-kappa B.

GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production

One of the proteins encoded by the riboflavin operon of Bacillus subtilis, RibA, was identified as the rate limiting enzyme in an industrial riboflavin producing strain. An additional single copy of the ribA gene was introduced into the sacB locus of the riboflavin production strain and was expressed constitutively from the medium strength vegI promoter. This led to improved riboflavin titers and yields of riboflavin on glucose of up to 25%. Both enzymatic activities of RibA, the 3,4-dihydroxy-2-butanone 4-phosphate synthase activity located in the N-terminal half of the protein and the GTP cyclohydrolase II activity of the C-terminal domain, are necessary for the improved riboflavin productivity.

Molecular cloning and characterisation of the ribC gene from Bacillus subtilis : A point mutation in ribC results in riboflavin overproduction

Abstract A mutation leading to roseoflavin resistance and deregulated riboflavin biosynthesis was mapped in the genome of the riboflavin-overproducing Bacillus subtilis strains RB52 and RB50 at map position 147°. The chromosomal location indicates that the deregulating mutation in RB52 and RB50 is an allele of the previously identified ribC mutation. We cloned the ribC gene and found that it encodes a putative 36-kDa protein. Surprisingly, RibC has significant sequence similarity to flavin kinases and FAD synthases from various other bacterial species. By comparing the deduced amino acid sequence of RibC from the wild-type parent strain of RB50 with the RibC sequence from the riboflavin-overexpressing RB50 mutant we identified a point mutation that resulted in a Gly to Ser exchange in the C-terminal region of the product

High-affinity receptor for interferon-gamma (IFN-gamma), a ubiquitous protein occurring in different molecular forms on human cells: blood monocytes and eleven different cell lines have the same IFN-gamma receptor protein.

High‐affinity receptors for human IFN‐γ were analyzed using 13 different cells, including blood monocytes. Scatchard analysis showed one high‐affinity binding site for each cell. One cross‐linked complex between IFN‐γ and the receptor was detected, although their apparent molecular masses were variable in different cells, as also confirmed in immunoblots of membrane extracts. Variations in molecular masses were abolished if N‐linked glycosylation was absent. Stable tryptic fragments contained the intact binding site for IFN‐γ and antibody epitopes characteristic of the extracellular domain of the IFN‐γ receptor of Raji cells and were of different sizes only if glycosylated. In addition, Northern analysis showed the same mRNA encoding the high‐affinity IFN‐γ receptor in each cell analyzed. Thus, all cells including blood monocytes express the same high‐affinity IFN‐γ receptor protein. N‐linked sugars may give structural stability to the IFN‐γ receptor and are unlikely to be directly involved in IFN‐γ binding.

Commercial riboflavin production by recombinant Bacillus subtilis: down-stream processing and comparison of the composition of riboflavin produced by fermentation or chemical synthesis

A novel process for riboflavin production using a recombinant Bacillus subtilis strain has been developed. Here we describe a down-stream processing procedure to obtain riboflavin qualities having a minimal content of 96% (‘feed-grade’) and 98% (‘food/pharma-grade’) riboflavin, respectively. Compared to riboflavin produced by chemical synthesis, products with improved chemical purity were obtained. All compounds representing more than 0.1% of the final products were identified. Feed-grade riboflavin material ex fermentation contained small amounts of amino acids and amino sugars and the biosynthetic riboflavin precursor dimethyl-ribityl-lumazine. All other side products found were derived from riboflavin, resulted from the purification procedure and were also found in riboflavin obtained by chemical synthesis. The Bacillus-produced riboflavin does not contain DNA. The data presented here were used to obtain product approval for the commercial application in the USA, Japan and the UK.

Cyclic AMP-independent activation of transcription factor NF-kappa B in HL60 cells by tumor necrosis factors alpha and beta.

No correlation exists in HL60 cells between NF-kappa B activation by tumor necrosis factor (TNF alpha) and TNF beta and intracellular levels of cyclic AMP. Cyclic AMP levels did not increase upon treatment of cells with each of these cytokines, although NF-kappa B was activated. Forskolin or 1-isobutyl-3-methylxanthine drastically increased intracellular levels of cyclic AMP, but neither activated NF-kappa B nor influenced TNF-induced NF-kappa B activation.

Characteristics of fungal phytases from Aspergillus fumigatus and Sartorya fumigata

Aspergillus fumigatus phytase has previously been identified as a phytase with a series of favourable properties that may be relevant in animal and human nutrition, both for maximising phytic acid degradation and for increasing mineral and amino acid availability. To study the natural variability in amino acid sequence and its impact on the catalytic properties of the enzyme, we cloned and overexpressed the phytase genes and proteins from six new purported A. fumigatus isolates. Five of these phytases displayed ≤2 amino acid substitutions and had virtually identical stability and catalytic properties when compared with the previously described A. fumigatus ATCC 13073 phytase. In contrast, the phytase from isolate ATCC 32239 (Sartorya fumigata, the anamorph of which was identified as A. fumigatus) was more divergent (only 86% amino acid sequence identity), had a higher specific activity with phytic acid, and displayed distinct differences in substrate specificity and pH-activity profile. Finally, comparative experiments confirmed the favourable stability and catalytic properties of A. fumigatus phytase.

Construction of yeast artificial chromosome (YAC) clone banks covering three genome equivalents and isolation of YACs containing the human gene encoding tumor necrosis factor receptor β

Yeast artificial chromosome (YAC) banks covering in total about three haploid genome equivalents were constructed using a human Epstein-Barr-virus-transformed B lymphocytic cell line. Two clone banks were made: 20,000 clones with average inserts of 350 kb in the pYAC4 vector and 9850 clones with average inserts of 180 kb using vectors pJS89 and pJS91. Direct comparison of pYAC4 with pJS89 and pJS91 showed pYAC4 to be the most suitable cloning vector. Two partial banks with average insert sizes of 220 kb for human endothelial cell DNA and epithelial HEp2 cell DNA were also constructed, each covering 10% of the haploid genome. A rapid, three-step PCR screening procedure for isolation of individual YAC clones was developed and used to identify two clones encoding TNF-R beta. These clones cover about 200 kb and have 170 kb in common. TNF-R beta is 9.3 kb long and contains two introns within the protein-coding sequence.