Two distinct extracellular bifunctional proteins with beta-L-arabinopyranosidase/alpha-D-galactopyranosidase activities were purified from the culture filtrate of Fusarium oxysporum 12S. The molecular masses of the enzymes were estimated to be 55 (Fo/AP1) and 73 kDa (Fo/AP2) by SDS-PAGE. They hydrolyzed both p-nitrophenyl beta-L-arabinopyranoside and p-nitrophenyl alpha-D-galactopyranoside with different specificities. Fo/AP1 also showed low activity towards alpha-D-galactopyranosyl oligosaccharides such as raffinose. Interestingly, both enzymes hydrolyzed larch wood arabinogalactan (releasing arabinose) but not carob galactomannan, which has alpha-D-galactopyranosyl side chains. When larch wood arabinogalactan was incubated with excess Fo/AP1 or Fo/AP2, both enzymes released approximately 10% of the total arabinose in the substrate. cDNAs encoding Fo/AP1 and Fo/AP2 (Foap1 and Foap2) were isolated by in vitro cloning. The coding sequences of Foap1 and Foap2 genes were 1,647 and 1,620 bp in length and encode polypeptides of 549 and 540 amino acids, respectively. The N-terminal halves of both proteins had high similarity to putative conserved domains of the melibiase superfamily (Pfam account number 02065). The deduced amino acid sequences of the two enzymes indicate that they belong to glycosyl hydrolase family 27. Moreover, the C-terminal regions of both proteins contain a putative family 35 carbohydrate-binding module.
A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a -1,6-galactanase that preferentially debranches -1,6-galactobiose from the substrate.Type II arabinogalactans in the Western larch consist of a -1,3-galactan backbone with many side chains at position 6 (15). About half of the main chain galactopyranose residues are replaced by -1,6-galactopyranobiose, and about a quarter are replaced by single galactopyranose units. The rest of the side chains contain three or more glycosidic residues. Arabinofuranose and arabinopyranose residues exist at the nonreducing ends of both the -1,3-galactan main chains and the side chains.Fungi and plants make several type II arabinogalactan-degrading enzymes. These include two endo--1,6-galactanases, one acting on grape arabinogalactans from Aspergillus niger (2) and one acting on radish arabinogalactans from Trichoderma viride (13), a -1,6-galactanase that acts on acid-treated larch wood arabinogalactan (LWAG) from A. niger (10), -galactosidases from radish seeds (19) and spinach (5) that act on -1,3 and -1,6 galactosyl sequences of arabinogalactans but not pectic -1,4 galactan, and two exo--1,3-galactanases (EC 3.2.1.145) from Irpex lacteus (22) and A. niger (14) that cleave the backbone of type II arabinogalactans in an exo-like manner. The latter-named enzymes are able to bypass the branching points of galactan backbones and to release the intact side chains of type II arabinogalactans and thus are very useful for the structural analysis of polysaccharides.To analyze the fine structures of polysaccharides enzymatically, recombinant enzymes are preferred, as they exclude the confounding influence of contaminating carbohydrases. However, only four genes encoding type II arabinogalactan-degrading enzymes have previously been characterized: two exo--1,3-galactanases from Phanerochaete chrysosporium (7) and Clostridium thermocellum (6), an endo--1,6-galactanase from T. viride (9), and a -galactosidase from radishes (8). In this paper, we describe the isolation and characterization of a -1,6-galactanase, termed FoGal1, that is produced by Fusarium oxysporum 12S. In addition, we report the nucleotide sequence of the gene that encodes FoGal1 (Fogal1) and describe the activity of the recombinant enzyme expressed in Escherichia coli.The F. oxysporum 12S strain was isolated from a rotten sugar beet as part of a large-scale screening program (unpublished data). The microorganism is deposited in the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (accession no. FERM P-20380; http://unit.aist.go.jp/ipod/cie/index.html). To purify the FoGal1, the F. oxysporum 12S strain was cultivated in 1 liter of LWAG medium consisting of 0.2% NH 4 NO 3 , 0.1% K 2 HPO 4 , 0.05% MgSO 4 · 7H...
To achieve quality control of therapeutic proteins, it is important to compare their biological potency with their clinical effects.1) Therefore, an animal-based assay is preferred for measuring their bioactivity. However, the use of laboratory animals poses ethical concerns, is time consuming and requires skilled staff and sophisticated experimental facilities. Furthermore, in vivo bioassays have low precision and accuracy, and hence, there is a need for an alternative assay method to estimate in vivo bioactivity.To date, attempts have been made to replace traditional bioassays with physicochemical techniques for potency determination of insulin and growth hormone because it is possible to design and validate manufacturing processes in the production of properly folded materials.2,3) Potency of these pharmaceutical preparations can be calculated using the content assay with HPLC, which quantifies the peak area of a product against that of a reference standard of known potency. The results of the HPLC assay have been shown to correlate well with those of the bioassay.2,3) In addition, the HPLC assay is rapid, precise and less labour intensive than the bioassay. However, for some glycoproteins, alternative assay methods using a quantitative charge-based separation technique, such as isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), have been shown to be feasible for estimating in vivo bioactivity 4,5) because negatively charged sialic acid residues are critical for in vivo bioactivity. For example, Mulders et al. 4) reported that quantitative assessment of the isoform distribution by IEF may provide a method for predicting in vivo biological potency of preparations containing recombinant human follicle-stimulating hormone (rhFSH). Close correlation between results of experimental in vivo bioactivities and IEF-predicted in vivo bioactivities suggest that the only factor affecting prediction of biological potency is the result of quantitative isoform distribution in rhFSH.Erythropoietin (EPO) is a glycoprotein hormone that regulates the red blood cell level by stimulating maturation of erythroid precursor cells. 6) Recombinant human EPO (rhEPO) produced in Chinese hamster ovary (CHO) cells is extensively used for serve anaemia therapy.7) EPO comprises a 165-amino acid protein with 40% of its molecular weight accounted for by carbohydrates.8,9) EPO contains three Nglycans located on Asn residues at positions 24, 38, and 83 and one mucin-type O-glycan located on the Ser-126 residue. The N-linked carbohydrates comprise bi-, tri-and tetraantennary oligosaccharides, which typically terminate with a negatively charged sialic acid residue. 10,11) Because of the variable number of sialic acid residues, the product of EPO usually exists as a mixture of isoforms with different isoelectric points. Several studies have reported that sialic acid residues are critical for in vivo bioactivity of EPO.12,13) Isoforms having a higher sialic acid content are seen to display higher in vivo bioactivity, longer seru...
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