Genome mining and motif modifications of glycoside hydrolase family 1 members encoded by Geobacillus kaustophilus HTA426 provide thermostable 6-phospho-ß-glycosidase and ß-fucosidase.

Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-ß-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a ß-glucosidase resulted in the unexpected generation of a thermostable, highly specific ß-fucosidase, concomitant with large increases in ß-glucosidase, ß-cellobiosidase, α-arabinofuranosidase, ß-mannosidase, ß-glucuronidase, ß-xylopyranosidase, and ß-fucosidase activities and a dramatic decline in 6-phospho-ß-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-ß-glycosidase and to generate a thermostable ß-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.

Potentiometric determination of α-L-fucosidase enzyme by using 2-chloro-4-nitrophenol-rhodamine B ion pair chemical recognition in PVC membrane sensor.

The activity of the α-L-fucosidase (AFU) enzyme represents an excellent test for diagnosis of hepatocellular carcinoma (HCC) and fucosidosis recognized in inborn disorder of metabolism and increases the sensitivity of detection to 95.5% in patients with HCC. Therefore, the determination of the activity of AFU enzyme is very important and can be used as a screening tool for the early diagnosis of tumors for HCC patients. A simple, accurate, and sensitive potentiometric method was developed for measuring the activity of AFU. The method was based upon measuring the concentration of 2-chloro-4-nitrophenol (2-chloro-4-NP) using a 2-chloro-4-NP-rhodamine B ion pair in a PVC membrane sensor. The electrode shows a linear, reproducible, and stable potentiometric response with an anionic Nernstian slope of -51.13 ± 0.6 mV/decade over a wide range of concentrations 10(-5)-10(-2) M and a detection limit of 1.0 × 10(-6) M of 2-chloro-4-NP. The membrane exhibits a fast response time of 30 s, over a pH range of 4.0-6.5. The selectivity coefficients indicate excellent selectivity for 2-chloro-4-NP over a number of interfering species, e.g., chloride, nitrate, sulfate, chromate urea, albumin, glucose, uric acid, and total protein. The prepared sensor has been used successfully for the determination of 2-chloro-4-NP produced from the hydrolysis of 2-chloro-4-NP-α-L-fucopyranoside substrate. It was also applied for the determination α-L-fucosidase enzyme of 33 serum samples of healthy subjects and patients. The average recoveries ± RSD for the healthy subjects, cirrhosis of chronic hepatitis C and B, and HCC serum samples were 102.6 ± 1.01%, 101.5 ± 0.95%, and 100.1 ± 1.1%, respectively. The results obtained are in good agreement with those obtained by standard methods.

Exoglycosidase purity and linkage specificity: assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection.

Simplified HPLC protocols to determine the activity and linkage specificity and to detect the most commonly-encountered contaminants in available exoglycosidase preparations (Jacob and Scudder, Methods Enzymol., 230, 280-300, 1994) were developed. Monosaccharides and oligosaccharides were analyzed in a single chromatographic step using high-pH anion-exchange chromatography with pulsed amperometric detection. All analyses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the chromatograph. The sialidase from Newcastle disease virus was found to release both alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac from a triantennary, lactosamine-type oligosaccharide. The activity of alpha-galactosidase from green coffee beans was assayed using Gal alpha(1-->3)[Fuc-alpha(1ar2)]Gal by detection of Gal and Fuc alpha(1-->3)Gal. The linkage specificities of beta-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Gal beta(1-->3 or 4)GlcNAc beta(1-->3)beta(1-->4)Glc as substrates. Contaminating beta-N-acetylhexosaminidase activity in the beta-galactosidase preparation was assayed using an agalactobiantennary oligosaccharide. The alpha(1-->3 or 4) linkage specificity of fucosidase III from almond meal was confirmed (Scudder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked alpha(1-->6). An alpha-fucosidase from chicken liver was found to cleave alpha(1-->2,3 or 6)-linked Fuc residues from oligosaccharides. The activity of jack bean (Canavalia ensiformis) alpha-mannosidase was assayed with a relatively resistant substrate, Man alpha(1-->3)- Man beta(1-->4)GlcNAc. A GlcNAc beta(1-->4)-terminated triantennary oligosaccharide was used to assay for contaminating beta-N-acetylhexosaminidase activity in alpha-mannosidase preparations and to determine the linkage and branch specificity of beta-N-acetylhexosaminidase at different enzyme concentrations.