Galactooligosaccharides (GOS) are currently attracting considerable interest as prebiotic substances and can be prepared by transgalactosylation reactions from lactose using β-galactosidase. We applied various combinations of the commercial β-galactosidases, such as Nola Fit 5500, Saphera 2600 L, Maxilact LGI 5000 and Maxilact A4 MG to achieve the highest yield of GOS and reduced lactose content. The combination of the Maxilact LGI 5000 and Nola Fit 5500 resulted in amount of GOS 105 g L-1 with lactose content lower than 5 g L-1, whilst the combination of the Maxilact A4 MG and Maxilact LGI 5000 enzymes led to an increase in GOS to 141,1 g L-1 and decrease of the lactose content to 46,9 g L-1. The combination of enzymes produced a higher yield of GOS, reduced the concentration of lactose, eventually, increases the efficiency of galactooligosaccharides purification that could be potentially used in the further investigations.

• Klíčová slova
• Galactooligosaccharide, HPLC, Lactose, Transgalactosylation, β-Galactosidase,
• MeSH
• beta-galaktosidasa metabolismus   MeSH
• časové faktory MeSH
• galaktosa biosyntéza   MeSH
• oligosacharidy biosyntéza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-galaktosidasa MeSH
• galaktosa MeSH
• oligosacharidy MeSH

Galactooligosaccharides (GOS) are lactose-derived functional ingredients applied in food products and have great potential in health protection. The conversion of lactose to GOS commonly occurs using β-galactosidases of mould, yeast and bacterial origin. The yield and structure of the resulting GOS depend on the enzyme used and the reaction conditions. This work focuses on the structural analysis of the products obtained with four commercial β-galactosidases Maxilact LGI 5000 (ML), Maxilact A4 MG (MA), Saphera 2600 L (SA) and NOLA Fit 5500 (NL) to evaluate their efficiency and specificity. HPLC, ESI-MS and NMR spectroscopy were applied to characterise the GOS preparations. GOS were separated from the reaction mixture using activated charcoal treatment. HPLC analysis confirmed that most of the monosaccharides and a part of the lactose, but also some other disaccharides, probably allolactose and 6-galactobiose, were retained by charcoal. In all the products, ESI-MS analysis detects oligosaccharides up to hexamers. NMR spectra confirmed the presence of GOS of various configurations and polymerisation degrees and evaluated the specificity of used enzymes. MA preferably forms 1,6- and 1,4-glycosidic bonds, and bacterial enzymes NL and SA also form 1,2- and 1,3- glycosidic bonds, while yeast enzyme ML cannot produce new 1,4-glycosidic bonds. The mould enzyme MA showed the highest trans-galactosylation activity, forming longer GOS oligomers than the other enzymes.

• Klíčová slova
• ESI-MS, Galactooligosaccharides, HPLC, NMR, Structure, β-Galactosidases,
• MeSH
• beta-galaktosidasa * metabolismus   chemie   MeSH
• galaktosa chemie   metabolismus   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací * metody   MeSH
• laktosa metabolismus   chemie   MeSH
• magnetická rezonanční spektroskopie * metody   MeSH
• oligosacharidy * chemie   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• beta-galaktosidasa * MeSH
• galaktosa MeSH
• laktosa MeSH
• oligosacharidy * MeSH

Polyacrylamide gel electrophoresis in an acidic buffer system was used to study the electrophoretic behaviour of two forms of alpha-D-galactosidase from seeds of soy bean (Glycine soja) and mung bean (Vigna radiata). The interaction of the enzymes with saccharides was monitored by affinity electrophoresis; for the preparation of affinity gels, water-soluble O-glycosyl polyacrylamide copolymers and polysaccharides were used. alpha-D-Galactosidases from both sources interact with immobilized alpha-D-galactosyl residues. On the basis of the results of affinity electrophoresis performed in the presence of various free sugars, dissociation constants for the complexes between alpha-D-galactosidase and free sugars were calculated.

• MeSH
• alfa-galaktosidasa analýza   MeSH
• chromatografie afinitní MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• galaktosidasy analýza   MeSH
• Glycine max enzymologie   MeSH
• koncentrace vodíkových iontů MeSH
• lektiny analýza   MeSH
• molekulová hmotnost MeSH
• rostlinné lektiny MeSH
• semena rostlinná enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alfa-galaktosidasa MeSH
• galaktosidasy MeSH
• lektiny MeSH
• rostlinné lektiny MeSH

• MeSH
• antisérum MeSH
• galaktosidasy analýza   MeSH
• imunoelektroforéza MeSH
• králíci MeSH
• krysa rodu Rattus MeSH
• střeva analýza   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisérum MeSH
• galaktosidasy MeSH

A simple, sensitive and reproducible method of detection of extracellular beta-galactosidase was worked out. beta-Galactosidase secreted by the callus culture, or the roots of sprouting plants, hydrolyzes the substrate (6-bromo-2-naphthyl-beta-D-galactopyranoside) to produce beta-D-galactose and 6-bromo-2-naphthol, which after simultaneous azocoupling with hexazotized p-rosaniline, or basic fuchsine, produce a reddish brown compound, nearly insoluble in water (a diazonium salt). The colouring under the callus culture and around it, as well as the colouring of the roots of the sprouting plants, is a manifestation of the activity of extracellular beta-galactosidase by the objects under study. The intensity of the colouring is a measure of their enzymatic activity. The share of intracellularly localized enzyme represents the majority and the share of extracellular beta-galactosidase the minority.

• MeSH
• beta-galaktosidasa biosyntéza   MeSH
• kořeny rostlin MeSH
• rostliny enzymologie   MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• Názvy látek
• beta-galaktosidasa MeSH

The activity of beta-D-galactosidase was studied in 13 strains of lactobacilli (groups Streptobacterium, Thermobacterium and Betabacterium). Using 2-nitrophenyl galactopyranoside as substrate, the enzyme activity varied with the strain. The values found in the Thermobacterium group were superior to those in the Streptobacterium group. The optimum pH for the species belonging to the Thermobacterium group was uniform, in contrast to the pH for those from the Streptobacterium which varied according to the species. The optimum temperature was quite uniform within each group and higher in the Streptobacterium. Lactose acted as a competitive inhibitor. MgCl2 protected the enzyme from thermal denaturation. The calcium ions inhibited the activity in all cases. The behaviour of the protectors of the SH groups varied according to the strain. 6-Phospho-beta-D-galactosidase activity was also determined, levels lower than beta-D-galactosidase were found, except in Lactobacillus plantarum ATCC 8014 and 14917.

• MeSH
• anionty MeSH
• beta-galaktosidasa metabolismus   MeSH
• druhová specificita MeSH
• galaktosidasy metabolismus   MeSH
• kationty MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• Lactobacillus enzymologie   MeSH
• teplota MeSH
• vápník farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• anionty MeSH
• beta-galaktosidasa MeSH
• galaktosidasy MeSH
• kationty MeSH
• vápník MeSH

UNLABELLED: The ability to predict the transglycosylation activity of glycosidases by in silico analysis was investigated. The transglycosylation abilities of 7 different β-d-galactosidases from GH family 2 were tested experimentally using 7 different acceptors and p-nitrophenyl-β-d-galactopyranoside as a donor of galactosyl moiety. Similar transglycosylation abilities were confirmed for all enzymes originating from bacteria belonging to Enterobacteriaceae, which were able to use all tested acceptor molecules. Higher acceptor selectivity was observed for all others used bacterial strains. Structure models of all enzymes were constructed using homology modeling. Ligand-docking method was used for enzymes-transglycosylation products models construction and evaluation. Results obtained by in silico analysis were compared with results arisen out of experimental testing. The experiments confirmed that significant differences in transglycosylation abilities are caused by small differences in active sites composition of analyzed enzymes. According to obtained result, it is possible to conclude that homology modeling may serve as a quick starting point for detection or exclusion of enzymes with defined transglycosylation abilities, which can be used for subsequent synthesis of e.g., pharmaceutically interesting glycosides. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02715-w.

• Klíčová slova
• Carbohydrate family, Catalysis, Homology modeling, Hydrolases, Ligand-docking,
• Publikační typ
• časopisecké články MeSH

The microflora of the chernozem soil mineralized 62% of the lactose retained on a column consisting of three 10-g layers of the soil at a daily flow of 48 mg of the sugar. Only 45% of the sugar was mineralized when the daily flow was 136 mg. The highest value of the beta-galactosidase activity in the system of heterocontinuous cultivation was two-fold with respect to batch cultivation. At a higher sugar concentration the enzyme activity in steady state was the same in the whole soil column. This value was reached first in the middle layer of the soil. At a lower concentration of the flowing sugar in steady state the highest enzyme activity was detected in the middle layer of the soil. In the upper layer the enzyme activity was one half, in the lower layer it began to decrease after reaching its maximum after 4 d of the incubation.

• MeSH
• Bacteria metabolismus   MeSH
• beta-galaktosidasa metabolismus   MeSH
• galaktosidasy metabolismus   MeSH
• houby metabolismus   MeSH
• laktosa metabolismus   MeSH
• půdní mikrobiologie * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-galaktosidasa MeSH
• galaktosidasy MeSH
• laktosa MeSH

An extracellular alpha-d-galactosidase from Talaromyces flavus CCF 2686 with extremely broad and unusual acceptor specificity is produced exclusively in the presence of the specific inducer--6-deoxy-D-glucose (quinovose). The procedure for the preparation of this very expensive substance has been modified and optimized. Surprisingly, any of other common alpha-D-galactosidase inducers or substrates, e.g., D-galactose, melibiose and raffinose, did not stimulate its production. The crude alpha-D-galactosidase preparation was purified by anion-exchange chromatography and three isoenzymes with different substrate specificities were identified. The main isoenzyme (alphaGal1) was further purified by cation-exchange chromatography and fully characterized. When compared with other alpha-galactosidases and also with other isoenzymes produced by T. flavus, it showed a markedly different regioselectivity and also negligible hydrolytic activity towards melibiose. Moreover, it was active on polymeric substrates (locust bean gum, guar gum) and significantly inhibited by alpha-D-galactopyranosyl azide, D-galactose, D-xylose, melibiose, methyl alpha- and beta-D-galactopyranoside and lactose.

• MeSH
• alfa-galaktosidasa biosyntéza   izolace a purifikace   metabolismus   MeSH
• deoxyglukosa analogy a deriváty   metabolismus   MeSH
• enzymová indukce MeSH
• kinetika MeSH
• substrátová specifita MeSH
• Talaromyces enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 6-deoxyglucose MeSH Prohlížeč
• alfa-galaktosidasa MeSH
• deoxyglukosa MeSH

Optimum conditions for beta-galactosidase production by K. fragilis were studied. Enzyme production has a maximum after 8-12 h of incubation. Composition of whey (from different sources) did not affect enzyme production. Different heart treatments also had no effect. Whey reconstituted to 8-12% total solids and adjusted to pH 4.0 afforded maximum enzyme production. Whereas inorganic nitrogen sources (specially ammonium salts) only slightly stimulated enzyme production, organic nitrogen sources (specially partially digested proteins) provided a nearly four-fold increase in enzyme production. Yeast extract and beef extract and industrial by-products like corn-steep liquor significantly stimulated enzyme production. Manganese and magnesium salts had a very little stimulation effect.

• MeSH
• Ascomycota metabolismus   MeSH
• beta-galaktosidasa biosyntéza   MeSH
• galaktosidasy biosyntéza   MeSH
• kultivační média * MeSH
• mléko * MeSH
• Saccharomycetales metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-galaktosidasa MeSH
• galaktosidasy MeSH
• kultivační média * MeSH