• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.40-44
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• 1998

A Penicillium strain which produces $eta$-galactosidase with high transgalactosylation activity, was isolated from soil and registered as Penicillium sp, KFCC 10888. When $eta$-galactosidase from Penicillium sp. KFCC 10855 reacted with 40% lactose, transgalactosylation ratio reached up to 70% at the 73% conversion of initial lactose. The biosynthesis of the enzyme in Penicillium sp. KFCC 10888 was not induced by lactose. The soybean meal was an effective component of the culture medium. The optimum pH and temperature for transgalactosylation were 4.0 and 55$^{\circ}C$, respectively. The production of galactooligosaccharides was in proportion to the initial lactose concentration. When the enzyme reacted with 40% lactose (pH 4.0) at 55$^{\circ}C$, the concentration of galactooligosaccharides increased up to 40% of total solid concentration.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.318-324
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• 1998

A ${\beta}$-galactosidase with high transgalactosylic activity was purified from a Bacillus species, registered as KFCC10855. The enzyme preparation showed a single protein band corresponding to a molecular mass of 150 kDa on SDS-PAGE and gave a single peak with the estimated molecular mass of 250 kDa on Sephacryl S-300 gel filtration, suggesting that the enzyme is a homodimeric protein. The amino acid and sugar analyses revealed that the enzyme is a glycoprotein, containing 19.2 weight percent of sugar moieties, and is much more abundant in hydrophilic amino acid residues than in hydrophobic residues, the mole ratio being about 2:1. The pI and optimum pH were determined to be 5.0 and 6.0, respectively. Having a temperature optimum at $70^{\circ}C$ for the hydrolysis of lactose, the enzyme showed good thermal stability. The activity of the enzyme preparation was markedly increased by the presence of exogenous Mg (II) and was decreased by the addition of EDTA. Among the metal ions examined, the most severely inhibitory effect was seen with Ag (I) and Hg (II). Further, results of protein modification by various chemical reagents implied that 1 cysteine, 1 histidine, and 2 methionine residues occur in certain critical sites of the enzyme, most likely including the active site. Enzyme kinetic parameters, measured for both hydrolysis and transgalactosylation of lactose, indicated that the enzyme has an excellent catalytic efficiency for formation of the transgalactosylic products in reaction mixtures containing high concentrations of the substrate.

• Applied Biological Chemistry
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• v.38 no.6
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• pp.502-506
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• 1995

A Bacillus strain which produces ${\beta}-galactosidase$ with high transgalactosylation activity, was isolated from soil and tentatively designated as Bacillus sp. A1. When ${\beta}-galactosidase$ from Bacillus sp. A1 reacted with 40% (w/w) lactose, transgalactosylation ratio reached up to 90% at the 70% conversion of the initial lactose. The biosynthesis of the enzyme in Bacillus sp. A1 required lactose as an inducer and was repressed by glucose. Observing that the addition of amino acids to culture medium resulted in enhancing, to a significant extent, both the growth and the enzyme production of the strain, yeast extract and commercially available hydrolysates of protein were examined for the suitability as amino acid source. As it turned out, SMP, an enzymatic hydrolysis product of soybean protein from Fuji Oil Co.(Japan), was the most suitable for optimization of the culture medium. When Bacillus sp. A1 was cultured in the presence of 0.5% SMP and 2% lactose, the enzyme activity increased up to $1.8\;U/m{\ell}-broth$.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.43 no.4
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• pp.373-379
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• 2017

We carried out the enzymatic synthesis of 1, 2-hexanediol galactoside (HD-gal) by transgalactosylation reaction using recombinant Escherichia coli ${\beta}-galactosidase$ (${\beta}-gal$). The amounts of ${\beta}-gal$ and 1, 2-hexanediol (HD), pH, and temperature, respectively, were first optimized (${\beta}-Gal$, 4.8 U/mL; HD, 75 mM; pH, 7.0; temperature, $37^{\circ}C$). Under these optimal conditions, about 96% HD was converted to HD-gal. When we investigated the water holding capacities (WHCs) of HD and HD-gal using pig epidermis in the concentrations of 84.4, 126.6, 168.8, 211.0 mM, WHC of HD-gal was superior to HD. In particular, at 168.8 mM HD and HD-gal, WHC of HD-gal showed about 20% greater than that of HD. However, it was observed that MIC values against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus of HD-gal were about three to ten times greater than those of HD, although MIC value of HD-gal against Enterococcus faecalis was almost the same as that of HD. Finally, it was concluded that the covalent bonding of a galactose molecule to HD (transgalactosylation) resulted in an increase in WHC of HD-gal and a decrease in anti-bacterial activity.

• Food Science of Animal Resources
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• v.33 no.5
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• pp.565-571
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• 2013

An enzyme ${\beta}$-galactosidase or ${\beta}$-galactohydrolase [EC3.2.1.23], commonly called lactase, mediates galacto-oligosaccharide (GOS) synthesis under conditions of high substrate concentrations. Also, lactase hydrolyzes ${\beta}$($1{\rightarrow}4$) lactose into glucose and galactose, the latter is successively transferred to free lactose to make various oligosaccharides via transgalactosylation. GOS is non-digestible to human digestive enzymes and has been used as a functional prebiotics. Among the 24 lactic acid bacteria (LAB) strains used, Lactobacillus paracasei YSM0308 was selected based on its exhibition of the highest ${\beta}$-galactoside hydrolysis activity, and the crude lactase was prepared for examination of reaction conditions to affect the GOS synthesis. Lactase activity was measured with a spectrophotometer using ONPG (o-nitropheyl ${\beta}$-D-galactopyranoside) method. Lactase activity was not detected in the culture supernatant and was mostly present in the cell pellet after centrifugation. Activity of the crude lactase preparation ranges from102 to 1,053 units/mL, with the highest activity determined for L. paracasei YSM0308. Optimal conditions for GOS synthesis are as follows: concentration of whey powder, pH, temperature, and time were 30%, pH 6.5-7.0, $30^{\circ}C$, and 4 h, respectively. The final GOS concentration was 19.41% (w/v) by the crude YSM0308 lactase, which was obtained from strain YSM0308 grown in the 10% (w/v) reconstituted whey-based medium.

• Applied Biological Chemistry
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• v.38 no.6
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• pp.507-511
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• 1995

In an attempt to improve the productivity of ${\beta}-galactosidase$ from Bacillus sp. A1, which was isolated from soil and has remarkably higher transgalactosylation activity than lactose hydrolysis activity, a chemical mutation procedure using N-methyl-N'-nitro-N-nitrosoguanidine followed by selection was conducted. The final selection, designated as Bacillus sp. A4442, turned out to show a substantially increased enzyme productivity. Catabolite repression by glucose and lactose requirement as an inducer for the enzyme biosynthesis, which were shown in the parent strain, was markedly diminished; instead it was found out that galactose acts as another inducer. Because pH of medium, one of the most important factors for cell growth as well as enzyme production, is closely related with the sugar concentration during culture, it was kept in the optimum range of $6.5{\sim}7.5$; for this the initial glucose concentration was adjusted to be 0.5% which was thereafter maintained by the controlled pumping-in of lactose using the pH-stat technique. By doing so, we were able to increase the productivity of ${\beta}-galactosidase$ with high transgalactosylation activity up to $44\;unit/m{\ell}-broth$.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.343-348
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• 1994

Enterobacter sp. producing -$\beta$-galactosidase with high transgalactosylation activity was isolated from dairy wastewater. The isolate had common biochemical features to E. aerogenes and E. cloacae. Enzyme production increased as the cell mass increased with optimum enzyme activity of 0.21 Unit/mg-protein (o-nitro-phenyl-$\beta$ -D-galactoside (ONPG) as substrate) until 8 hr of culture. Whole cells permeabilized by toluene were used to produce galacto-oligosaccharide. Optimum toluene concentration, temperature and pH for -$\beta$-galactosidase activity of permeabilized whole cells were 10% (v/v), $50^{\circ}C$ and 6.0, respectively. A maximum of 38% (w/w) of galacto-oligosaccharide was obtained with lactose concentration of 20% (w/w) at $40^\{\circ}C$ and pH 6.0.

• KSBB Journal
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• v.11 no.3
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• pp.317-322
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• 1996

Galactooligosaccharlde (GOS) is a kind of functional oligosaccharides that can be used as a food ingredient and a cosmetic additive. In this paper, characteristics of GOS synthesis by cellulase, using lactose as a substrate, were investigated. Penicillium funiculosum cellulose was found to be the most efficient for GOS production among six cellulose tested. The optimum pH and temperature for GOS production were 5.0 and $50^{\circ}C$, respectively. There was an optimum ratio of lactose concentration to enzyme loading; the value was 10 (w/w). The reaction pattern of P. funiculosum cellulase is consistent with that of microbial ${\beta}$-galactosidase which shows transgalactosylation activity. Amounts of GOS produced from 20% (w/v) lactose after 6 h incubation at $50^{\circ}C$, were 23% (w/w) based on total saccharide in the reaction medium. The GOS % increased with initial lactose concentration in the range of 5 to 20%. The products mainly consisted of a trisaccharide and tetrasaccharide from HPLC and TLC analysis. Among enzymes involved in transgalactosylation reaction, high molecular weight fractions over 50,000 Da, presumably ${\beta}$-glucosldase, were considered to be responsible for GOS production. Using this cellulose, a direct synthesis of galactosyl g1ucoside including GOS could be readily achieved with lactose as a galactosyl donor.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.333-337
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• 1996

For continuous production of galactooligosaccharides(GOS), $\beta-galactosidase$ with h1gh transgalactosylation activity from Bacillus sp. A 4442 was Immobilized onto $Diaion^{TM}$ HPA 75(styrene-divinylbenzene resin). The parameters influencing enzyme immobilization were scrutinized in order to maximize immobilization yield while minimizing enzyme inactivation. The optimum conditions turned out to be: Tris buffer concentration 30 mM, pH 8.0, contact time at room temperature 3 hr, and enzyme loading 25 mg protein/g resin. Both the thermal stability and the operational stability of immobilized enzyme were markedly enchanced by the treatment with 0.5% glutaraldehyde as a cross-linker. Under the experimental conditions established, the yield of ${\beta}-galactosidase$ immobilization was 40% or more and the activity of the immobilized enzyme ca. 200 U/g resin. When a packed-bed reactor was employed to continuously convert lactose to GOS, the specific production, which refers to as the amount of commercially valuable GOS produced by a unit amount of immobilized ${\beta}-galactosidase$, was found to be ca. 300 g GOS/g carrier.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.550-558
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• 2021

Lactobacillus kefiranofaciens contains two types of β-galactosidase, LacLM and LacZ, belonging to different glycoside hydrolase families. The difference in function between them has been unclear so far for practical application. In this study, LacLM and LacZ from L. kefiranofaciens ATCC51647 were cloned into constitutive lactobacillal expression vector pMG36e, respectively. Furtherly, pMG36n-lacs was constructed from pMG36e-lacs by replacing erythromycin with nisin as selective marker for food-grade expressing systems in Lactobacillus plantarum WCFS1, designated recombinant LacLM and LacZ respectively. The results from hydrolysis of o-nitrophenyl-β-galactopyranoside (ONPG) showed that the β-galactosidases activity of the recombinant LacLM and LacZ was 1460% and 670% higher than that of the original L. kefiranofaciens. Moreover, the lactose hydrolytic activity of recombinant LacLM was higher than that of LacZ in milk. Nevertheless, compare to LacZ, in 25% lactose solution the galacto-oligosaccharides (GOS) production of recombinant LacLM was lower. Therefore, two β-galactopyranosides could play different roles in carbohydrate metabolism of L. kefiranofaciens. In addition, the maximal growth rate of two recombinant strains were evaluated with different temperature level and nisin concentration in fermentation assay for practical purpose. The results displayed that 37℃ and 20-40 U/ml nisin were the optimal fermentation conditions for the growth of recombinant β-galactosidase strains. Altogether the food-grade Expression system of recombinant β-galactosidase was feasible for applications in the food and dairy industry.