• Korean Journal of Microbiology
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• v.45 no.2
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• pp.215-221
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• 2009

Site-specific incorporation of unnatural amino acids into proteins in vivo can be achieved by co-expression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to establish this technique, here we generated an Escherichia coli reporter strain DH10B(Tn:lacZam) by integrating amber mutated lacZ gene into the chromosome of E. coli DH10B strain. In vivo expression of E. coli amber suppressor $tRNA^{Gln}$ produced blue colonies in culture plates containing X-Gal as well as dramatically increased $\beta$-galactosidase activity. In addition, expression of an orthogonal pair of Saccharomyces cerevisiae suppressor $tRNA^{Tyr}$ and tyrosyl-tRNA synthetase also produced blue colonies as well as moderate increase of $\beta$-galactosidase activity. These data demonstrate that our reporter strain will provide an efficient method to assess amber suppression in both qualitative and quantitative manners.

• 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.

• Food Science and Preservation
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• v.8 no.1
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• pp.66-73
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• 2001

This study was carried out to know whether $\beta$-galactosidase is directly important or not on fruit softening during the development of peach fruits compared to those in the stage stage. It was investigated that the flesh firmness, cell wall components, and the glycosidase activities of the peach fruits with a fast softening cultivar, 'Mibeakdo', a slow softening cultivar,'Yumyung'and a middle softening cultivar, 'Okubo$\beta$, at different developmental stages, on 13 May, 16 June, 16 July, and 5 August and on 28 August which harvested only 'Yumyung' fruits. In order to investigate the amounts of total sugar and non-cellulosic neutral sugar, the cell wall materials of each fruit were solubilized in distilled water, 0.05M CDTA, 0.05M Na$_2$CO$_3$, 4% KOH, and 24% KOH sequentially. During the fruit development, the fruit firmness of three cultivars decreased and the fruit firmness of 'Yumyung' was higher than that fo 'Mibeakdo' and 'Okubo' in the overall period. During the fruit development, the changes of total sugar amounts of each measured fractions were similar among peach cultivars. Arabinose and galactose were the predominant non-cellulosic neutral sugars in all the fractions including cell wall material of the three cultivars. There was an active relationship between the changes of flesh firmness in three cultivars and the mol % changes of rhamnose on 5 August which was the harvest date of 'Mibeakdo' and 'Okubo' fruits. The activity of soluble $\beta$-galactosidase was high at the early developmental stage and then dropped to a very low activity level in all cultivars. The activity of cell wall-bound $\beta$-galactosidase was high at the early developmental stage and then decreased continuously through the harvest date. In addition the changes of other glycosidase activities were similar among cultivars.

• Applied Biological Chemistry
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• v.39 no.1
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• pp.60-66
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• 1996

To investigate a possible application of three strains of lactic acid bacteria(strain No. 49. No. 61. No. 75) from kimchi in milk fermentation industry, the optimal condition for production of intracellular ${\beta}-galactosidase$ from Lactobacillus(L.) plantarum and its enzymatic properties were examined. The preferable carbon source of the medium for strain No. 49 in production of ${\beta}-galactosidase$ was MRS broth with 1.0% lactose instead of dextrose of pH 65. for strain No. 75 with 1.0% galactose and for strain No. 61 with 3.0% lactose at pH 7.5, respectively. The maximum enzyme production from strain No. 49, No. 75 was observed after 48 hours culture at $30^{\circ}C$ in a medium containing the appropriate carbon source, from strain No. 61 after 48 hours culture at room temperature. The optimum temperature for ${\beta}-galactosidase$ activity from L. plantarum was $60^{\circ}C$ for strain No. 49, $37^{\circ}C$ for strain No. 61 and $50^{\circ}C$ for strain No. 75, respectively. The heat stability of enzyme activities for all three strains remained 90% at $45^{\circ}C$. The optimal pH was pH 6.5 and enzyme activities were most stable at pH for all three bacteria.

• Microbiology and Biotechnology Letters
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• v.11 no.1
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• pp.59-66
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• 1983

Cultural conditions for the production of extracellar $\beta$-galactosidase by Loctobacillus sporogenes, a spore forming lactic acid bacterium, were investigated with shaken flask and jar fermenter cultures. The fermentation medium giving maximum $\beta$-galactosidase yield was found to consist of 1 % lactose as a carbon source, 1.5% peptone as an organic nitrogen source. 0.2% ammonium sulfate as an inorganic nitrogen source, 0.8% ammonium phosphate dibasic as a phosphorus source, and 0.05% potassium chloride and 0.001% ferric chloride as mineral source. Optimal initial pH of the medium was 7.0 and the highest enzyme excretion was observed after 40 hours of cultivation at 37$^{\circ}C$. In this experiment, the 500$m\ell$ conical flask containing 50-200$m\ell$ of medium was shaken at 140 strokes per minute with 7cm amplitude in a reciprocating shaker. The maximum enzyme value attained was 38 U/$m\ell$ of the culture broth which was found to be slightly higher than the highest intermolecular enzyme activity (30 U/$m\ell$) observed after 24 hours of incubation. In the fermentor culture, the fermentation profile was shown to be similar to that observed in the shaken flask experiment. But the maximum extracellular enzyme activity was 45 U/$m\ell$ to be even higher than the value obtained with the shaken flask culture.

• Microbiology and Biotechnology Letters
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• v.11 no.3
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• pp.205-210
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• 1983

Extracellular $\beta$-galactosidase was prepared from a culture of Lactobacillus sporogenes, a spore-forming lactic acid bacterium. The enzyme functioned optimally at pH 6.8 and at 6$0^{\circ}C$ o-nitrophenyl-$\beta$-D-galactopyranoside (ONPG) in 0.05M sodium phosphate buffer. The activation energy of the enzymatic hydrolysis of ONPG was about 16,000 cal/mole below $50^{\circ}C$ and 11,300 cal/mole above the temperature. It was fairly stable over a pH range from 4.0 to 8.0 losing only less than 30% of its activity after hearting at 6$0^{\circ}C$ and pH 6.8 for 3 hours. Metal ions showed no significant effect on the enzyme activity, whereas L-cysteine exerted a slight stimulatory effect at the concentration of 10mM. The km values were 1.48mM for ONPG and 64.5mM for lactose. Hydrolysis of ONPG by the enzyme was product-inhibited by galactose (Ki=13.3mM, competitive inhibition) and by glucose(Ki= 11.4mM, uncompetitive type). The enzyme activity was also noncompetitively inhibited in the presence of lactose (Ki= 17.8mM).

• BMB Reports
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• v.40 no.3
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• pp.419-425
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• 2007

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli $\beta$-galactosidase and $\beta$-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more $\beta$-glucuronidase activity than wild-type $\beta$-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high $\beta$-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.355-359
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• 1985

The purified $\beta$-galactosidase from L. sporogenes was most active at pH 7.0 and 6$0^{\circ}C$ with O-nitrophenyl-$\beta$-D-galactopyranoside (ONPG) in 0.05 M phosphate buffer. It was stable over a pH range from 5.0 to 9.0 and lost less than 10％ of its activity after heating for 30 minutes at 6$0^{\circ}C$ and pH 7.0. All the mineral ions examined in this work showed no significant activating effect, whereas L-cysteine exerted a great stimnlatory effect on the enzyme activity at the concentration of 10 mM. The Km values were 1.2 mM for ONPG and 33.3 mM for lactose. Approximately 85％ of lactose in cow's milk, in 10％ skim milk and in 5％ lactose solution was hydrolyzed after 4 hours incubation at 6$0^{\circ}C$ with 2 units of the purified $\beta$-galactosidase per $m\ell$ of the substrate solutions. The $\beta$-galactosidase from L. sporogenes, therefore, is considered to be suitable for hydrolysis of lactose in milk and other dairy products.

• 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 $44\;unit/m{\ell}-broth$.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1979.04a
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• pp.113.2-114
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• 1979

$\beta-Galactosidase$ is an enzyme which catalizes hydrolysis of lactose, a natural substrate, to glucose and galctose and transferring some monosac-charide units to active acceptors as sugar or alcohol. The occurence of $\beta-Galactosidase$ is known in various microorganisms, animals and higher plants and has been studied by many investigatigators. Especially, a great deal of articles for the enzyme of E. coli have been presented in genetic control mechanism and induction-repression effects of proteins, On the other hand, in the dairly products industry, it is important to hydrolyes lactosd which is the principal sugar of milk and milk products. During the last few years, the interest in enzymatic hydrolysis of milk lactose has teen increased, because of the lactose intolerence in large groups of the population. Microbial $\beta-Galactosidases$ are considered potentially most suitable for processing milk to hydrolyse lactose and, in recent years, the immobilized enzyme from yeast has been examined. Howev, most of the microbial $\beta-Gal$ actosidase are intracellular enzymes, except a few fungal $\beta-Gala-$ ctosidases, and extracellular $\beta-Galactosidase$ which may be favorable to industrial applieation is not so well investigated. On this studies, a mold producing a potent extracellular $\beta-Galactosidase$ was isolated from soil and identified as an imperfect fungus, Beauveria bassians. In this strain, both extracellular and intracellular $\beta-Galactosidases$ were produced simultaneously and a great increase of the extracellular production was acheved by improving the cultural conditions. The extracellular enzyme was purified more than 1, 000 times by procedures including Phosphocellulose and Sephadex G-200 chromatographies. Several characteristics of the enzymewas clarified with this preparation. The enzyme has a main subunit of molecular weight of 80, 000 which makes an active aggregate. And at neutral pH range, it has optimum pH for activity and stability. The Km value was determined to be 0.45$\times$10$^{-3}$ M for $o-Nitrophenyl-\beta-Galactoside.$ In any event, it is interesting to sttudy the $\beta-Galactosidase$ of B. bassiana for the mechanism of secretion and conformational structure of enzyme.