• Title/Summary/Keyword: Beta-galactosidase

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Production of $\beta$-Galactosidase from Alkalophilic Bacillus sp. (II) (호알카리성 Bacillus sp.로부터 $\beta$-Galactosidase의 생산(II))

  • 유주현;윤성식
    • Microbiology and Biotechnology Letters
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    • v.17 no.5
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    • pp.524-528
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    • 1989
  • A $\beta$-Galactosidase producing strain, Alkalophilic Bacillus sp, YS-309, has been isolated from soil sample. The strain was capable of producing large amount of intracellular $\beta$-galactosidase in the alkaline media rather than in the neutral media. The preferable medium composition has been determined to be as follows: 0.5% lactose, 0.5% yeast extract, 0.5% soybean meal, 0.1% KH$_2$PO$_4$, 0.02% MgSO$_4$7$H_2O$ 0,0.6% Na$_2$CO$_3$ (pH 9.9). The enzyme was produced by lactose or IPTG as in-ducer. But both Enzyme synthesis and cellular growth were decreased when lactose was added at the higher concentrations than 1.5% (v/v).

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Molecular Cloning of a $\beta$-D-Galactosidase Gene from Lactococcus lactis subsp. lactis 7962

  • CHANG, HAE-CHOON;YANG-DO CHOI;HYONG-JOO LEE
    • Journal of Microbiology and Biotechnology
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    • v.6 no.6
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    • pp.386-390
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    • 1996
  • The ${\beta}$-galactosidase gene from Lactococcus lactis subsp. lactis ATCC 7962 was cloned and its enzymatic properties were characterized, with a view to assessing its potential use as a selection marker in the food-grade cloning vector. Chromosomal DNA from L. lactis subsp. lactis 7962 was cleaved with PstI and ligated into pBR322 for transformation into Escherichia coli TGl. Transformants showing ${\beta}$-galactosidase activity possessed the pBR322 plasmid containing a 10 kilobase (kb) PstI fragment and this plasmid was named pCKL11. The cloned ${\beta}$-galactosidase gene came from the chromosomal DNA of L. lactis subsp. lactis 7962 was confirmed by Southern hybridization. A restriction map of pCKL11 was constructed from the cleavage of both pCKL11 and the purified 10kb insert fraqment. The. optimum pH of the ${\beta}$-galactosidase determined with the E. coli harboring the pCKL11 was 7.0. The optimum temperature was $50^{\circ}C$, while the pI of the enzyme was 7.4. These values were the same as those of the enzyme from the parent strain.

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Characterization of the Nar Promoter Modified by Site-directed Mutagenesis to Use as an Expression Promoter (특정부위돌연변이화에 의해 변형된 nar 프로모터를 발현 프로모터로 이용하기 위한 특성연구)

  • 이종원
    • KSBB Journal
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    • v.11 no.4
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    • pp.431-437
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    • 1996
  • The nar promoter of Escherichta coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an expression promoter. The modified nar promoter, in which several bases in the -10 region was mutated to the consensus sequence by site-directed mutagenesis, was characterized in E. coli, on which chromosome the fnr gene affecting expression of the nar promoter according to dissolved oxygen level was mutated. The E. coli lacZ gene was used as a reporter gene. The following effects were investigated to find optimal conditions to induce the modified nar promoter: induction methods, optimal nitrate concentrations, the amount of ${\beta}$-galactosidase expressed at the different growth conditions, and induction characteristics. The following results were obtained from the experiments : expression of ${\beta}$-galactosidase from the modified nar promoter was not affected much by nitrate concentrations. The maximal specific ${\beta}$-galactosidase activity was obtained when E. coli was grown under aerobic conditions, and then the modified nar promoter was induced at OD600=2.2 under microaerobic conditions (DO=1∼2%), under which conditions the maximal specific ${\beta}$-galactosidase activity was 13,000 Miller units. However, the specific ${\beta}$-galactosidase activity was approximately 6,000 Miller units even before the modified nar promoter was induced. Therefore, the modified nar promoter seemed to be useful when the cloned gene wants to be expressed in E. coli constitutively.

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The Activity Changes and Properties of ${\beta}$-Galactosidase in Ripening Tomato Fruits (성열중(成熱中) Tomato 과실(果實)의 ${\beta}$-Galactosidase의 활성변화(活性變化)와 그 특성(特性))

  • Kweon, Sang Oh;Moon, Kwang Deok;Sohn, Tae Hwa
    • Current Research on Agriculture and Life Sciences
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    • v.7
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    • pp.153-163
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    • 1989
  • The activity changes and biochemical properties of ${\beta}$-gal in tomato fruits during ripening were investigated. The total activity was increased during ripening and three isoenzymes (${\beta}$-gal I, II and III) were purified through DEAE Sephadex A-50 and Sephadex G-100 column chromatography. The activities of ${\beta}$gal isoenzymes (${\beta}$-gal I, II and III) during ripening were 69.8, 31.8 and 170.0 units in mature green phase, while those were 48.7, 88.4 and 136.8 units in Red phase, respectively. As the ripening proceeded the activities of ${\beta}$-gal I and III were some what decreased but the activity of ${\beta}$-gal II was incresed more than 2.8 fold. The optimum pH of ${\beta}$-gal I, II and III were 3.9, 4.2 and 3.9 and the optimum temperature of those were $60^{\circ}C$, $56^{\circ}C$ and $60^{\circ}C$, respectively. All isoenzymes were stable at pH 3.6~6.0 and lost their activity about 50% when it heated at $55^{\circ}C$ for 5 minute. $Mg^{{+}{+}}$-activated the three isoenzymes but $Ca^{{+}{+}}$ and SDS inhibited about 30~40%. $Hg^{{+}{+}}$ inhibited completely. The km value of ${\beta}$-gal I, II and III was 0.36mM, 0.63mM and 0.45mM, reaction rate was rapidly increased until the concentration of substrate was $6.0{\times}10^{-5}M$.

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Repeated-Batch Operation of Immobilized ${\beta}$-Galactosidase Inclusion Bodies-Containing Escherichia coli Cell Reactor for Lactose Hydrolysis

  • Yeon, Ji-Hyeon;Jung, Kyung-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.21 no.9
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    • pp.972-978
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    • 2011
  • In this study, we investigated the performance of an immobilized ${\beta}$-galactosidase inclusion bodies-containing Escherichia coli cell reactor, where the cells were immobilized in alginate beads, which were then used in repeated-batch operations for the hydrolysis of o-nitrophenyl-${\beta}$-D-galactoside or lactose over the long-term. In particular, in the Tris buffer system, disintegration of the alginate beads was not observed during the operation, which was observed for the phosphate buffer system. The o-nitrophenyl-${\beta}$-D-galactoside hydrolysis was operated successfully up to about 80 h, and the runs were successfully repeated at least eight times. In addition, hydrolysis of lactose was successfully carried out up to 240 h. Using Western blotting analyses, it was verified that the ${\beta}$-galactosidase inclusion bodies were sustained in the alginate beads during the repeated-batch operations. Consequently, we experimentally verified that ${\beta}$-galactosidase inclusion bodies-containing Escherichia coli cells could be used in a repeated-batch reactor as a biocatalyst for the hydrolysis of o-nitrophenyl-${\beta}$-D-galactoside or lactose. It is probable that this approach can be applied to enzymatic synthesis reactions for other biotechnology applications, particularly reactions that require long-term and stable operation.

Production, Purification and Characterization of $\beta$-Galactosidase from Streptococcus thermophilus 510 (Streptococcus thermophilus 510에 의한 $\beta$-Galactosidase의 생산, 정제 및 특성)

  • 강국희;박신인
    • Microbiology and Biotechnology Letters
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    • v.17 no.1
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    • pp.35-45
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    • 1989
  • Streptococcus thermophilus 510 was investigated as n potential source of $\beta$-galactosidase. Optimum cultural conditions for maximum enzyme production were 0.5% loctose as carbon source, initial pH 7.0, 37 $^{\circ}C$, and 18 hours of cultivation. The enzyme was purified to homogeneity by ammonium sulfate fractionation, protamine sulfate precipitation, Sephadex G-200 gel filtration, and DEAE-Sephadex A-50 ion exchange chromntography. The purified enzyme exhibited an optimum pH at 1.0, and an optimum temperature of 5$0^{\circ}C$. Metal ions such as Mn$^{2+}$ and $K^+$, dithiothreitol, and 2-mercaptoethanol stimulated $\beta$-galactosidase activity. Ethylenediamine tetraacetic add, 8-hydroxyquinoline, Hg$^2+$, Zn$^{2+}$, Co$^{2+}$, $Ca^{2+}$, and galactose were inhibitory. The $K_m$ and V$_{max}$ for o-nitrophenyl $\beta$-D-galactopyranoside were 1.25mM and 88.50$\mu$moles/min.mg protein, respectively. The molecular weight was estimated to be 520,000, and the amino acid composition indicated relatively high contents of glutamic acid, aspartic acid, leucine, and valine.

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β-Galactosidase Gene of Thermus thermophilus KNOUC112 Isolated from Hot Springs of a Volcanic Area in New Zealand: Identification of the Bacteria, Cloning and Expression of the Gene in Escherichia coli

  • Nam, E.S.;Choi, J.W.;Lim, J.H.;Hwang, S.K.;Jung, H.J.;Kang, S.K.;Cho, K.K.;Choi, Y.J.;Ahn, J.K.
    • Asian-Australasian Journal of Animal Sciences
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    • v.17 no.11
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    • pp.1591-1598
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    • 2004
  • To isolate the $\beta$-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of $\beta$-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72$^{\circ}C$, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding $\beta$-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of $\beta$-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase(KNOUC112$\beta$-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112$\beta$-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b.

High Production of Thermostable Beta-galactosidase of Bacillus stearothemophilus in mesophiles

  • Okada, Hirosuke;Hirata, Haruhisa;Negoro, Seiji
    • Proceedings of the Korean Society for Applied Microbiology Conference
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    • 1986.12a
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    • pp.509.1-509
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    • 1986
  • Recent advances in recombinant DNA techniques have provided a tool for breeding of microorganisms of hyper production. Enzyme production by cloned microorganism has some advantages. They are ⅰ) Enzymes can be produced by a microorganism easily cultured ⅱ) Hyper production. ⅲ) In some cases, such as thermophilic enzyme gene is cloned in a mesophilic bacteria, the enzyme purification procedure can be simplified. One example, production of thermophilic ${\beta}$-galactosidase in B. subtilis will be presented. Bacillus stearothermophilus IAM 11001 produced three ${\beta}$-galactosidases, ${\beta}$-galactosidase I, II and III (${\beta}$-gal-I, II and III). By connecting restriction fragments of the chromosomal DNA to plasmid vector, followed by transformation of Escherichia coli, two ${\beta}$-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were cloned.

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Effect of Heating Temperature on Viable Yoghurt Culture and $eta$-Galactosidase in Yoghurt (Yoghurt의 가열처리가 젖산균 생균수 및 $eta$-galactosidase의 활성에 미치는 영향)

  • 이광희
    • The Korean Journal of Food And Nutrition
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    • v.6 no.3
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    • pp.208-210
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    • 1993
  • In order to know the influences of heat treatment of yoghurt on pH, $\beta$-galactosidase and viable cells, yoghurt sample was made by general method with Lactobacillus bulgaricus and Streptococcus thermophilus, and the changes in pH, $\beta$-galactosidase-activity and viable cell-count were determined during heating at 55$^{\circ}C$ and 7$0^{\circ}C$. The pH of yoghurt was not changed when the yoghurt was heated at 7$0^{\circ}C$, but at 55$^{\circ}C$ it decreased slightly. The stability of $\beta$-galactosidase was not affected markedly by heat treatment at 55$^{\circ}C$, but was rapidly inactivated at 7$0^{\circ}C$. The heat treatment of yoghurt at 55$^{\circ}C$ had the halb of viable cell in 1 hour, but the heat treatment at 7$0^{\circ}C$ had considerable effect on viable cell in 5 minutes.

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Formation of galactooligosaccharides by ${\beta}-galactosidase$ from Streptococcus thermophilus 510 (Streptococcus thermophilus 510의 ${\beta}-galactosidase$에 의한 galactooligosaccharides의 생성에 관한 연구)

  • Park, Shin-In;Kang, Kook-Hee
    • Korean Journal of Food Science and Technology
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    • v.21 no.1
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    • pp.164-172
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    • 1989
  • The formation of galactooligosaccharides by transgalactosidation reactions during hydrolysis of lactose by the ${\beta}-galactosidase$ from Streptococcus thermophilus 510 was investigated. Three oligosaccharides were detected during hydrolysis. It was found that the optimum conditions for the production of oligosaccharides was 40% lactose treated with ${\beta}-galactosidase(50\;ONPG\;units/ml)$ at $37^{\circ}C$ for 4 hours. The oligosaccharides formed accounted for 30% of the total sugars when the lactose had been 94% hydrolysed. 69% of the oligosaccharides were identified as $6-o-{\beta}-D-galactopyranosyl-D-glucose(allolactose)$ and 23% as $6-o-{\beta}-D-galactopyranosyl-D-galactose(isogalactobiose)$. The separation of galactooligosaccharides by the use of Bio-Gel P-2 gel permeation chromatography was also studied.

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