• Journal of Microbiology and Biotechnology
• /
• 제16권8호
• /
• pp.1325-1329
• /
• 2006

Structural modeling of $\beta$-galactosidase from L. lactis ssp. lactis 7962 has shown that the residues Cys-472 and His-509 are located in the wall of the active-site cavity. To examine the functions of Cys-472 and His-509, we generated five site-specific mutants: Cys-472-Ser, Cys-472-Thr, Cys-472-Met, His-509-Asn, and His-509-Phe. $\beta$-Galactosidase substituted at Cys-472 with Met or His-509 with Phe had <3% of the activity of the native enzyme when assayed using ONPG as substrate. The other mutants Cys-472-Ser, Cys-472-Thr, and His-509-Asn had ca. 10-15% of the native enzyme activity. The V$_max$ values of the five mutated enzymes were lower (60-7,000-fold) than that of native enzyme. These results show that the catalytic ability of $\beta$-galactosidase is significantly affected by mutations at Cys-472 or His-509.

• Journal of Microbiology and Biotechnology
• /
• 제13권1호
• /
• pp.134-138
• /
• 2003

The secondary and tertiary structures of ${\beta}$-galactosidase from L. lactis ssp. lactis 7962 were designed using Nnpredict and Sybyl version 6.3. By using site-directed mutagenesis, the mutated enzymes, Tyr-475-phe and Glu-506-Asp, were generated based on the structural modeling of L. lactis ssp. lactis 7962. The enzymes Tyr.-475-Phe and Glu-506-Asp had <$1\%$ of the activity of the native enzyme with ONPG as substrate. The $V_{max}$ values of the mutated enzymes were greatly reduced (1,800~40,000-1314) compared with the value for the native ${\beta}$-galactosidase. However, the $K_m$ values of Tyr-475-Phe and Glu-506-Asp with ONPG, PNPG, PNPF, and PNPA were not significantly different from those of the native enzyme. The results obtained support the suggestion that Tyr-475 and Glu-506 constitute very important parts of the catalytic machinery of the ${\beta}$-galactosidase.

• 분석과학
• /
• 제5권4호
• /
• pp.471-475
• /
• 1992

유당분해효소를 유지방으로 미세캡슐화시켰으며, 이때의 미세캡슐화 수율을 정량적으로 분석하는 방법을 제시하였다. 본 연구에서 적용한 미세캡슐화 공정과정에 의한 유당분해효소의 불활성화 정도는 초기 효소활성도의 5.2%였으며 이러한 불활성화 정도를 고려하여 유당분해효소의 미세캡슐화 수율을 여러 가지 방법에 의하여 계산하였을 때 각각 92.6% (간접적인 측정방법), 88.6% (열처리에 의한 방법) 및 94.1% (효소처리에 의한 방법)였다.

• 한국미생물·생명공학회지
• /
• 제25권2호
• /
• pp.144-150
• /
• 1997

After DNA damage, umuDC is the only SOS operon that must be induced to promote SOS mutagenesis in Escherichia coli. The recombinant plasmid pBC401 and pBC402 were constructed to fuse the lac structural genes with promoter region of umuDC operon to induce the expression of lacZ gene by DNA damage. We transformed the plasmid pBC401 and pBC402 into E. coli MC1061, lacZ deleted strain and determined the activity of $\beta$-galactosidase for various mutagen; UV, mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroqunoline-1-oxide (NQO), ethyl methanesulfonate (EMS). The $\beta$-galactosidase activities of PBC401 and pBC402 for UV, MMC, and NQO were increased in proportion to expression time until 3 hours thereafter, the activities were constant or slightly decreased. The activities for MNNG and EMS were not so high as for UV, MMC, and NQO. When MNNG and EMS were treated, $\beta$-galactosidase activity of pBC402 was slightly lower than pBC401 but when UV, MMC, and NQO were treated in pBC402, $\beta$-galactosidase activity was slightly higher than in pBC401. Therefore, the pBC402 was better than the pBC401 in terms of sensitivity for frameshift mutagen. We suggest that the plasmid pBC401 and pBC402 are easy to detect mutagens which cause frameshift mutation rather than point mutation.

• BMB Reports
• /
• 제34권5호
• /
• pp.395-401
• /
• 2001

Schizosaccharomyces pombe gene encoding redox enzymes, such as thioltransferase (TTase) and thioredoxin (TRX), were previously cloned and induced by oxidative stress. In this investigation, their expressions were examined using $\beta$-galactosidase fusion plasmids. The expression of the two cloned genes appeared to be growth-dependent. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was increased in the medium with the low glucose level, whereas it was significantly decreased in the medium without glucose or with galactose. It was also decreased in the nitrogen-limited medium. The synthesis of galactosidase from the TRX-lacZ fusion was unaffected by galactose or low glucose. However, it was lowered the absence of glucose. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was shown to be enhanced in a higher medium pH. Our findings indicate that S. pombe TTase and TRX genes may be regulated by carbon and nitrogen sources, as well as medium pH.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제5권3호
• /
• pp.215-218
• /
• 2000

Sialytrisaccharides based on $\beta$-galactosyldisaccharides were synthesized using $\beta$-galactosidase and trans-sialidase in one pot. Using $\beta$-galactosidase from Bacillus Ciculans and trans-sialidase from Trypanosoma cruzi simulaneously, 6mM sialyltrisaccharides composed of about 95% NeuAc$\alpha$(2,3)Gal$\beta$(1,4)GlcNAc and 5% NeuAc$\alpha$(2,3)Gal$\beta$(1,6)GlcNAc were produced from a reaction mixture containing 25mM o-nitropheny1-$\beta$-D-galsctolneuraminic acid. One beauty of this reaction was that a secondary hydrolysis of the disaccharide intermediate occurring between the activated galactopyranoside and N-acetylgucosamine was prevented. Using $\beta$-galactosidase from Escherichia cloi and the same trans-sialidase, 15mM sialyltrisaccharides composed of about 90% NeuAc$\alpha$(2,3)Gal$\beta$(1,6)GlcNac and 10% NeuAc$\alpha$(2,3)Gal$\beta$(1,4)GlcNAc were produced from a reaction misture containing 400nM galactose, 800nM N-acetylglucosylation rection between galactose and N-actylgucosamine was diminant since the disaccharide intermediate mainly resulted sreulted in the silylated product.

• Journal of Microbiology and Biotechnology
• /
• 제1권2호
• /
• pp.90-95
• /
• 1991

A ${\alpha}-galactosidase{\;}({\alpha}-D-galactoside$ galactohydrolase; EC 3.2.1.22) was purified from the culture filtrate of Penicillium purpurogenum by DEAE-cellulose column chromatography, gel filtration of Bio gel p-l00, and subsequent SP-Sephadex C-25 chromatography. The final preparation thus obtained showed a single band on polyacrylamide disc-gel and SDS-polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were determined to be 63,000 and pH 4.0 by SDS-polyacrylamide gel electrophoresis and isoelectric focusing, respectively. The galactosidase exhibited maximum activity at pH 4.5 and $55^{\circ}C$, and was stable between pH 2 and 5, and also stable up to $40^{\circ}C$. The enzyme activity was not affected considerably by treatment with other metal compounds except mercuric chloride and silver nitrate. Copra galactomannan was finally hydrolyzed to galactose, mannose and mannobiose through the sequential actions of the purified galactosidase and mannanase from the same strain. The enzyme hydrolyzed melibiose and raffinose, but not lactose.

• Journal of Microbiology and Biotechnology
• /
• 제17권9호
• /
• pp.1430-1436
• /
• 2007

[ ${\alpha}$ ]-Galactosidase was immobilized in a mixture of k-carrageenan and locust bean gum. The properties of the free and immobilized enzyme were then determined. The optimum pH for both the soluble and immobilized enzyme was 4.8. The optimum temperature for the soluble enzymes was $50^{\circ}C$, whereas that for the immobilized enzyme was $55^{\circ}C$. The immobilized enzyme was used in batch, repeated batch, and continuous modes to degrade the raffinose-family sugars present in soymilk. Two hours of incubation with the free and immobilized ${\alpha}$-galactosidases resulted in an 80% and 68% reduction in the raffinose oligo saccharides in the soymilk, respectively. In the repeated batch, a 73% reduction was obtained in the fourth cycle. A fluidized bed reactor was also designed to treat soymilk continuously and the performance of the immobilized ${\alpha}$-galactosidase tested at different flow rates, resulting in a 90% reduction of raffinose-family oligosaccharides in the soymilk at a flow rate 40 ml/h. Therefore, the present study demonstrated that immobilized ${\alpha}$-galactosidase in a continuous mode is efficient for reducing the oligosaccharides present in soymilk, which may be of considerable interest for industrial application.

• 한국축산식품학회지
• /
• 제27권1호
• /
• pp.102-109
• /
• 2007

Lactobacillus salivarius subsp. salivarius CNU27 possessed a high level of ${\alpha}$-galactosidase activity. Purified ${\alpha}$-galactosidase was obtained after sonication of harvested cell pellet followed by DEAE-Sephadex A-50 and Mono Q anion exchange chromatography. The specific activity of the purified enzyme was 8,994 units/mg protein which is 17.09 times higher than that in crude extract. The native enzyme was a monomer with a molecular mass of 56,397.1 dalton. The optimum temperature and pH for the enzyme were $40^{\circ}C$ and 6.0, respectively. The enzyme was stable between 25 and $50^{\circ}C$. However, ${\alpha}$-galactosidase activity was lost rapidly below pH 4.5 and above pH 8.5. The enzyme activity decreased to 6.73% and 4.30% of the original activity by addition of $Cu^{2+}$ and $Hg^{2+}$, respectively. Other metal compounds did not affect the enzyme activity significantly. The enzyme liberated galactose from melibiose, raffinose, and stachyose. The rate of substrates hydrolysis was measured by HPLC. Raffinose, stachyose and melibiose were completely decomposed after 24 hr at $40^{\circ}C$.

• 한국식품위생안전성학회지
• /
• 제2권2호
• /
• pp.67-73
• /
• 1987

Kluyveromyces fragilis로부터 $\beta$-galactosidase의 생성조건과 조효소액의 성질 그리고 당 전이 반응에 의한 oligosaccharide 생성을 조사한 결과 다음과 같은 결과를 얻었다. \circled1 Peptone-Yeast extract 배지에 6% lactose를 첨가하였을 때 최대 효소생산을 보였다. \circled2 0.05 M potassium phosphate buffer (pH 7.0)에서 3% toluene를 첨가하여 37$^{\circ}C$ 5시간 배양하였을 때 K. fragilis로부터 효소가 최대로 추출되었다. \circled3 효소의 최적온도는 4$0^{\circ}C$이고 4$0^{\circ}C$ 이상의 열처리에서는 효소가 파괴되었으며, pH6-7에서는 상당히 안정하였다. \circled4 ONPG 기질로 사용하였을 때 Km 값은 2.5mM이었다. \circled5 당 전이 반응의 결과, 7개의 oligosaccharide가 생성되었다. 이상의 실험결과로 볼 때, 본 실험에 사용한 K. fragilis SKD 7001은 Beta-galactosidase의 생산을 위해서 이용 가치가 인정되었으며, 특히, 이 효소의 활성이 중성 pH에서 강하고 안정한 상태를 보이는 것은 시유나 원료우유의 lactose를 중성에서 해야 한다는 점을 고려할 때, 실용적 가치가 있다고 본다. 또, 이 효소의 작용과정에서 생성되는 oligosacchride는 장내 bifidus균의 생육을 촉진시키는 효과가 인정되고 있기 때문에 이용가치를 더욱 높여주는 것으로 생각된다.