• Journal of Microbiology and Biotechnology
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• 제2권1호
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• pp.56-65
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• 1992

The complete nucleotide sequence of the Bacillus circulans F-2 RSDA gene, coding for raw starch digesting a-amylase (RSDA), has been determined. The RSDA structure gene consists of an open reading frame of 2508 bp. Six bp upstream of the translational start codon of the RSDA is a typical gram-positive Shine-Dalgarno sequence and the RSDA encodes a preprotein of 836 amino acids with an Mr of 96, 727. The gene was expressed from its own regulatory region in E. coli and two putative consensus promoter sequences were identified upstream of a ribosome binding site and an ATG start codon. Confirmation of the nucleotide sequence was obtained and the signal peptide cleavage site was identified by comparing the predicted amino acid sequence with that derived by N-terminal analysis of the purified RSDA. The deduced N-terminal region of the RSDA conforms to the general pattern for the signal peptides of secreted prokaryotic proteins. The complete amino acid sequence was deduced and homology with other enzymes was compared. The results suggested that the Thr-Ser-rich hinge region and the non-catalytic domain are necessary for efficient adsorption onto raw substrates, and the catalytic domain (60 kDa) is necessary for the hydrolysis of substrates, as suggested in previous studies (8, 9).

• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.230-233
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• 1999

A plasmid vector was constructed for the expression and secretion of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in Bacillus subtilis. The vector, pUBACGT, was composed of the ribosome-binding sequence, signal sequence, and cgt gene from B. macerans under the control of amyR2, the promoter of amyE gene coding for $\alpha$-amylase from B. subtilis var. natto. Bacillus subtilis LKS88, a mutant strain lacking genes for an amylase and two proteases, was used as a host for the transformation of the plasmid vector. The transformants were selected on kanamycin-containing Luria-Bertani plates. The starch hydrolyzing activity was observed on the starch-containing plates by the iodine method and cyclodextrin-forming activity was detected in the culture medium. A SDS-PAGE analysis showed that most of the expressed CGTase in the recombinant B. subtilis was secreted into the medium at a high expression level.

• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1975-1983
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• 2008

The neuropeptide ${\alpha}$-melanocyte-stimulating hormone (${\alpha}$-MSH) has anti-inflammatory property by down regulating the expressions of proinflammatory cytokines. Because ${\alpha}$-MSH elicits the anti-inflammatory effect in various inflammatory disease models, we examined the therapeutic effect of oral administration of recombinant Lactobacillus casei, which secretes ${\alpha}$-MSH (L. casei-${\alpha}$-MSH), on dextran sulfate sodium (DSS)-induced colitis in Balb/c mice. Thus, we constructed the ${\alpha}$-MSH-secreting Lactobacillus casei by the basic plasmid, pLUAT-ss, which was composed of a PldhUTLS promoter and ${\alpha}$-amylase signal sequence from Streptococcus bovis strain. Acute colitis was induced by oral administration of 5% DSS in drinking water for 7 days. To investigate the effect of L. casei-${\alpha}$-MSH on the colitis, L. casei or L. casei-${\alpha}$-MSH was orally administered for 7 days and their effects on body weight, mortality rate, cytokine production, and tissue myeloperoxidase (MPO) activity were observed. Administration of L. casei-${\alpha}$-MSH reduced the symptom of acute colitis as assessed by body weight loss (DSS alone: $14.45{\pm}0.2\;g$; L. casei-${\alpha}$-MSH: $18.2{\pm}0.12\;g$), colitis score (DSS alone: $3.6{\pm}0.4$; L. casei-${\alpha}$-MSH: $1.4{\pm}0.6$), MPO activity (DSS alone: $42.7{\pm}4.5\;U/g$; L. casei-${\alpha}$-MSH: $10.25{\pm}0.5\;U/g$), survival rate, and histological damage compared with the DSS alone mice. L. casei-${\alpha}$-MSH-administered entire colon showed reduced in vitro production of proinflammatory cytokines and $NF-{\kappa}B$ activation. The ${\alpha}$-MSH-secreting recombinant L. casei showed significant anti-inflammatory effects in the murine model of acute colitis and suggests a potential therapeutic role for this agent in clinical inflammatory bowel diseases.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.402-406
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• 2005

Lipomyces starkeyi produces a novel glucanhydrolase containing endo-dextranase and ${\alpha}-amylase$ activities. A cDNA from L. starkeyi encoding a dextranase was isolated and characterized. The 2,052 kb cDNA fragment (lsd1) carrying dextranase gene showed one open reading frame (ORF) composed of 1,824 bp flanked by a 41 bp 5'-UTR and a 184 bp 3'-UTR including a poly(A) tail of 27 bp. The ORF encodes for a 608 amino acid with a predicted molecular mass of 67.6 kDa. There was 77% deduced amino acid sequence identity between the LSD1 dextranase and the dextranase from Penicillium minioluteum. The primary structure of the dextranase from L. starkeyi has distant similarity with enzymes belonging to glycosyl hydrolase family 49. The lsd1 protein was expressed in the Saccharmyces cerevisiae under control of GAL1 promoter and active dextranase was produced.

• Journal of Microbiology and Biotechnology
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• 제20권8호
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• pp.1215-1218
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• 2010

The recombinant DNA pLR5cat_PSAB, in which pediocin PA-1 structural and immunity genes (pedAB) fused with the promoter and deduced signal sequence of an ${\alpha}$-amylase gene from a bifidobacterial strain were inserted in Escherichia coli-lactobacilli shuttle vector pLR5cat, was transferred to Lactobacillus reuteri KCTC 3679 and the transformant presented bacteriocin activity. The recombinant L. reuteri KCTC 3679 transformed with the shortened pLR5cat(S)_PSAB, where a nonessential region for the lactobacilli replicon was removed, also showed bacteriocin activity. The molecular mass of the secreted pediocin PA-1 from the recombinant bacteria was the same as that of native pediocin PA-1 (~4.6 kDa) from Pediococcus acidilactici K10 on a sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. In cocultures with Listeria monocytogenes, the recombinant L. reuteri KCTC 3679 effectively reduced the viable cell count of the pathogenic bacterium by a 3 log scale compared with a control where L. monocytogenes was incubated alone.

• Preventive Nutrition and Food Science
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• 제15권4호
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• pp.360-363
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• 2010

To enhance the expression and secretion of pediocin PA-1 from heterologous bacterial hosts, the promoter and deduced signal sequence (PS) of an $\alpha$-amylase gene from a Bifidobacterium adolescentis strain was fused with pediocin PA-1 structural and immunity genes (AB) and the resulting functions were evaluated in Escherichia coli. Two recombinant PCR products were created-one with just the deduced signal sequence and one with the sequence plus the Ser and Thr sequences that are the next two amino acids of the signal sequence. These two products, the PSAB (---AQA::KYY---) and PSABST (---AQA$\underline{ST}$::KYY---), respectively, were inserted into a TA cloning vector (yT&A) and named pPSAB, which was previously reported, and pPSABST. The two recombinant plasmid DNAs were transferred into E. coli JM109 and the transformants displayed antimicrobial activity, where the activity of E. coli JM109 (pPSAB) was stronger than that of E. coli JM109 (pPSABST), indicating that the ST amino acid residues were not necessary for secretion and might have even decreased the antimicrobial activity of recombinant pediocin PA-1.

• Journal of Microbiology and Biotechnology
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• 제13권3호
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• pp.451-456
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• 2003

An expression vector system was developed for the secretory production of recombinant Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae. The mature L1 lipase gene was fused to ${\alpha}-amylase$ signal sequence from Aspergillus oryzae for the effective secretion into the culture broth and the expression was controlled under GAL10 (the gene coding UDP-galactose epimerase of S. cerevisiae) promoter. S. cerevisiae harboring the resulting plasmid successfully secreted L1 lipase into the culture broth. To examine an optimum condition for L1 lipase expression in the fed-batch culture, L1 lipase expression was induced at three different growth phases (early, mid, and late-exponential growth phases). Maximum product on of L1 lipase (1,254,000 U/l, corresponding to 0.65/1) was found when the culture was induced at an early growth phase. Secreted recombinant L1 lipase was purified only through CM-Sepharose chromatography, and the purified enzyme showed 1,963 U/mg of specific activity and thermoalkalophilic properties similar to those reported for the enzyme expressed in Escherichia coli.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.576-581
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• 2005

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast. Saccharomyces cerevisiae, by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) of Aspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast ${\alpha}-agglutinin$, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced into S. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.

• Journal of Microbiology
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• 제40권2호
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• pp.140-145
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• 2002

The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMHl turned out to have a replication origin and two open reading frames (ORFs) of the putative γ-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMHl all constructed vectors were stable over 100 generations In a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a navel secretion vector, Using the $\alpha$-amylase promoter/signal sequence of B. subtilils the novel plasmid pJSN was constructed. When $\beta$-glucosidase was expressed using pJSN, we found $\beta$-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

• 한국식품과학회지
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• 제49권6호
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• pp.610-616
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• 2017

본 연구를 통해 BAGGT를 재조합 B. subtilis를 이용하여 대량 생산하기 위하여 유전자 클로닝, 발현 시스템 구축 및 배지 최적화를 진행하였다. 이중프로모터 시스템을 이용하여 야생형 균주에 비해 42배 효소 생산성이 향상된 발현 시스템을 구축하였다. 또한 PBD 분석을 통해 당밀과 CSL이 재조합 B. subtilis 시스템에서 BAGGT의 생산성에 큰 영향을 주는 인자임을 확인하였으며, 염류의 첨가에 의한 효소 생산성 증대 효과는 미비하거나 부정적이었다. 탄소원으로 당밀을 선택하고 고가의 질소원인 트립톤을 저가의 CSL로 교체한 후 CCD 분석을 통해서 결정된 최적배지 사용 시 최적화 이전의 LB 배지 대비 4.3배의 생산성 증대를 이루었으며, 이는 LB 배지에서 야생형 균주의 BAGGT 생산성 대비 180배의 효소 생산성 개선에 해당하였다. 본 연구를 통해 식품용 효소로서 BAGGT의 대량생산을 위한 공정을 구축하였으며, 이후 정미성 소재 생산에 활용할 수 있을 것으로 기대한다.