• 한국미생물·생명공학회지
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• 제23권5호
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• pp.544-549
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• 1995

The alkaline elastase is an extracellular serine protease of the alkalophilic Bacillus strain Ya-B. To increase the gene copy number and the production level of the alkaline elastase Ya-B, we designed, on the B. subtilis chromosome, a gene amplification of the 10.6 kb repeating unit containing amyE, aleE (alkaline elastase Ya-B gene) and tmrB. The aleE was inserted between amyE and tmrB, and B. subtilis APT119 strain was transformed with this amyE-aleE-tmrB-junction region fragment. As a result, we succeeded in obtaining tunicamycin-resistant (Tm$^{r}$) transformants (Tf-1, Tf-2) in which the designed gene amplification of 10.6 kb occurred in chromosome. The transformants showed high productivity of $\alpha $-amylase and alkaline elastase Ya-B. The copy number of the repeating unit (amyE-aleE-tmrB) was estimated to be 25, but plasmid vector (pUC19) was not integrated. The amplified aleE of chromosome was more stable than that of plasmid in absence of antibiotics.

• Journal of Microbiology and Biotechnology
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• 제1권1호
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• pp.22-30
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• 1991

In order to secrete the Bacillus subtilis cytidine deaminase (CDase, cytidine/2'-deoxycytidine deaminase) encoded by the B. subtilis cdd gene in E. coli by the aid of signal sequences, the cdd gene was fused in-frame to either amyE or penP signal sequences and the gene expression and CDase localization were examined. For the penP signal sequence::cdd fusion, the cdd gene with 9 amino acids truncated from the 5'-terminus was fused in-frame to the signal sequence, then the $cdd^{+}$ colonies were not occurred from the minimal plate by cdd complementation. The result suggests that 9 amino acids on the $NH_2-terminal$ of CDase have an essential function in the enzyme activity. The hybrid protein obtained by fused gene amyE signal sequence::cdd structural gene gave $cdd^{+}$ phenotype and about half of the total CDase activity was found to be secreted in the periplasm of E. coli transformant JF611/pSO202. The periplasmic CDase activity of JF611 harboring pSO52 containing the intact cdd gene was considerablely lower than that of the cells harboring pSO202 carrying the hybrid cdd gene. This suggests that the CDase was secreted to the periplasm through the cytoplasmic membrane by the aid of the amyE signal sequence in the E. coli transformant.

• 생명과학회지
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• 제15권6호
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• pp.1013-1021
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• 2005

미생물 메타게놈은 특이한 생체촉매의 다양한 원료로 제공된다. 한우의 반추위에서 게놈 DNA를 분리한 후 메타게놈 은행을 구축하고 $\alpha$-amlylase를 암호화하는 유전자를 클로닝하여 DNA 및 아미노산 서열을 밝히고 생화적 특징을 조사하였다. amyA유전자는 1,893 bp로 631개의 아미노산 잔기를 가진 단백질을 암호화였으며 효소의 분자량은 단백질 전기영동 결과 약 71,000 Da으로 확인되었다. 이 효소를 다른 아밀라제와 비교한 결과 $21-59\%$의 상동성을 보였다. AnyA는 pH $6.40\%$에서 최적 활성을 나타내었고, pl값은 5.87이었다. E. coliDH5$\alpha$에서 발현된 AmyA의 활성은 $\Mg^{2+}$(20mM), $Ca^{2+}$ (30 mM) 존재 시 그 활성이 증가하였고, $Fe^{2+}$, $Cu^{2+}$ 존재 시 저해되었다. amyA 유전자의 internal primer를 사용하여 인공적으로 배양할 수 있는 49종의 반추세균에서 분리한 게놈 DNA을 주형으로 PCR분석한 결과 해당하는 벤드를 확인할 수 없었다. AmyA는 현재로 배양할 수 없는 반추 미생물에서 온 것으로 추정된다.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1863-1870
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• 2015

Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy-, Spec^S) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner.

• 한국미생물·생명공학회지
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• 제14권6호
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• pp.479-485
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• 1986

Bacillus amyloliquefaciens가 생성하는 액화형 $\alpha$-amylase의 구조유전자를 클로닝하기 위하여 이 균주 염색체의 2 - 5kb 획분을 분리하여 Mbo I으로 부분분해한 후 pUB110플라스미드의 BamHI 부위에 삽입하여 hybrid vector pSKS3 를 제작하였다. 이 재조합플라스미드를 세한수식결손 세포인 Bacillus subtilis ED 107에서 subcloning한 후 당화형 $\alpha$-amylase를 생성하는 Bacillus subtilis NA 64의 $\alpha$-amylase결실변이주 Bacills subtilis KM 107 주에서 형질 발현시켰다. 이때 형질전환빈도는 $10^{-5}$ - $10^{-7}$정도였으며 그중 약 50%가 $\alpha$-amylase 분비능이 있었으나 거의 대부분이 쉽게 실활되었다. 최종선별과정에서 $\alpha$-amylase를 가장 강하게 생성하는 형질전환주 Bacillus subtilis KM213으로부터 재 추출한 재조합플라스미드 pSKS3는 5.7kb 삽입된 단편이 BamH I/Mbo I 부위에 결합되어 있었다. pSKS3에 클로닝된 유전자에 의해 발현되는 $\alpha$-amylase 활성은 B. amyloliquefaciens 활성의 약 25%였으며, 이 pSKS3에 의해 발현되는 $\alpha$-amylase 단백은 B.amyloliquefaciens의 $\alpha$-amylase와 동일한 전기영동성을 나타내었음을 볼 때 pSKS3에 클로닝된 유전자는 B. amyloliquefaciens에서 유래함을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제5권5호
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• pp.257-263
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• 1995

As a first step for developing Lactobacillus strains capable of fermenting starch directly, the $\alpha$-amylase gene (amyL) from Bacillus licheniformis (Kim et al., 1988. Kor. J. Appl. Microbiol. Bioeng. 16: 369-373) was introduced into Lactobacillus casei strains and the level of $\alpha$-amylase expression in transformants was examined. 3 kb EcoRI fragments encompassing amyL were subcloned into the suitable lactococcal cloning vectors (pSA3, pMG36e, and p1L2530) and then recombinant plasmids were introduced into E. coli and L. casei strains by electroporation. Only one recombinant plasmid, $pIL2530\alpha$ was able to transform few L. casei strains tested at low efficiencies. The transformation efficiencies with the plasmid into L. casei YIT 9018 and L. casei A Tee 4646 were less than $10^2/\mu$ g pIL2530\alpha$. The level of amylase activities in L. casei was five to ten-fold lower than that in E. coli cells. $p1L2530\alpha$ was stably maintained in Lactobacillus strains in the presence of Em (5 $\mu $g/ml) but without antibiotic selection, it was unstable so more than 95$%$ of cells lost plasmids after a week of daily subculturing.

• International Journal of Industrial Entomology and Biomaterials
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• 제1권2호
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• pp.123-129
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• 2000

Expression of the crylAcl gene of an acrystalliferous Bacillus thuringiensis strain under the control of the native or $\alpha$-amylase gene promoter was investigated. The crylAcl gene was cloned in a B. thuringiensis - E. coli shutle vector, pHT3101, undder the control of either the native promoter (pProAc) or the $\alpha$-amylase promoter from Bacillus subtilis (pAmyAc). These two recombinant plasmids were successfully expressed in B. thuringiensis subsp. kurstaki Cry B. The first transformant (ProAc/CB), harboring pProAc, expressed an about 130 kDa protein begining 24 hr after inoculations just as in the case of the wild type of B. thuringiensis subsp. kurstaki HD-73. The second pAmyAc-transformant (AmyAc/CB) began to express the gene just 6 hr after inoculation, but Western analysis showed that the activity of the $\alpha$-amylase promoter was relatively weaker than that of the native promoter. As expected, their toxicity against Plutella xylostella larvae was dependent on the amount of Cry1Acl protein expressed.

• Preventive Nutrition and Food Science
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• 제13권3호
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• pp.190-195
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• 2008

The $\alpha$-amylase gene, amyL, from Bacillus licheniformis was expressed in Lactobacillus brevis 2.14 and Escherichia coli $DH5{\alpha}$ using two different shuttle vectors, pCW4 and pSJE. E. coli transformants (TFs) harboring either $pCW4T{\alpha}$ or $pSJET{\alpha}$ produced active $\alpha$-amylase but L. brevis TFs did not, as determined by enzyme assays and zymography. But amyL transcripts were synthesized in L. brevis TFs. In terms of plasmid stability, pSJE, a theta-type replicon, was more stable than pCW4, an RCR (rolling circle replication) plasmid, in L. brevis without antibiotic selection.

• Asian-Australasian Journal of Animal Sciences
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• 제9권4호
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• pp.405-410
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• 1996

Phylogenetic analyses were carried out using four Indonesian native chicken breeds; Kampung, Bangkok, Pelung and Kedu. Gene frequencies of four blood group (A, B, D and E) and eight electrophoretic loci (akp, Akp-2, Es-1, Amy-1, Alb, Tf, Pas and Pa-1) were examined. Geographical and breed specific trends in the gene frequencies were not found in the local population of Kampung breed or in four native breeds. The values of average heterozygosity were estimated as 0.35-0.45. Genetic distances among the local populations of Kampung breed and other native breeds were comparatively small. In a cluster analysis, the Bangkok breed and Kampung E population showed distance from another cluster. The coefficient of gene differentiation for local populations of Kampung breed was estimated as 0.099.

• 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.