• 한국미생물·생명공학회지
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• 제17권2호
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• pp.160-164
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• 1989

알카리성 Bacillus sp. AL-8의 알카리성 amylase 유전자를 포함하는 5.7Kb의 EcoRI 단편을 pUB 110을 vector로 하여 amylase를 생산하지 못하는 B. subtilis sta-1에서 발현시켰다. 재조합 plasmid pMB802와 pMB809는 숙주세포인 B. subtilis에서 매우 안정하게 유지되었으며 amylase 생산이 공여균 주에서 보다 1.8배 증가하였다. 형질전환주에서 생산된 amylase는 공여균주와 같은 효소적 성질을 나타내었다. 5.7Kb 단편을 E. coli에 subcloning한 결과 3.7Kb의 EcoRI 단편에 알카리성 amylase 유전자가 존재하였다.

• 한국미생물·생명공학회지
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• 제15권6호
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• pp.441-445
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• 1987

알카리성 amylase를 내는 Bacillus sp. AL-8의 알카리성 amylase 유전자를 amylase를 생성하지 않는 Escherichia coli HB101에 pBR322를 vector로 하여 형질전환하였다. E. coli도 알카리성 amylase를 생성하여 5$0^{\circ}C$에서 최적 활성온도를 가지며 5$0^{\circ}C$까지 열안정성을 갖고, pH10에서 최적 활성 pH를 나타내는 동시에 pH8-10에서 안정하였다. 알카리성 amylase 유전자가 E. coli에 형질전환되어 donor 세포와 같은 효소 성질을 갖고 있으며 pBR322로 삽입된 유전자는 pJW8에서 5.8kb이며 pJW200에서는 3.0kb로 E. coli에서 안정하게 발현되었다.

• 미생물학회지
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• 제31권2호
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• pp.175-178
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• 1993

Alkaline .alpha.-amylase expressed in the transformant, Baciollus subtills MB809, containing alkaline amylase gene cloned from alkalophilic Bacillus sp. AL-8, was purified through for step separation processes. The purified alkaline .alpha.-amylase had molecular weight of app[roximately 59, 000 daltons on SDS-PAGE and Sephaex G-100 gel filtration. Amino acid sequence of terminal portion of the enzyme was analyzed with pure amylase eluted form the SDS-PAGE gel. N-terminal amino acid sequence of .alpha.-amylase was determined by the Edman degradation method and resulted in $NH_{2}$-ser-thr-ala-pro-ser-(ile)-lys-ala-gly-thr-(ile)-leu. For C-terminal amino acid sequencing, purified .alpha.-amylase was digested with carboxypuptidase A and B, and reverse-phase HPLC gradient elution system resulted in -thr-trp-pro-lys-COOH.

• Journal of Microbiology and Biotechnology
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• 제2권3호
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• pp.166-173
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• 1992

The expression of Bacillus subtilis $\alpha$-amylase from the phoA-amyE fusion gene in recombinant E. coli was investigated under various environmental conditions. The overexpression of cloned $\alpha$-amylase caused retardations in cell growth and synthesis of alkaline phosphatase (AP) from the chromosomal phoA gene. The change of culture temperature from $37^\circ{C}$ to $30^\circ{C}$ increased the specific activities of both $\alpha$-amylase and $\beta$-lactamase by six and two times, respectively, whereas the AP activity remained unchanged. The experiments with chlorampenicol (a translation inhibitor) suggested the enhancement of $\alpha$-amylase activity at $30^\circ{C}$, and this was partly due to the stability of $\alpha$-amylase itself. The further decrease of the temperature to $25^\circ{C}$ slowed down both the cell growth and cloned-gene expression rate. The $\alpha$-amylase activity showed a maximum at pH of 7.4 while alkaline phosphatase was most effectively produced at pH of 8.3.

• 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.

• 한국미생물·생명공학회지
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• 제14권3호
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• pp.239-244
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• 1986

새로운 알카리내성 숙주균주를 개발하기 위하여 토양으로부터 알카리조건하에서 생육하는 미생물을 분리하고 이들 중에서 amylase활성과 Protease활성 및 항균력을 동시에 가지며 plasmid pUB 110이 형질전환 되어 이 plasmid가 안정하게 유지되는 균주를 선정하여 알카리내성 Bacillus sp. YA-14로 동정하였다. 분리균주의 amylase와 protease는 각각 pH 8.0과 pH 7.5에서 가장 활성이 높았으며 피검균으로 사용한 B. subtilis와 Sarcina lutea에 항균력을 나타내었다. 분리균주의 형질전환에는 0.4% MgSO$_4$ 가 함유된 modified SPI배지 (MSPI, pH8.0)를 사용하였으며 말기대수증식기의 균체가 적당하였다. Transformant는 20세대 계대배양 후에도 pUB,110plasmid가 안정하게 유지되어 형질발현되었다. 이 결과로부터 분리균주는 Bacillus속의 host-vector계에서 새로운 알카리내성 숙주균주로서 이용할 수 있는 가능성을 보여주었다.

• 미생물학회지
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• 제18권2호
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• pp.51-58
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• 1980

Mutational experiments were performed to improved to improve the protease productivity of Aspergillus flavus KU 153, which is selected among the wild strains. A UV-induced mutant strain having high protease productivity was obtained by the use of the clear zone method as a simple criterion for a primary screening test. Neutral and alkaline protease activities of hte mutant strain were higher than 1.8 times, comopared with those of the parental strain, respectively, while in the case of acid protease, it was 2.7 times. The mutant strain selected was more powerful in the production of cellulase and amylase, as well s protease in wheat bran, compared with those of the parental strain. protease production of the parental strain has reached maximum level at 3 days culture, while alkaline nad neutral protease production of the mutantstrain has reached at 2 days culture. On the other hand, the mutant strain formed the spore slowly, compared with the parental strain. Column chromatography of the neutral protease on DEAE-Sephadex A-50 showed that the mutant strain was not induced the formation of another neutral protease isozyme, but induced the variation in the function of regulatory gene.

• 한국잠사곤충학회지
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• 제39권2호
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• pp.119-133
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• 1997

Isozyme was used to characterize general protein patterns of genetic relationships among 303 silkworm stocks preserved in National Sericultural and Entomology Research Institute, RDA. Six isozymes (esterase, acid phosphatase, alkaline phosphatase, amylase, glucose-6-phosphate dehydrogenase and sucrase) from hemolymph, midgut, and digestive juice were employed to construct dendograms(UPGMA method) using a polycrylamide gel electrophoresis. A cluster analysis revealed four major group, which were divided into several subgroups within each group, contained assemglages of Japanese and Chinese races. Especially, genetic differentiation in the first and second group was greatest rather than within Japanese and Chinese races repectively and was concordant with the hypothesis of phyletic sorting of initial variability in China many years ago. Hypothesized recent introgression between groups was also plausible, but the eviednce suggested bidirectional gene flow between the Chinese and the Japnaese lineages. Interpreting the results in light of evidence from the current study, the genetic diversity and relationship showed in Korean silkworm race, Hansammyun reflected early and independent evolution from the Chinese ancestor, limited addition of new variability and phyletic sorting within Korean peninsula more than 4,000 years.

• 한국가금학회지
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• 제23권3호
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• pp.135-144
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• 1996

This study was carried out to clarify the genetic constitution of biochemical polymorphic loci controlling blood protein and enzymes as genetic rnarkers in Korean native chicken(KNG) population Blood samples were collected from 230 KNG representing three colored-lines(reddish-, yellowish- and blackish- brown) raised in Daejeon branch of National Livestock Research Institute. Eight blood marker loci, transferrin(Tf), post-albumin(Pas), albumin(Alb), amylase-1(Arny-1), es-terase-1(Es-1), alkaline phosphatase(Akp), catalase(Cat) and hemoglobin(Hh) were analyzed by using starch, agarose and polyacrylamide gel electrophoresis. Based on the gene frequencies of polymorphic marker loci, the genetic characteristics of KNF population was analyzed, and the genetic ariability within population was quantified. The genetic relationships between KNC and other native fowls or improved breeds were also estimated. The gene frequencies of Tf, Pas and AIb loci were similar to those of improved breeds among the seven biochemical polymorphic loci, while gene frequencies of Cat and Es-i loci were remarkably different between KNC and improved breeds. Gene frequencies of amy-i and Akp loci were similar to those of New Hampshire and Rhode Island Red and White Leghorn, respectively. However in comparison with other improved breeds, great differences were observed in gene frequencies of these loci The average heterozygosity, effective number of alleles and homogeneity index for the seven loci combined were estimated to be .334, 1.639 and .373, respectively. Based on the dendrogram and genetic distances, the KNC was genetically closer to New Hampshire, Plymouth Rock and Rhode Island Red breeds than to the White Leghorn breed.

• 한국식품과학회지
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• 제48권4호
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• pp.341-346
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• 2016

HPA는 식품으로 섭취되는 녹말을 분해하는데 있어서 매우 중요한 역할을 수행하는 효소이기 때문에 HPA 효소 활성의 억제는 당뇨와 비만과 같은 질환의 예방과 치료에 있어서 의미를 가진다. 따라서 HPA는 당뇨병 치료와 비만 예방을 위한 새로운 식 의약품 소재 개발을 위한 주요 타깃 효소 중 하나이며, 새로운 소재의 개발을 위해서는 HPA의 반응 메커니즘을 비롯하여 천연 기질 분해 특성에 대한 이해가 반드시 필요하다. 본 연구에서는 HPA의 동질효소 중 연구가 거의 진행되지 않은 HPA II에 대한 효소 특성화를 진행하고자 P. pastoris 시스템을 이용하여 재조합 HPA II를 생산하였으며, 녹말 분해와 관련된 효소적 특성을 분석하였다. HPA II는 10 mM NaCl까지 농도 의존적으로 효소활성이 증가하였으며, 최적 활성을 위한 pH는 0 mM NaCl 조건에서 pH 6.5이었으나 10 mM NaCl조건에서 pH 7.5로 이동하는 특성을 보였으며, 이는 HPA I을 포함하는 염소이온 의존형 아밀레이스가 나타내는 전형적인 특징이다. 염소이온 존재 시 최적 pH가 염기성 pH 영역으로 이동하는 것은 염소 이온과 효소의 결합에 의해 HPA II의 산/염기 촉매 잔기의 $pK_a$값이 커진다는 것을 의미하며, 염소이온을 첨가하였을 때 녹말에 대한 가수분해 활성의 증대 정도가 산성 pH 영역보다 염기성 pH 영역에서 두드러지게 나타났다는 점이 이를 뒷받침하였다. 반응속도론적 분석 결과에 따르면 염소이온 존재 시 효소활성의 증대는 대부분 전환수(turnover number)의 증대에 의한 것으로 나타났으며, 가용성 녹말 보다 곡류 녹말에 대한 전환수의 증대가 크게 나타났다. 염소이온은 활성의 증대뿐만 아니라 고농도의 기질 조건에서 기질에 의한 효소 활성 억제 양상을 심화시키는 것으로 나타났다. 결론적으로 HPA II의 특징은 HPA I과 거의 유사한 경향을 나타내었으며, 염소이온 첨가여부에 따른 HPA II의 가수분해활성 결과를 바탕으로 향 후 HPA에 대한 곡류 녹말 가수분해 활성 억제제 개발을 위한 연구를 추진할 계획이다.