• Biotechnology and Bioprocess Engineering:BBE
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• 제7권3호
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• pp.121-129
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• 2002

Existing methods for optimization of sequences by random mutagenesis generate libraries with a small number of mostly deleterious mutations, resulting in libraries containing a large fraction of non-functional clones that explore only a small part of sequence space. Large numbers of clones need to be screened to find the rare mutants with improvements. Library display formats are useful to screen very large libraries but impose screening limitations that limit the value of this approach for most commercial applications. By contrast, in both classical breeding and in DNA shuffling, natural diversity is permutated by homologous recombination, generating libraries of very high quality, from which improved clones can be identified with a small number of complex screens. Given that this small number of screens can be performed under the conditions of actual use of the product, commercially relevant improvements can be reliably obtained.

• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.2004-2007
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• 2006

An efficient method for the in vitro reassembly of homologous DNA sequences is presented. The proposed method involves obtaining single strands of homologous genes and hybridizing them to obtain partially hybridized heteroduplex DNA; cleaving the single-stranded regions of the heteroduplex DNA using S1 nuclease to generate double-strand DNA fragments; denaturing the double-strand DNA fragments to generate single-strand DNA fragments; conducting a series of polymerase chain reactions (PCR) using the single-strand DNA fragments as internal primers and a mixture of homologous DNA as templates to obtain elongated reassembled DNA; and finally, amplifying the reassembled DNA by a PCR using terminal primers. As a result, DNA reassembly could be achieved between homologous genes with a sequence similarity as low as 78%.

• 미생물학회지
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• 제39권2호
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• pp.76-82
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• 2003

Alcaligenes faecalis T1의 Poly(3-hydroxybutyrate)(PHB) depolymerase활성 증진을 위해 DNA shuffling방법을 이용하였다. 제조된 A. faecalis T1의 PHB depolymerase 돌연변이 유전자의 library를 Pseudomonas syringae의 icenucleation protein유전자를 포함하는 발현벡터 pJHCll에 클로닝하여 약 7,000개의 형질전환체를 얻었다. 탄소원으로 PHB또는 poly(3-hydroxybutyrate-co-3-hydroxyvalerate)를 포함하는 M9최소배지를 이용하여 형질전환체들로부터 활성이 서로 다른 돌연변이주들을 선별하였다. 이들의 PHB depolymease 활성은 평판배지에서의 halo형성 및 배양 상등액을 이용한 탁도 감소 실험으로 확인하였으며,형질전환체들 중에서 shuffling전의 대조군에 비하여 사용된 기질에 따라 효소활성이 1.8-3.2배 증진된 II-4 돌연변이주를 얻었다. DNA 염기서열의 분석을 통하여 II-4의 PHB depolymease에는 3개의 아미노산 치환(A1a209Va1, Leu258Phe, Asp263Thr)이 이루어졌음을 확인하였다. 여러 가지 돌연변이주의 아미노산 서열의 변화를 분석한 결과, PHB depolymerase의 catalytictriad주위에 기존 아미노산에 비하여 보다 소수성인 아미노산으로의 치환이 소수성 기질인 PHB에 대한분해 활성 중진에 기여하는 것으로 추정되었다.

• Journal of Microbiology and Biotechnology
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• 제32권6호
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• pp.800-807
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• 2022

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

• BMB Reports
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• 제40권3호
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• pp.419-425
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• 2007

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli $\beta$-galactosidase and $\beta$-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more $\beta$-glucuronidase activity than wild-type $\beta$-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high $\beta$-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1536-1541
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• 2009

The DNA shuffling technique has been used to generate libraries of evolved enzymes in thermostability. We have shuffled two thermostable cytidine deaminases (CDAs) from Bacillus caldolyticus DSM405 (T53) and B. stearothermophilus IFO12550 (T101). The shuffled CDA library (SH1067 and SH1077 from the first round and SH2426 and SH2429 from the second round) showed various patterns in thermostability. The CDAs of SH1067 and SH1077 were more thermostable than that of T53. SH2426 showed 150% increased halftime than that of T53 at $70^{\circ}C$. The CDA of SH2429 showed about 200% decreased thermostability than that of T53 at $70^{\circ}C$. A single amino acid residue replacement that presented between SH1077 and SH2429 contributed to dramatic changes in specific activity and thermostability. On SDS-PAGE, the purified CDA of SH1077 tetramerized, whereas that of SH2429 denatured and became almost monomeric at $80^{\circ}C$. A simulated three-dimensional structure for the mutant CDA was used to interpret the mutational effect.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.153-157
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• 2004

Tyrosine phenol-lyase (TPL) is a useful enzyme for the synthesis of pharmaceutical aromatic amino acids. In the current study, sequential DNA shuffling and screening were used to enhance the stability of TPL. Twenty-thousand mutants were screened, and several improved variants were isolated. One variant named A13V, in which the $13^{th}$ amino acid alanine was substituted by valine, exhibited a higher temperature and denaturant stability than the wild-type TPL. The purified mutant TPL, A13V, retained about 60% of its activity at $76^\circ{C}$, whereas the activity of the wild-type TPL decreased to less than 20% at the same temperature. Plus, A13V exhibited about 50% activity with 3 M urea, while the wild-type TPL lost almost all its catalytic activity, indicating an increased denaturant tolerance in the mutant A13V. It is speculated that the substitution of Val for the Ala in the $\beta$-strand of the N-terminal arm was responsible for the heightened stabilization, and that the current results will contribute to further research on the structural stability of TPL.

• 미생물학회지
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• 제46권4호
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• pp.383-388
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• 2010

알칼리성 단백질 분해효소 고생산 돌연변이 균주 Vibrio metschnikovii L12-23, N4-8, KS1으로부터 알칼리성 단백질 분해효소를 암호화하는 vapK (Vibrio alkaline protease K) 유전자들을 PCR에 의하여 분리한 다음 DNA shuffling, error-prone PCR 방법과 같은 분자진화 기술을 통해 고활성 단백질 분해효소를 생산하는 재조합 V. metschnikovii 균주를 제작하였다. DNA shuffling 방법을 통해 변형시킨 vapK-1 유전자와 이 유전자를 주형으로 error-prone PCR 기법을 통해 재 변형된 vapK-2 유전자를 cloning한 후 V. metschnikovii KS1 균주에 역도입하여 재조합 균주를 제조하였다. 재조합 균주들의 단백질 분해 능력을 조사한 결과 vapK-2 유전자가 2 copy 도입된 재조합 균주의 경우 야생형 균주인 V. metschnikovii RH530에 비해 43.6배 높은 단백질 분해활성을 보였으며 숙주인 V. metschnikovii KS1에 비해 약 3.9배 향상된 단백질 분해 활성을 확인할 수 있었다. 변형된 vapK-1과 vapK-2 유전자를 야생형 vapK 유전자의 염기서열을 비교 분석한 결과 단백질 분해 능력의 활성에 영향을 미치는 active site를 제외한 부분에서 변화가 일어났음을 확인 할 수 있었다. 변형된 유전자 vapK-1을 two copy를 포함한 재조합 플라스미드를 가진 V. metschnikovii KS1을 30 L fermentor로 배양 하였을 때 배양 후 35 시간에 18,000 PU/ml의 활성을 보였으며, 이는 향후 산업용 균주로서 사용될 수 있는 가능성을 제시하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.52-52
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• 2002

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.47-57
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• 2001