• International Journal of Industrial Entomology and Biomaterials
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• 제25권1호
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• pp.99-110
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• 2012

Comparison on the genomic structure and phylogenetic relationship of the Hsp88 genes from P. tenuipes Jochoen-1, P. tenuipes, C. militaris and C. pruinosa was described. The Hsp88 genes from the three entomopathogenic strains, P. tenuipes Jocheon-1(strain), P. tenuipes(original species), and C. militaris contain the identical genomic structure, namely 5 introns and 6 exons with the length of 13, 62, 32, 1,438, 306, 288 nucleotides encoding 713 amino acid residues, whereas in case of C. pruinosa, it contains 4 introns and 5 exons with the length of 13, 62, 32, 1,744, 288 nucleotides encoding 713 amino acid residues. The genomic DNA length of the Hsp88 genes from P. tenuipes Jocheon-1 and P. tenuipes are both 2,600 nucleotides long in size. The Hsp88 genes from C. militaris and C. pruinosa are 2,582, 2,576 nucleotides long in size, respectively. Hsp88 genes of the P. tenuipes Jochoen-1, P. tenuipes, C. militaris and C. pruinosa also contain the conserved ATP-binding domain. Phylogenetic analysis of the Hsp genes of the four strains tested in this study showed that the fungal Hsp88 is divided into two separate clades, ascomycetes and deutromycete. Within the ascomycetes fungal clade, the P. tenuipes Jochoen-1 and P. tenuipes formed a subgroup, on the other hand, C. militaris and C. pruinosa formed another subgroup. Pair-wise comparison of P. tenuipes Jocheon-1 Hsp88 with those of P. tenuipes, C. militaris and C. pruinosa Hsp88s revealed significant identity in deduced amino acid sequence among these strains. The P. tenuipes Jocheon-1 Hsp88 showed 99% identity with the P. tenuipes, 97% identity with the C. militaris, and 98% identity with the C. pruinosa.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.387-397
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• 2023

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

• 생명과학회지
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• 제28권4호
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• pp.408-414
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• 2018

Actein은 널리 알려진 승마 추출물의 주요 생리 활성 효능 성분이다. 본 연구에서는 acetylcholine 수용체의 활성을 억제하는 것으로 활용된 AchBP 단백질 길항제(antagonist) tubocurarine과 승마 추출물의 효능 성분 actein 및 actein 유도체(27-deoxyactin, (26S)-actein, (26R)-actein)들의 AchBP 단백질 B와 C domain 활성 부위에 대한 친화도 분석 실험을 컴퓨터 분자결합 분석 방법을 통해 비교하였다. AchBP 단백질 B와 C domain의 3차원 구조정보는 PDB database (PDB ID: 2XYT)를 활용하였다. In silico 결합 분석을 수행하기 위해 PyRx, Autodock Vina, Discovery Studio Version 4.5, and NX-QuickPharm 프로그램을 각 분석 조건에 따라 활용하였다. AchBP 단백질 B와 C domain 활성 부위에 대한 actein의 최대 결합친화도는 -10.50 kcal/mol으로 나왔으며 이는 -9.80 kcal/mol으로 분석된 tubocurarine의 결합 친화도 보다 훨씬 더 높고 효율적인 것으로 분석되었다. Tubocurarine에 비하여 결합친화도 값이 높게 분석된actein, 27-deoxyactein, (26R)-actein 유도체 성분들과 상호작용 하는 AchBP 단백질 활성 부위의 아미노산들 가운데 tryptophan 84와 tyrosine 147이 높은 결합친화도를 형성하는데 매우 중요한 역할을 하는 아미노산으로 예상이 되었다. Tubocurarine의 AchBP 단백질 활성 부위에 대한 X,Y,Z Grid 값은 X=38.300689, Y=112.053467, Z=51.991022으로 나왔으나 actein과 actein 유도체들은 대부분 X=26.4, Y=127.3, Z=43.7 값 주변에 centroid grid를 형성하였다. 즉, tubocurarine이 결합하는 부위와는 다른 부위에 결합하여 AchBP의 활성에 영향을 주는 것으로 사료되었다. 이상의 연구 결과들을 분석해 볼 때, 아세틸콜린 수용체 길항제 tubocurarine보다 승마 추출물 생리 활성 물질인 actein과 그 유도체들이 보다 더 효율적인 아세틸콜린 수용체 길항제로 작용할 수 있음을 확인하였다. 결론적으로 승마 추출물 또는 actein 성분은 피부 주름 개선 효능을 지닌 보톡스를 대체하거나 또는 주름 개선용 화장품 신물질 연구 개발 분야에 효율적으로 활용할 수 있을 것으로 사료된다.

• 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.

• The Plant Pathology Journal
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• 제34권2호
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• pp.150-156
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• 2018

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass (Zostera marina) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae. They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses.

• Parasites, Hosts and Diseases
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• 제57권1호
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• pp.55-60
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• 2019

This study was undertaken to determine the complete mitochondrial DNA sequence and structure of the mitochondrial genome of Spirometra ranarum, and to compare it with those of S. erinaceieuropaei and S. decipiens. The aim of this study was to provide information of the species level taxonomy of Spirometra spp. using the mitochondrial genomes of 3 Spirometra tapeworms. The S. ranarum isolate originated from Myanmar. The mitochondrial genome sequence of S. ranarum was compared with that of S. erinaceieuropaei (GenBank no. KJ599680) and S. decipiens (GenBank no. KJ599679). The complete mtDNA sequence of S. ranarum comprised 13,644 bp. The S. ranarum mt genome contained 36 genes comprising 12 protein-coding genes, 22 tRNAs and 2 rRNAs. The mt genome lacked the atp8 gene, as found for other cestodes. All genes in the S. ranarum mitochondrial genome are transcribed in the same direction and arranged in the same relative position with respect to gene loci as found for S. erinaceieuropaei and S. decipiens mt genomes. The overall nucleotide sequence divergence of 12 protein-coding genes between S. ranarum and S. decipiens differed by 1.5%, and 100% sequence similarity was found in the cox2 and nad6 genes, while the DNA sequence divergence of the cox1, nad1, and nad4 genes of S. ranarum and S. decipiens was 2.2%, 2.1%, and 2.6%, respectively.

• 한국해양생명과학회지
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• 제7권2호
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• pp.163-170
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• 2022

본 연구에서는 해상가두리 양식장에서 북방전복과 둥근전복속 교잡종(왕전복♀^*둥근전복♂)의 생물지표를 분석하여 교잡육종의 결과를 평가하고자 하였다. 생존율은 북방전복과 유사하였으나, 성장(각장)은 교잡종에서 약 10% 빠른 것으로 분석되었다. 패각의 호흡공 기형율은 교잡종이 북방전복보다 약 6% 낮았으며, 패각 함몰 및 부식율은 교잡종이 약 15% 낮았다. 생화학적 조성에서는 조단백질의 경우 교잡종에서 약 3.1% 높았으며, 이를 제외한 나머지에서 유사한 값을 나타냈다. 소화, 흡수 및 해독기능을 수행하는 간췌장의 조직학적 평가에서는 교잡종에서 좋은 결과를 보였다. 이러한 결과로 보아 둥근전복속 교잡종은 추후 양식 환경에 접목하였을 때 높은 양식 생산력을 가질 것으로 판단된다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.512.1-512
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• 1986

The cdd gene of Bacillus subtilis, encoding the deoxycytidinecytidine deaminase of pyrimidine nucleotide biosynthesis has been cloned into the EcoRl site of pBR322. The recombinant plasmid, pSol, promoted the synthesis of 100-140 fold elevated levels of the enzyme. A comparison of the polypeptides encoded by cdd complementing and non-complementing plasmids in the mini cell showed the gene product to have a molecular mass of approximately 14 kDa. The nucleotide sequence of the gene and 460 base pairs upstream from the coding region was determined. An open-reading frame, encoding a protein with a calculated molecular mass of 14337 Da, was deduced to be the coding region for cdd. However, the enzyme has an apparent molecular mass of 54 kDa as determined by gel filteration, whereas sucrose density gradient centrifugation shows 58 kDa. It means that the enzyme could be forming a tetramer in a physiological state. About 28 amino acids of the N-tetramer in a physiological state. About 28 amino acids of the N-terminal presumably form a signal for membrane translocation and six cystein residues are contained in the structure. S1 nuclease mapping indicated that transcription of cdd is initiated 17 base pairs upstream from the translational start. The structural characterization of the odd gene was performed.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.321-329
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• 2005

Ornithine decarboxylase (ODC) antizyme is a key regulatory protein in the control of cellular polyamines. We have isolated two distinct ODC antizyme cDNA clones (AZS and AZL) from a flounder (Paralichthys olivaceus) brain cDNA library. Their sequences revealed that both clones required translational frameshifting for expression. Taking + 1 frameshifting into account, AZS and AZL products were 221 and 218 amino acid residues long, respectively, and shared $83.3\%$ amino acid sequence identity. Comparison of the structure and nucleotide sequence of the antizyme genes showed that the genes were highly conserved in flounder, zebrafish, mouse, and human. A phylogenetic tree was constructed, based on the antizyme amino acid sequences from various species. The presence of the two types of antizyme mRNA species in brain, kidney, liver, and embryo was confirmed by using the reverse transcription­polymerase chain reaction (RT-PCR) and Northern blot analysis. Recombinant proteins of flounder ODC antizymes, containing His-Nus-S tag at the amino-terminus, were overexpressed as His-AZL and His-AZS fusion proteins in Escherichia coli BL21 (DE3) pLys by using the pET­44a(+) expression vector.

• Archives of Pharmacal Research
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• 제18권5호
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• pp.308-313
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• 1995

This study has been undertaken to examine the acetylcholinesterase (AChE) of electric organ from korean electtric ray(Narke japonica). Korean electric ray was caughted at Chungmu sea and transported to the laboratory, where electric organs were removed and stored at $-70^{\circ}C$ until used. Acelycholinesterase(AChE) of electric organ was purified by affinity column that was prepared with dicaproyl-methylpyridinium linked to Sepharose 4B. Upon purification, the specific activities in Ellman unit were increased by 52 and 39 times for high salt soluble AChE (HSSE, 870.86 $\DeltaOD/min/geam$ of tissue) and detergent soluble AChE(DSE, 105.42 .$\DeltaOD/min/geam$ of tissue), respectively. Each subunit of AChE separated by SDS polyacrylamide gel electrophoresis(SDS-PAGE)was transferred to immonilon P by western boltting and detected by mAbs raised against each subunit of AChE from electric organ og Torpedo califomica. Collagenic tails of AChE from Torpedo califomica, likewise 103Kd protein of AChE from Narke japonica was detected by monoclonal antibody specific to 103Kd of AChE from Torpedo califomica. However, molar ratio of three subunits of AChE from Narke japonica is different from that of Torpedo calicormica. Furthermore, catalytic subunit of AChE from Narke japonica was not identified by monoclnal antibody specific to catalytic subunit of AChE from Torpedo californica. These results showed differences in molecular structure of AChE from Narke japonica and AChE from Torpedo califormica eventhough they showed same enzymatic activities.