• Journal of Microbiology and Biotechnology
• /
• 제30권2호
• /
• pp.216-225
• /
• 2020

An esterase gene, estA1, was cloned from Alteromonas sp. 39-G1 isolated from the Beaufort Sea. The gene is composed of 1,140 nucleotides and codes for a 41,190 Da protein containing 379 amino acids. As a result of a BLAST search, the protein sequence of esterase EstA1 was found to be identical to Alteromonas sp. esterase (GenBank: PHS53692). As far as we know, no research on this enzyme has yet been conducted. Phylogenetic analysis showed that esterase EstA1 was a member of the bacterial lipolytic enzyme family IV (hormone sensitive lipases). Two deletion mutants (Δ20 and Δ54) of the esterase EstA1 were produced in Escherichia coli BL21 (DE3) cells with part of the N-terminal of the protein removed and His-tag attached to the C-terminal. These enzymes exhibited the highest activity toward p-nitrophenyl (pNP) acetate (C₂) and had little or no activity towards pNP-esters with acyl chains longer than C₆. Their optimum temperature and pH of the catalytic activity were 45℃ and pH 8.0, respectively. As the NaCl concentration increased, their enzyme activities continued to increase and the highest enzyme activities were measured in 5 M NaCl. These enzymes were found to be stable for up to 8 h in the concentration of 3-5 M NaCl. Moreover, they have been found to be stable for various metal ions, detergents and organic solvents. These salt-tolerant and chemical-resistant properties suggest that the enzyme esterase EstA1 is both academically and industrially useful.

• KSBB Journal
• /
• 제24권3호
• /
• pp.312-320
• /
• 2009

치료용 단백질을 생산하는 생물의약품 산업이나 세포치료제 및 이식용 세포를 다루는 재생의학 분야 등의 세포기반 산업에서 안정적인 세포의 보존은 필수적인 요소이다. 본 연구에서는 인간 피부 섬 유아세포의 $4^{\circ}C$ 저온보존에서 우수한 성능을 나타내는 개선된 저온보존액을 개발하고, 저온에 의한 세포 손상을 보호함으로써 보다 안정적인 세포 저온보존 기술을 제공하고자 하였다. 세포의 저온보존에서 우수한 효능을 나타내는 핵심 성분을 탐색한 결과, yeast hydrolysate 등의 단백질 가수분해물을 첨가한 보존액에서 월등히 뛰어난 보존효과가 나타남을 확인하였다. 단백질 가수분해 물은 미생물, 식물, 동물유래 단백질 가수분해 물에서 모두 우수한 효과를 나타냈으며, 특히 단백질 가수분해물 성분 중 분자량 10kDa 이하의 펩타이드를 첨가한 저온보존에서 우수한 보존효과가 나타났다. 저온에 의한 세포손상에 대해 단백질 가수분해물은 세포내 ATP level의 감소를 막아주고 ROS 생성을 억제하는 것으로 나타났으며, 항산화제 및 삼투압 조절물질을 단백질 가수분해 물과 함께 첨가하였을 때 더욱 우수한 세포 보존효과를 보였다. 최종적으로 본 연구에서 개발한 KUL261 저은보존액 (DMEM/F12 1 : 1 medium, yeastolate 1%, $\alpha$-tocopherol $100{\mu}M$, dextran 2.5%)은 기존의 저온 보존액에 비해 세포 생존을 및 성장률에서 월등히 우수한 성능을 나타내었다. 결론적으로, 핵심 유효성분으로 단백질 가수분해물을 포함하는 개선된 저온보존액은 기존의 보존액보다 월등히 우수한 보존효과를 제공하며, 세포치료제 및 재생의학 분야의 발전과 글로벌 상업화에 기여할 수 있을 것이다.

• The Plant Pathology Journal
• /
• 제25권1호
• /
• pp.47-53
• /
• 2009

Sequence analysis of the Chlorella virus SS-2 revealed one putative nuclease gene that is 807 bp long and encodes a 31kDa protein. Multiple sequence alignment analysis reveals the presence of highly conserved PD-(D/E)XK residues in the encoded protein. The gene cloned into an expression vector was expressed as a His-tagged fusion protein in chaperone containing pKJE7 cells. The recombinant protein was purified using a His-Trap chelating HP column and used for functional analysis. Exonuclease activity of the SS-2 nuclease was detected when the DNA substrates, such as linear ssDNA, PCR amplicon, linear dsDNA with 5'-overhang ends, 3'-overhang ends, or blunt ends were used. Covalently closed circular DNA was also degraded by the SS-2 recombinant protein, suggesting that the SS-2 nuclease has an endonuclease activity. Stable activity of SS-2 nuclease was observed between $10^{\circ}C$ and $50^{\circ}C$. The optimum pH concentrations for the SS-2 nuclease were pH 6.0-8.5. Divalent ions inhibited the SS-2 nuclease activity.

• Preventive Nutrition and Food Science
• /
• 제10권1호
• /
• pp.40-45
• /
• 2005

Age-related changes in bone metabolism are well established by biochemical markers of bone matrix in serum and urine, but analysis of the residual bone matrix, which is still turning over, has not been investigated. In the present study, we measured in vivo rates of bone protein synthesis using a precursor-product method based on the exchange of ²H from ²H₂O into amino acids. Four percent ²H₂O was administered to mice in drinking water after intraperitonial (i.p) bolus injection of 99.9% ²H₂O. Mice were divided into the two groups: growing young mice were administered 4% ²H₂O for 12 weeks after an i.p bolus injection at 5 week of age, whereas weight stable adult mice started drinking 4% ²H₂O 8 weeks later than the growing group and continued 4% ²H₂O drinking for 8 weeks. Mass isotopomer abundance in alanine from bone protein was analyzed by gas chromatography/mass spectrometry. Body ²H₂O enrichments were in the range of 1.88-2.41% over the labeling period. The fractional synthesis rates (ks) of bone protein were 2.000±0.071%/d for growing mice and 0.243±0.014%/d for adult mice. These results demonstrate that the bone protein synthesis rate decreases with age and present direct evidence of age-related changes in bone protein synthesis.

• Genomics & Informatics
• /
• 제21권3호
• /
• pp.41.1-41.11
• /
• 2023

The Dengue virus M protein is a 75 amino acid polypeptide with two helical transmembranes (TM). The TM domain oligomerizes to form an ion channel, facilitating viral release from the host cells. The M protein has a critical role in the virus entry and life cycle, making it a potent drug target. The oligomerization of the monomeric protein was studied using ab initio modeling and molecular dynamics simulation in an implicit membrane environment. The representative structures obtained showed pentamer as the most stable oligomeric state, resembling an ion channel. Glutamic acid, threonine, serine, tryptophan, alanine, isoleucine form the pore-lining residues of the pentameric channel, conferring an overall negative charge to the channel with approximate length of 51.9 Å. Residue interaction analysis for M protein shows that Ala94, Leu95, Ser112, Glu124, and Phe155 are the central hub residues representing the physicochemical interactions between domains. The virtual screening with 165 different ion channel inhibitors from the ion channel library shows monovalent ion channel blockers, namely lumacaftor, glipizide, gliquidone, glisoxepide, and azelnidipine to be the inhibitors with high docking scores. Understanding the three-dimensional structure of M protein will help design therapeutics and vaccines for Dengue infection.

• 한국미생물·생명공학회지
• /
• 제1권2호
• /
• pp.99-104
• /
• 1973

A study on the active center of the acid protease from Penicillium sp. was conducted, and also the milk clotting activity of acid prorease was measured. 1. PCMB failed to influence the proteolytic activity of acid protease, indicating that a reactive sulfhydryl group is not required for the enzymatic activity. 2. $\varepsilon$-amino caproic acid did not show any inhibitory effect on tile proteolytic activity of acid protease. 3. Also 2, 4-dinitro phenol did not show any inhibitory effect on the enzyme activity. 4. Acid protease from Penicillium sp. showed a strong milk clotting activity in the presence of Ca ion. 5. This enzyme had a strong proteolytic activity on various substrate, such as casein, denatured hemoglobin, ovalbumin, denatured bovine muscle protein, denatured percine muscle protein and denatured chicken muscle protein.

• Genomics & Informatics
• /
• 제21권1호
• /
• pp.7.1-7.11
• /
• 2023

The gram-positive bacterium Listeria monocytogenes is an important foodborne intracellular pathogen that is widespread in the environment. The functions of hypothetical proteins (HP) from various pathogenic bacteria have been successfully annotated using a variety of bioinformatics strategies. In this study, a HP Imo0888 (NP_464414.1) from the Listeria monocytogenes EGD-e strain was annotated using several bioinformatics tools. Various techniques, including CELLO, PSORTb, and SOSUIGramN, identified the candidate protein as cytoplasmic. Domain and motif analysis revealed that the target protein is a PemK/MazF-like toxin protein of the type II toxin-antitoxin system (TAS) which was consistent with BLASTp analysis. Through secondary structure analysis, we found the random coil to be the most frequent. The Alpha Fold 2 Protein Structure Prediction Database was used to determine the three-dimensional (3D) structure of the HP using the template structure of a type II TAS PemK/MazF family toxin protein (DB ID_AFDB: A0A4B9HQB9) with 99.1% sequence identity. Various quality evaluation tools, such as PROCHECK, ERRAT, Verify 3D, and QMEAN were used to validate the 3D structure. Following the YASARA energy minimization method, the target protein's 3D structure became more stable. The active site of the developed 3D structure was determined by the CASTp server. Most pathogens that harbor TAS create a crucial risk to human health. Our aim to annotate the HP Imo088 found in Listeria could offer a chance to understand bacterial pathogenicity and identify a number of potential targets for drug development.

• Journal of Microbiology and Biotechnology
• /
• 제27권3호
• /
• pp.507-513
• /
• 2017

Bacillus subtilis spores can be used for protein display to engineer protein properties. This method overcomes viability and protein-folding concerns associated with traditional protein display methods. Spores remain viable under extreme conditions and the genotype/phenotype connection remains intact. In addition, the natural sporulation process eliminates protein-folding concerns that are coupled to the target protein traveling through cell membranes. Furthermore, ATP-dependent chaperones are present to assist in protein folding. CotA was optimized as a whole-cell biocatalyst immobilized in an inert matrix of the spore. In general, proteins that are immobilized have advantages in biocatalysis. For example, the protein can be easily removed from the reaction and it is more stable. The aim is to improve the pH stability using spore display. The maximum activity of CotA is between pH 4 and 5 for the substrate ABTS (ABTS = diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). However, the activity dramatically decreases at pH 4. The activity is not significantly altered at pH 5. A library of approximately 3,000 clones was screened. A E498G variant was identified to have a half-life of inactivation ($t_{1/2}$) at pH 4 that was 24.8 times greater compared with wt-CotA. In a previous investigation, a CotA library was screened for organic solvent resistance and a T480A mutant was found. Consequently, T480A/E498G-CotA was constructed and the $t_{1/2}$ was 62.1 times greater than wt-CotA. Finally, E498G-CotA and T480A/E498G-CotA yielded 3.7- and 5.3-fold more product than did wt-CotA after recycling the biocatalyst seven times over 42 h.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제11권3호
• /
• pp.268-272
• /
• 2006

A dense, pellicular UpFront adsorbent ($p=1.5 g/cm^3$, UpFront Chromatography, Cophenhagen, Denmark) was characterized in terms of hydrodynamic properties and protein adsorption performance in expanded bed chromatography. Cibacron Blue 3GA was immobilised into the adsorbent and protein adsorption of bovine serum albumin (BSA) was selected to test the setup. The Bodenstein number and axial dispersion coefficient estimated for this dense pellicular adsorbent was 54 and $1.63{\times}10^{-5}m^2/s$, respectively, indicating a stable expanded bed. It could be shown that the BSA protein was captured by the adsorbent in the presence of 30% (w/v) of whole-yeast cells with an estimated dynamic binding capacity $(C/C_o = 0.01)$ of approximately 6.5 mg/mL adsorbent.

• BMB Reports
• /
• 제55권12호
• /
• pp.627-632
• /
• 2022

Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the canonical WNT/β-catenin pathway, which regulates osteoblast differentiation. However, the role of DKK1 on osteoblast differentiation has not yet been fully clarified. Here, we investigate the functional role of DKK1 on osteoblast differentiation. Primary osteoprogenitor cells were isolated from human spinal bone tissues. To examine the role of DKK1 in osteoblast differentiation, we manipulated the expression of DKK1, and the cells were differentiated into mature osteoblasts. DKK1 overexpression in osteoprogenitor cells promoted matrix mineralization of osteoblast differentiation but did not promote matrix maturation. DKK1 increased Ca⁺ influx and activation of the Ca⁺/calmodulin-dependent protein kinase II Alpha (CAMK2A)-cAMP response element-binding protein 1 (CREB1) and increased translocation of p-CREB1 into the nucleus. In contrast, stable DKK1 knockdown in human osteosarcoma cell line SaOS2 exhibited reduced nuclear translocation of p-CREB1 and matrix mineralization. Overall, we suggest that manipulating DKK1 regulates the matrix mineralization of osteoblasts by Ca⁺-CAMK2A-CREB1, and DKK1 is a crucial gene for bone mineralization of osteoblasts.