• Journal of Microbiology and Biotechnology
• /
• v.26 no.10
• /
• pp.1708-1716
• /
• 2016

Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (gdhB) was cloned from Bacillus thuringiensis subsp. kurstaki, and wild-type (GDH-BT^WT) and His-tagged (GDH-BT^N-His, GDH-BT^C-His) enzymes were produced in Escherichia coli BL21 (DE3). All enzymes were produced in the soluble forms from E. coli. GDH-BT^WT and GDH-BT^N-His showed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BT^C-His showed a very low specific enzymatic activity of 0.020 U/mg. These results suggest that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BT^WT was stable up to 65℃, whereas GDH-BT^N-His and GDH-BT^C-His were stable up to 45℃. Gel permeation chromatography showed that GDH-BT^WT is a dimer, whereas GDH-BT^N-His and GDH-BT^C-His are monomeric. These results suggest that the intact N- and C-termini are required for GDH-BT to maintain thermostability and to form its dimer structure. The homology model of the GDH-BT^WT single subunit was constructed based on the crystal structure of Bacillus megaterium GDH (PDB ID 3AY6), showing that GDH-BT^WT has a Rossmann fold structure with its N- and C-termini located on the subunit surface, which suggests that His-tagging affected the native dimer structure. GDH-BT^WT and GDH-BT^N-His regenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.2
• /
• pp.231-236
• /
• 2004

The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at $26^{\circ}C$. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70％. Both 23 kDa ($\alpha$) and 62 kDa ($\beta$) subunits were recovered by using IMAC with just C-terminus tagging of the $\beta$ subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.

• Korean Journal of Veterinary Research
• /
• v.46 no.1
• /
• pp.13-19
• /
• 2006

Generally known psittacosis or ornithosis is a disease of birds caused by the bacterium Chlamydia psittaci. Humans are accidential hosts and are most commonly infected from avian sources. It raises hepatitis or neurosis. As major outer membrane protein (MOMP) of Chlamydia psittaci has been known to play a role in the avoidance of host immune defenses, research on developing a Chlamydia vaccine has focused on the MOMP. In this study, the gene encoding the major outer membrane protein (MOMP) of the Chlamydia psittaci strain 6BC was cloned and expressed in Escherichia coli strain M-15. The recombinant DNA was cloned by fusion prokaryotic expression vector pQE30-GFPII. Expression of the recombinant protein was performed in E. coli and was induced by IPTG. The size of expressed recombinant protein is 74.220 kDa (MOMP, 43.260 kDa; GFP expression region, 30 kDa; $6{\times}His$ tag, 960Da). This protein was purified by using his-tagging-inclusion body. Recombinant protein was reconfirmed through ELISA test and western blot with antibody against pQE30-GFPII. It will be useful antibody development.

• Korean Journal of Microbiology
• /
• v.53 no.4
• /
• pp.297-303
• /
• 2017

Leucine-responsive regulatory protein (LRP) is a regulatory protein of molecular weight 18.8 kDa and is widely known to regulate many metabolic and functional activities of operons in Escherichia coli. The gene for Lrp from Escherichia coli in pQE system of 6 ${\times}$ His-tagging was expressed and $^3H$-labeled protein, as well as the wild type Lrp, was purified. The crosslink experiments were performed to analyze the quaternary structure of Lrp at high of $5{\mu}M$ and at low concentrations below $0.3{\mu}M$ with cross linkers, such as glutaraldehyde, 1, 2, 3, 4-diepoxy-butane (DEB), and ethylene glycol bis (succinimidyl succinate) (EGS). In the experiments, we found that the Lrp protein can be formed higher conformation states of tetramer, hexamer, octamer, as well as dimeric state when incubated with the above cross linkers.

• BMB Reports
• /
• v.40 no.4
• /
• pp.539-546
• /
• 2007

The lysozymes encoded by bacteriophage T7 and K11 are both bifunctional enzymes sharing an extensive sequence homology (75%). The constructions of chimeric lysozymes were carried out by swapping the N-terminal and C-terminal domains between phage T7 and K11 lysozymes. This technique generated two chimeras, T7K11-lysozyme (N-terminal T7 domain and C-terminal K11 domain) and K11T7-lysozyme (N-terminal K11 domain and C-terminal T7 domain), which are both enzymatically active. The amidase activity of T7K11-lysozyme is comparable with the parental enzymes while K11T7-lysozyme exhibits an activity that is approximately 45% greater than the wild-type lysozymes. Moreover, these chimeric constructs have optimum pH of 7.2-7.4 similar to the parental lysozymes but exhibit greater thermal stabilities. On the other hand, the chimeras inhibit transcription comparable with the parental lysozymes depending on the source of their N-terminals. Taken together, our results indicated that domain swapping technique localizes the N-terminal region as the domain responsible for the transcription inhibition specificity of the wild type T7 and K11 lysozymes. Furthermore, we were able to develop a simple and rapid purification scheme in purifying both the wild-type and chimeric lysozymes.

• Korean Journal of Microbiology
• /
• v.49 no.2
• /
• pp.205-210
• /
• 2013

Lumazine protein is a fluorescent protein isolated from the bioluminescent bacteria of Photobacterium species. To generate minimal size of lumazine protein with possessing fluorescent characteristic, the gene coding for the wild type N-terminal domain of lumazine protein (N-LumP 118) containing amino acids up to 118 from Photobacterium leiognathi was produced. In addition, the genes coding for the variant proteins of N-LumP 118, replaced with one tryptophan amino acid (N-LumP 118 V41W, S48W, T50W, D64W, and A66W), were also constructed by Polymerase Chain Reaction and Site Directed Mutagenesis. These proteins were expressed in Escherichia coli by transformation with recombinant plasmids and purified by 6X-His tagging system. Spectroscopic studies have show that the purified proteins are capable of binding to the fluorescent ligand 6,7-dimethyl-8-ribityllumazine, resulted in showing of fluorescent characteristic with the minimal size of protein. From these studies, the mutant proteins containing single tryptophan amino acid residue, possessing its own intrinsic flouophore character at the different position, will be able to the use as a probe for further studies to deduce their three dimensional structure and the binding modes.

• Asia pacific journal of information systems
• /
• v.21 no.2
• /
• pp.89-116
• /
• 2011

In collaborative tagging systems such as Delicious.com and Flickr.com, users assign keywords or tags to their uploaded resources, such as bookmarks and pictures, for their future use or sharing purposes. The collection of resources and tags generated by a user is called a personomy, and the collection of all personomies constitutes the folksonomy. The most significant need of the folksonomy users Is to efficiently find useful resources or experts on specific topics. An excellent ranking algorithm would assign higher ranking to more useful resources or experts. What resources are considered useful In a folksonomic system? Does a standard superior to frequency or freshness exist? The resource recommended by more users with mere expertise should be worthy of attention. This ranking paradigm can be implemented through a graph-based ranking algorithm. Two well-known representatives of such a paradigm are Page Rank by Google and HITS(Hypertext Induced Topic Selection) by Kleinberg. Both Page Rank and HITS assign a higher evaluation score to pages linked to more higher-scored pages. HITS differs from PageRank in that it utilizes two kinds of scores: authority and hub scores. The ranking objects of these pages are limited to Web pages, whereas the ranking objects of a folksonomic system are somewhat heterogeneous(i.e., users, resources, and tags). Therefore, uniform application of the voting notion of PageRank and HITS based on the links to a folksonomy would be unreasonable, In a folksonomic system, each link corresponding to a property can have an opposite direction, depending on whether the property is an active or a passive voice. The current research stems from the Idea that a graph-based ranking algorithm could be applied to the folksonomic system using the concept of mutual Interactions between entitles, rather than the voting notion of PageRank or HITS. The concept of mutual interactions, proposed for ranking the Semantic Web resources, enables the calculation of importance scores of various resources unaffected by link directions. The weights of a property representing the mutual interaction between classes are assigned depending on the relative significance of the property to the resource importance of each class. This class-oriented approach is based on the fact that, in the Semantic Web, there are many heterogeneous classes; thus, applying a different appraisal standard for each class is more reasonable. This is similar to the evaluation method of humans, where different items are assigned specific weights, which are then summed up to determine the weighted average. We can check for missing properties more easily with this approach than with other predicate-oriented approaches. A user of a tagging system usually assigns more than one tags to the same resource, and there can be more than one tags with the same subjectivity and objectivity. In the case that many users assign similar tags to the same resource, grading the users differently depending on the assignment order becomes necessary. This idea comes from the studies in psychology wherein expertise involves the ability to select the most relevant information for achieving a goal. An expert should be someone who not only has a large collection of documents annotated with a particular tag, but also tends to add documents of high quality to his/her collections. Such documents are identified by the number, as well as the expertise, of users who have the same documents in their collections. In other words, there is a relationship of mutual reinforcement between the expertise of a user and the quality of a document. In addition, there is a need to rank entities related more closely to a certain entity. Considering the property of social media that ensures the popularity of a topic is temporary, recent data should have more weight than old data. We propose a comprehensive folksonomy ranking framework in which all these considerations are dealt with and that can be easily customized to each folksonomy site for ranking purposes. To examine the validity of our ranking algorithm and show the mechanism of adjusting property, time, and expertise weights, we first use a dataset designed for analyzing the effect of each ranking factor independently. We then show the ranking results of a real folksonomy site, with the ranking factors combined. Because the ground truth of a given dataset is not known when it comes to ranking, we inject simulated data whose ranking results can be predicted into the real dataset and compare the ranking results of our algorithm with that of a previous HITS-based algorithm. Our semantic ranking algorithm based on the concept of mutual interaction seems to be preferable to the HITS-based algorithm as a flexible folksonomy ranking framework. Some concrete points of difference are as follows. First, with the time concept applied to the property weights, our algorithm shows superior performance in lowering the scores of older data and raising the scores of newer data. Second, applying the time concept to the expertise weights, as well as to the property weights, our algorithm controls the conflicting influence of expertise weights and enhances overall consistency of time-valued ranking. The expertise weights of the previous study can act as an obstacle to the time-valued ranking because the number of followers increases as time goes on. Third, many new properties and classes can be included in our framework. The previous HITS-based algorithm, based on the voting notion, loses ground in the situation where the domain consists of more than two classes, or where other important properties, such as "sent through twitter" or "registered as a friend," are added to the domain. Forth, there is a big difference in the calculation time and memory use between the two kinds of algorithms. While the matrix multiplication of two matrices, has to be executed twice for the previous HITS-based algorithm, this is unnecessary with our algorithm. In our ranking framework, various folksonomy ranking policies can be expressed with the ranking factors combined and our approach can work, even if the folksonomy site is not implemented with Semantic Web languages. Above all, the time weight proposed in this paper will be applicable to various domains, including social media, where time value is considered important.

• Korean Journal of Microbiology
• /
• v.47 no.1
• /
• pp.14-21
• /
• 2011

Riboflavin synthase catalyzes the formation of one molecule of each riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrates, 6,7-dimetyl-8-ribityllumazine. The most remarkable feature is the sequence similarity between the N-terminal half (1-97) and the C-terminal half domain (99-213). To investigate the structure and fluorescent characteristics of the N-terminal half of riboflavin synthase (N-RS) in Escherichia coli, more than 10 mutant genes coding for the mutated N-terminal domain of riboflavin synthase were generated by polymerase chain reaction. The genes coding for the proteins were inserted into pQE vector designed for easy purification of protein by 6X-His tagging system, expressed, and the proteins were purified. Almost all mutated N-terminal domain of riboflavin synthases bind to 6,7-dimethyl-8-ribityllumazine and riboflavin as fluorescent ligands. However, N-RS C47D and N-RS ET66,67DQ mutant proteins show colorless, indicating that fluorescent ligands were dissociated during purification. In addition, most mutated proteins show low fluorescent intensity comparing to N-RS wild type, whereas N-RS C48S posses stronger fluorescent intensity than that of wild type protein. Based on this result, N-RS C48S can be used as the tool for high throughput screening system for searching for the compound with inhibitory effect for the riboflavin synthase.

• Journal of Intelligence and Information Systems
• /
• v.17 no.4
• /
• pp.143-155
• /
• 2011

Lately electronic tagging policy for the sexual offenders was introduced in order to reduce and prevent sexual offences. However, most sexual offences against children happening these days are committed by the tagged offenders whose identities have been released. So, for the crime prevention, we need measures with which we could minimize the suffers more promptly and actively. This paper suggests a new system to relieve the sexual abuse related anxiety of the children and solve the problems that electronic bracelet has. Existing bracelets are only worn by serious criminals, and it's only for risk management and positioning, there is no way to protect the children who are the potential victims of sexual abuse and there actually happened some cases. So we suggest also letting the students(children) wear the LBS(Location Based Service) and USN(Ubiquitous Sensor Network) technology based electronic bracelets to monitor and figure out dangerous situations intelligently, so that we could prevent sexual offences against children beforehand, and while a crime is happening, we could judge the situation of the crime intelligently and take swift action to minimize the suffer. And by checking students' attendance and position, guardians could know where their children are in real time and could protect the children from not only sexual offences but also violent crimes against children like kidnapping. The overall system is like follows : RFID Tag for children monitors the approach of offenders. While an offender's RFID tag is approaching, it will transmit the situation and position as the first warning message to the control center and the guardians. When the offender is going far away, it turns to monitoring mode, and if the tag of the child or the offender is taken off or the child and offender stay at one position for 3~5 minutes or longer, then it will consider this as a dangerous situation, then transmit the emergency situations and position as the second warning message to the control center and the guardians, and ask for the dispatch of police to prevent the crime at the initial stage. The RFID module of criminals' electronic bracelets is RFID TAG, and the RFID module for the children is RFID receiver(reader), so wherever the offenders are, if an offender is at a place within 20m from a child, RFID module for children will transmit the situation every certain periods to the control center by the automatic response of the receiver. As for the positioning module, outdoors GPS or mobile communications module(CELL module)is used and UWB, WI-FI based module is used indoors. The sensor is set under the purpose of making it possible to measure the position coordinates even indoors, so that one could send his real time situation and position to the server of central control center. By using the RFID electronic roll book system of educational institutions and safety system installed at home, children's position and situation can be checked. When the child leaves for school, attendance can be checked through the electronic roll book, and when school is over the information is sent to the guardians. And using RFID access control turnstiles installed at the apartment or entrance of the house, the arrival of the children could be checked and the information is transmitted to the guardians. If the student is absent or didn't arrive at home, the information of the child is sent to the central control center from the electronic roll book or access control turnstiles, and look for the position of the child's electronic bracelet using GPS or mobile communications module, then send the information to the guardians and teacher so that they could report to the police immediately if necessary. Central management and control system is built under the purpose of monitoring dangerous situations and guardians' checking. It saves the warning and pattern data to figure out the areas with dangerous situation, and could help introduce crime prevention systems like CCTV with the highest priority. And by DB establishment personal data could be saved, the frequency of first and second warnings made, the terminal ID of the specific child and offender, warning made position, situation (like approaching, taken off of the electronic bracelet, same position for a certain time) and so on could be recorded, and the data is going to be used for preventing crimes. Even though we've already introduced electronic tagging to prevent recurrence of child sexual offences, but the crimes continuously occur. So I suggest this system to prevent crimes beforehand concerning the children's safety. If we make electronic bracelets easy to use and carry, and set the price reasonably so that many children can use, then lots of criminals could be prevented and we can protect the children easily. By preventing criminals before happening, it is going to be a helpful system for our safe life.