• 한국미생물·생명공학회지
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• 제42권2호
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• pp.190-193
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• 2014

CMP-Neu5Ac synthetase는 sialyated 된 glycoconjugates의 전구체로 사용되는 CMP-Neu5Ac를 합성하는데 관여하는 주요 효소이다. Escherichia coli K1에서 유래한 CMP-Neu5Ac synthetase 유전자 (neuA)는 평소 E. coli BL21(DE3)에서 비수용성으로 생성되는데, 이를 수용성 단백질로 생산하고자 여러 가지 샤페론 단백질 동시 발현기술을 이용하였다. 이를 위해, GroEL-ES와 DnaK-DnaJ-GrpE를 암호화하는 pG-KJE8 plasmid와 neuA를 동시 형질전환 시켰고 0.01 mM IPTG와 0.005 mg/ml의 L-arabinose로 유도하여 $20^{\circ}C$에서 발현시켰다. 그 결과, E. coli에서의 수용성 CMP-Neu5Ac Synthetase 생산이 현저하게 증가하였다.

• 한국미생물·생명공학회지
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• 제43권3호
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• pp.212-218
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• 2015

Fucosyltransferase는 퓨코실화된 올리고당을 생성하는데 필수적인 효소로서, GDP-β-L-fucose로 부터 fucose를 수용체로 전이시켜 알파 글리코사이드 결합을 형성하는 과정을 촉매한다. 하지만 Escherichia coli 에서 발현시켰을 때, 대부분의 경우 inclusion body를 형성하여 비활성으로 생성되었다. 따라서 본 연구에서는 Helicobacter pylori 26695에서 유래한 α1,2-fucosyltransferase (FucT2) 유전자의 수용성 발현을 위하여, E. coli에서 샤페론 단백질인 GroEL, GroES, DnaK, DnaJ, GrpE과 함께 동시발현시켰다. SDS-PAGE 분석결과, 5가지 샤페론 단백질과 함께 발현되었으며, 수용성 FucT2 단백질이 증가하였고 효소활성은 5배 증가하였다. 결론적으로, 샤페론 동시발현기술을 이용하여 대장균에서 FucT2 수용성 생산을 증가시킬 수 있었으며 본 수용성 효소는 퓨코실화된 올리고당을 효율적으로 생산하는데 이용될 수 있다.

• Journal of Microbiology and Biotechnology
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• 제20권8호
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• pp.1243-1250
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• 2010

A cDNA encoding a cysteine protease inhibitor (CPI) was isolated from the cDNA library of clamworm Perinereis aibuhitensis Grube. The deduced amino acid sequence analysis showed that the protein had 51%, 48%, and 48% identity with Zgc:153129 from Danio rerio, cystatin B from Theromyzon tessulatum, and the ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Escherichia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Ni-charged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PA-CPI was 16 kDa as deduced by SDS-PAGE. Activity analysis showed that the recombinant protein could inhibit the proteolytic activity of papain. A constitutive and secretive expression vector was also constructed, and the cDNA encoding CPI was subcloned into the vector for extracellular expression. Western blotting analysis results showed that the PA-CPI was secreted into the medium. Bioassay demonstrated that E. coli DH5${\alpha}$ harboring pUC18ompAcat-CPI showed a significant difference in mortality to the Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5${\alpha}$ and control.

• Asian-Australasian Journal of Animal Sciences
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• 제23권10호
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• pp.1268-1275
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• 2010

Post-weaning diarrhoea (PWD) and oedema disease (ED) caused by E. coli F18 always result in economic losses to pig producers, and no effective methods of controlling PWD and ED are presently available. FUT1 has been identified as a candidate gene controlling the expression of E. coli F18 receptor. This study examined the correlation between F18ab and F18ac adhesion phenotypes and the polymorphism at position M307 of the FUT1 gene in three pig breeds (231 Large White, 107 Landrace and 109 Songliao Black). The results showed: i) Both the susceptible genotypes (GG and GA) and the adhesion phenotypes (adhesive or weekly adhesive) were dominant in all three breeds with frequencies over 95%. ii) Three adhesion patterns of the two F18 variants F18ab and F18ac, i.e., ($ab^+$, $ac^+$), ($ab^+$, $ac^-$) and ($ab^-$, $ac^-$), were found in all three breeds, and there was no significant difference in the distribution of adhesion phenotypes of the two variants (separately or jointly) among the three breeds (p>0.05). iii) The FUT1 M307 genotypes were completely associated with the F18ab adhesion phenotypes and very strongly associated with the F18ac adhesion phenotypes. All individuals of genotype AA were non-adhesive to both F18ab and F18ac. All individuals of genotype GG or GA were adhesive to F18ab, whereas 11% of them were non-adhesive to F18ac. These results suggest that the polymorphism at FUT1 M307 can be used for marker-assisted selection of PWD and ED resistant pigs.

• Journal of Microbiology and Biotechnology
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• 제34권2호
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• pp.467-475
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• 2024

ρ-Hydroxyacetophenone is an important and versatile compound that has been widely used in medicine, cosmetics, new materials, and other fields. At present, there are two ways to obtain ρ-hydroxyacetophenone. One is to extract it from plants, such as Artemisia capillaris Thunb and Cynanchum otophyllum Schneid, and the other is to synthesize it by using chemical methods. Of these two methods, the second is the main one, although it has problems, such as flammable and explosive reagents, difficult separation of by-products, and harsh reaction conditions. To solve these issues, we adopted genetic engineering in this study to construct engineered Escherichia coli containing Hped gene or EbA309 gene. Whole-cell biotransformation was conducted under the same conditions to select the engineered E. coli with the higher activity. Orthogonal tests were conducted to determine the optimal biotransformation condition of the engineered E. coli. The results showed that the optimal condition was as follows: substrate concentration of 40 mmol/l, IPTG concentration of 0.1 mmol/l, an induction temperature of 25℃, and a transformation temperature of 35℃. Under this condition, the effects of transformation time on the ρ-hydroxyacetophenone concentration and cell growth were further studied. We found that as the transformation time extended, the ρ-hydroxyacetophenone concentration showed a gradually increasing trend. However, when the ρ-hydroxyacetophenone concentration increased to 1583.19 ± 44.34 mg/l in 24 h, cell growth was inhibited and then entered a plateau. In this research, we realized the synthesis of ρ-hydroxyacetophenone by biotransformation, and our findings lay a preliminary foundation for further improving and developing this method.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.419-428
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• 2019

Phytases are enzymes that can hydrolyze phytate and its salts into inositol and phosphoric acid, and have been utilized to increase the availability of nutrients in animal feed and mitigate environmental pollution. However, the enzymes' low thermostability has limited their application during the feed palletization process. In this study, a combination of B-value calculation and protein surface engineering was applied to rationally evolve the heat stability of Escherichia coli phytase. After systematic alignment and mining for homologs of the original phytase from the histidine acid phosphatase family, the two models 1DKL and 1DKQ were chosen and used to identify the B-values and spatial distribution of key amino acid residues. Consequently, thirteen potential amino acid mutation sites were obtained and categorized into six domains to construct mutant libraries. After five rounds of iterative mutation screening, the thermophilic phytase mutant P56214 was finally yielded. Compared with the wild-type, the residual enzyme activity of the mutant increased from 20% to 75% after incubation at $90^{\circ}C$ for 5 min. Compared with traditional methods, the rational engineering approach used in this study reduces the screening workload and provides a reference for future applications of phytases as green catalysts.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1751-1760
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• 2015

Mucin2 (MUC2), an important regulatory factor in the immune system, plays an important role in the host defense system against bacterial translocation. Probiotics known to regulate MUC2 gene expression have been widely studied, but the interactions among probiotic, pathogens, and mucin gene are still not fully understood. The aim of this study was to investigate the role of MUC2 in blocking effects of probiotics on meningitic E. coli-induced pathogenicities. In this study, live combined probiotic tablets containing living Bifidobacterium, Lactobacillus bulgaricus, and Streptococcus thermophilus were used. MUC2 expression was knocked down in Caco-2 cells by RNA interference. 5-Aza-2'-deoxycytidine (5-Aza-CdR), which enhances mucin-promoted probiotic effects through inducing production of Sadenosyl-L-methionine (SAMe), was used to up-regulate MUC2 expression in Caco-2 cells. The adhesion to and invasion of meningitic E. coli were detected by competition assays. Our studies showed that probiotic agents could block E. coli-caused intestinal colonization, bacteremia, and meningitis in a neonatal sepsis and meningitis rat model. MUC2 gene expression in the neonatal rats given probiotic agents was obviously higher than that of the infected and uninfected control groups without probiotic treatment. The prohibitive effects of probiotic agents on MUC2-knockdown Caco-2 cells infected with E44 were significantly reduced compared with nontransfected Caco-2 cells. Moreover, the results also showed that 5-Aza-CdR, a drug enhancing the production of SAMe that is a protective agent of probiotics, was able to significantly suppress adhesion and invasion of E44 to Caco-2 cells by upregulation of MUC2 expression. Taken together, our data suggest that probiotic agents can efficiently block meningitic E. coli-induced pathogenicities in a manner dependent on MUC2.

• BMB Reports
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• 제44권4호
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• pp.244-249
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• 2011

The quality of a phage-displayed antibody library deteriorates with clonal variations, which are caused by differentially expressed Escherichia coli antibody genes. Using the human Fab SP114 against the pyruvate dehydrogenase complex-E2 (PDCE2), we created four E. coli TOP10F' clones with a pCMTG phagemid encoding Fab-pIII (pCMTG-Fab), Fd ($V_H+C_{H1}$)-pIII (pCMTG-Fd), or light chain (L) (pCMTG-L), or the vector only (pCMTG-${\Delta}Fab$) to investigate the effect of clonal variations in a defined manner. Compared to the others, the E. coli clone with pCMTG-Fab was growth retarded in liquid culture, but efficiently produced phage progenies by Ex12 helper phage superinfection. Our results suggest that an antibody library must be cultured for a short duration before helper phage superinfection, and that the Ex12 helper phage helped to alleviate the detrimental effect of clonal variation, at least in part, by preferentially increasing functional phage antibodies during phage amplification.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권4호
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• pp.378-384
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• 2005

Vanillin is one of the world's principal flavoring compounds, and is used extensively in the food industry. The potential vanillin production of the bacteria was compared to select and clone genes which were appropriate for highly productive vanillin production by E. coli. The fcs (feruloyl-CoA synthetase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104 and Delftia acidovorans were introduced to pBAD24 vector with $P_{BAD}$ promoter and were named pDAHEF and pDDAEF, respectively. We observed 160 mg/L vanillin production with E. coli harboring pDAHEF, whereas 10 mg/L of vanillin was observed with pDAHEF. Vanillin production was optimized with E. coli harboring pDAHEF. Induction of the fcs and ech genes from pDAHEF was optimized with the addition of 13.3 mM arabinose at 18 h of culture, from which 450 mg/L of vanillin was produced. The feeding time and concentration of ferulic acid were also optimized by the supplementation of $0.2\%$ ferulic acid at 18 h of culture, from which 500 mg/L of vanillin was obtained. Under the above optimized condition of arabinose induction and ferulic acid supplementation, vanillin production was carried out with four different types of media, M9, LB, 2YT, and TB. The highest vanillin production, 580 mg/L, was obtained with LB medium, a 3.6 fold increase in comparison to the 160 mg/L obtained before the optimization of vanillin production.

• Asian-Australasian Journal of Animal Sciences
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• 제17권3호
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• pp.401-409
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• 2004

Two experiments were conducted to evaluate the effects of a complex Lactobacilli preparation on performance, resistance to E. coli infection and gut microbial flora of weaning pigs. In exp. 1, twelve pigs (7.65$\pm$1.10 kg BW), weaned at 28 d, were randomly allotted into 2 groups and placed in individual metabolic cages. During the first 7 d, one group of pigs was provided ad libitum access to water containing $10^5$ colony forming units (CFU) Lactobacilli per ml and the control group was provided tap water. The Lactobacilli preparation included Lactobacillus gasseri, L. reuteri, L. acidophilus and L. fermentum, which were isolated from the gastrointestinal (GI) tract mucosa of weaning pigs. On d 8, 20 ml of $10^8$ CFU/ml E. coli solution (serovars K99, K88 and 987P at the ratio of 1:1:1) was orally administered to each pig. Diarrhea scores and diarrhea incidence were recorded from d 7 to 14. On d 14, pigs were euthanized and digesta and mucosa from the stomach, duodenum, jejunum, ileum, cecum and colon were sampled using aseptic technique to determine microflora by culturing bacteria in selective medium. The results showed that Lactobacilli treatment significantly decreased E. coli and aerobe counts (p<0.01) but increased Lactobacilli and anaerobe counts (p<0.01) in digesta and mucosa of most sections of the GI tract. A 66 and 69.1% decrease in diarrhea index and diarrhea incidence, respectively, was observed in the Lactobacilli treated group. In exp. 2, Thirty-six crossbred Duroc$\times$Landrace$\times$Yorkshire piglets, weaned at 28$\pm$2 days, were selected and randomly allocated into 2 groups. There were 18 piglets in each group, 3 piglets in one pen and 6 replicates in each treatment with 3 pens of barrow and 3 pens of female piglet in each treatment. Piglets had ad libitum access to feed and water. The initial body weight of piglet was 7.65$\pm$1.09 kg. Dietary treatments included a non-medicated basal diet with Lactobacilli ($10^5$ CFU/g diet) or carbadox (60 mg/kg) as control. On d 21, six pigs per group (one pig per pen) were euthanized. Ileal digesta was collected to determine apparent amino acid digestibility. Microflora content was determined similarly to exp.1. The results showed that Lactobacilli treatment significantly improved average daily feed intake (ADFI) of pigs compared to carbadox (p<0.05) during the first 2 wks after weaning and average daily gain (ADG) and ADFI increased significantly (p<0.05) from d 8 to 14. Nitrogen and total phosphorus digestibility also increased (p<0.05). Bacterial counts were similar to exp. 1. The results indicate that the complex Lactobacilli preparation improved performance for 2 wks after weaning, enhanced resistance to E. coli infection, and improved microbial balance in the GI tract.