• Title/Summary/Keyword: Bacterial Enzyme

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High-Level Expression of T4 Endonuclease V in Insect Cells as Biologically Active Form

  • Kang, Chang-Soo;Son, Seung-Yeol;Bang, In-Seok
    • Journal of Microbiology and Biotechnology
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    • 제16권10호
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    • pp.1583-1590
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    • 2006
  • T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

무작위로 클로닝한 Porphyromonas endodontalis ATCC 35406 지놈 DNA의 제한절편 hybridization법에 의한 세균동정 (BACTERIAL IDENTIFICATION WITH RANDOM-CLONED RESTRICTION FRAGMENT OF Porphyromonas endodontalis ATCC 35406 GENOMIC DNA)

  • 엄원석;윤수한
    • Restorative Dentistry and Endodontics
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    • 제20권2호
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    • pp.645-654
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    • 1995
  • Porphyromonas endodontalis is a black-pigmented anaerobic Gram negative rod which is associated with endodontal infections. It has been isolated from infected dental root canals and submucous abscesses of endodontal origin. DNA probe is an available alternative, offering the direct detection of a specific microorganism. Nucleic-acid probes can be off different types: whole different: whole-genomic, cloned or oligonucleotide probes. Wholegenomic probes are the most sensitive because the entire genome is used for possible hybridization sites. However, as genetically similar species of bacteria are likely to be present in specimences, cross-reactions need to be considered. Cloned probes are isolated sequences of DNA that do not show cross-reactivity and are produced in quantity by cloning in a plasmid vector. Cloned probes can approach the sensitivity found with whole-genomic probes while avoiding known cross-reacting species. Porphyromonas endodontalis ATCC 35406 (serotype $O_1K_1$) was selected in this experiment to develop specific cloned DNA probes. EcoR I-digested genomic DNA fragments of P. endodontalis ATCC 35406 were cloned into pUC18 plasmid vector. From the E. coli transformed with the recombinant plasmid 4 clones were selected to be tested as specific DNA probes. Restriction-digested whole-genomic DNAs prepared from P. gingivalis 38(serotype a), W50(serotype b), A7A1-28(serotype C), P. intermedia 9336(serotype b), G8-9K-3(serotype C), P. endodontalis ATCC 35406(serotype $O_1K_1$), A. a Y4(serotype b), 75(serotype a), 67(serotype c), were each seperated on agarose gel electrophoresis, blotted on nylon membranes, and were hybridized with digoxigenin-dUTP labeled probe. The results were as follows: 1. Three clones of 1.6kb(probe e), 1.6kb(probe f), and 0.9kb(probe h) in size, were obtained. These clones were identified to be a part of the genomic DNA of P. endodontalis ATCC 35406 judging from their specific hybridization to the genomic DNA fragments of their own size on Southern blot. 2. The clones of 4.9kb(probe i) was identified to be a part of the genomic DNA of P. endodontalis ATCC 35406. but not to specific for itself. It was hybridized to P. gingivalis A7A1-28, P. intermedia G89K-3.

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Biotinoyl Domain of Human Acetyl-CoA Carboxylase;Structural Insights into the Carboxyl Transfer Mechanism

  • Lee, Chung-Kyung;Cheong, Hae-Kap;Ryu, Kyoung-Seok;Lee, Jae-Il;Jeon, Young-Ho;Cheong, Chae-Joon
    • 한국자기공명학회논문지
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    • 제12권1호
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    • pp.1-13
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    • 2008
  • Acetyl-CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis: the synthesis of malonyl-CoA from acetyl-CoA. As essential regulators of fatty acid biosynthesis and metabolism, ACCs are regarded as therapeutic targets for the treatment of metabolic diseases such as obesity, In ACC, the biotinoyl domain performs a critical function by transferring an activated carboxyl group from the biotin carboxylase domain to the carboxyl transferase domain, followed by carboxyl transfer to malonyl-CoA. Despite the intensive research on this enzyme, only the bacterial and yeast ACC structures are currently available, To explore the mechanism of ACC holoenzyme function, we determined the structure of the biotinoyl domain of human ACC2 and analyze its characteristics using NMR spectroscopy. The 3D structure of the hACC2 biotinoyl domain has a similar folding topology to the previously determined domains from E. coli and P. Shermanii, however, the 'thumb' structure is absent in the hACC2 biotinoyl domain. Observations of the NMR signals upon the biotinylation indicate that the biotin group of hACC2 does not affect the structure of the biotinoyl domain, while the biotin group for E. coli ACC interacts directly with the thumb residues that are not present in the hACC2 structure. These results imply that, in the E. coli ACC reaction, the biotin moiety carrying the carboxyl group from BC to CT can pause at the thumb of the BCCP domain. The human biotinoyl domain, however, lacks the thumb structure and does not have additional non-covalent interactions with the biotin moiety; thus, the flexible motion of the biotinylated lysine residue must underlie the "swinging arm" motion. This study provides insight into the mechanism of ACC holoenzyme function and supports the "swinging arm" model in human ACCs.

Bacillus subtilis JK-56이 생산하는 chitinase isozyme의 정제와 특성 규명 (Purification and characterization of the chitinase from Bacillus subtilis JK-56)

  • 전홍기;김낙원;정영기
    • 생명과학회지
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    • 제12권1호
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    • pp.77-86
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    • 2002
  • 토양으로부터 chitinase를 생성하는 균주를 분리하여 동정한 결과 Bacillus subtilis로 판명되었으며, 분리한 균주를 Bacillus subtilis JK-56이라 명명하였다. B. subtilis JK-56의 chitinase 생산 최적 조건을 검토한 결과 1% chitin, 0.5% polypeptone, 0.1% KCI, 0.05% MnS $O_4$.4$H_2O$이며 초발 pH 7.0, 배양온도 37$^{\circ}C$에서 가장 많은 효소를 생산하였다. 본 균주가 생산하는 chitinase를 정제하기 위해서 native-PAGE를 이용해 효소활성 band를 확인한 결과, 1개의 강한 활성 band와 2개의 약한 활성 band를 가지는 isozyme으로 확인되었다. 확인된 isozyme을 정제한 결과, isozyme 중 1개의 강한 활성 band를 정제하였고 정제된 효소를 Chi-56A라고 명명하였다 Chi-56A의 효소 특성에 관해서 실험한 결과 분자량은 약 53kDa, pI는 4.3으로 확인되었다. 본 효소는 $65^{\circ}C$까지 상당히 안정하였으며 효소의 최대활성 온도도 $65^{\circ}C$로 확인되는 등 열에 대해 상당히 안정한 효소로 확인되었다. Collidal chitin에 대한 정제효소 Chi-56A의 $K_{m}$ 값은 17.33g/L였다. 그리고 pH 6.0에서 최대의 활성을 나타내었고, 산성범위보다 알칼리범위에서 안정한 것으로 나타났다. 또한 $Mn^{2+}$ 존재 하에서 높은 활성을 나타내었고 C $O^{2+}$$Mg^{2+}$ 존재 하에서도 활성이 약간 증가한 반면에 H $g^{2+}$ 존재 하에서는 상당한 저해를 받았다. Chito 올리고당에 대한 분해 산물을 HPLC로 확인해 본 결과 짝수개의 올리고당의 분해산물은 (GlcNAc)$_2$만을 생산하였고 홀수개의 올리고당에 대해서는 GlcNAc와 (GlcNAc)$_2$를 생산하는 것으로 비환원성 말단으로부터 이당체인 diacetyl chitobiose ((GlcNAc)$_2$)를 생산하는 exo형 chitinase로 추정 된다.

동물모델을 이용한 짚신나물 물 추출물의 위염 예방효과 및 유전독성 평가 (Preventive Effect of the Water Extract of Agrimonia pilosa Ledeb and Micronucleus Assay-Based Evaluation of Genotoxicity in Gastritis Animal Models)

  • 노종현;장지훈;이현주;양버들;우경완;김아현;서재완;황태연;조현우;정호경
    • 한국약용작물학회지
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    • 제27권2호
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    • pp.136-142
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    • 2019
  • Background: Agrimonia pilosa Ledeb has been used as a traditional medicine for the treatment of hematuresis and uterine bleeding in Korea. It has been reported to have anti-obesity, anti-diabetes and anti-inflammaotry effect by regulating the inflammatory signaling pathway. However, the preventive effect of Agrimonia pilosa Ledeb on gastritis has not been elucidated. Thus, in the present study, we evaluated the effects of the water extract of Agrimonia pilosa Ledeb (APW) using HCl/EtOH-induced gastritis rat models. Method and Results: Gastritis was induced in rats by HCl/EtOH administration. The rats in each group were orally administered with two doses of APW (100 and 500 mg/kg). Omeprazole was used as a positive control drug. An enzyme-linked immunosorbent assay (ELISA) was used to measure the prostaglandin $E_2$ ($PGE_2$) levels in stomach. The treatment with 500 mg/kg APW reduced the gastric ulcer area. The APW treatment prevented a decreased in $PGE_2$ concentration induced by HCl/EtOH in rats. In the micronucleus test, the ratio of micronucleated polychromatic erythrocytes to polychromatic erythrocytes showed no significant change in the APW-treated group compared with the control group. Conclusions: These results indicate that APW could be used to prevent the gastritis caused by the HCl/EtOH-induced damage to stomach lining. In addition, the APW treatment showed no significant change in results of the micronucleus test. However, further experiments are required to determine how APW influenced the secretion of mucus and gastric acid using the chromosome aberration test and bacterial reverse mutation assay.

Impact of different shades of light-emitting diode on fecal microbiota and gut health in broiler chickens

  • Ianni, Andrea;Bennato, Francesca;Di Gianvittorio, Veronica;Di Domenico, Marco;Martino, Camillo;Colapietro, Martina;Camma, Cesare;Martino, Giuseppe
    • Animal Bioscience
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    • 제35권12호
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    • pp.1967-1976
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    • 2022
  • Objective: The aim of this study was to characterize the fecal microbiota of broiler chickens reared in the presence of different shades of light-emitting diode (LED) lights, correlating this information with biochemical and molecular evidence that allowed drawing conclusions on the state of health of the animals. Methods: Overall, the metagenomic approach on fecal samples was associated with evaluations on enzymes involved in the cellular response to oxidative stress: glutathione peroxidase (GPX), superoxide dismutase and catalase; while the inflammatory aspect was studied through the dosage of a proinflammatory cytokine, the interleukin 6 (IL-6), and the evaluation of the matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9). Specifically, analysis was performed on distinct groups of chickens respectively raised in the presence of neutral (K = 3,300 to 3,700), cool (K = 5,500 to 6,000), and warm (K = 3,000 to 2,500) LED lightings, and a direct comparison was performed with animals reared with traditional neon lights. Results: The metagenomic analysis highlighted the presence of two most abundant bacterial phyla, the Firmicutes and the Bacteroidetes, with the latter characterized by a greater relative abundance (p<0.05) in the group of animals reared with Neutral LED light. The analysis on the enzymes involved in the antioxidant response showed an effect of the LED light, regardless of the applied shade, of reducing the expression of GPX (p<0.01), although this parameter is not correlated to an effective reduction in the tissue amount of the enzyme. Regarding the inflammatory state, no differences associated with IL-6 and MMP-9 were found; however, is noteworthy the significant reduction of MMP-2 activity in tissue samples obtained from animals subjected to illumination with neutral LED light. Conclusion: This evidence, combined with the metagenomic findings, supports a potential positive effect of neutral LED lighting on animal welfare, although these considerations must be reflected in more targeted biochemical evaluations.

이탄 토양으로부터 식물생육촉진세균 Pseudomonas sp. SH-26의 분리 및 특성 (Isolation and Characterization of Plant Growth Promoting Bacteria Pseudomonas sp. SH-26 from Peat Soil)

  • 신호용;김다솜;이창호;이동석;한송이
    • 한국응용과학기술학회지
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    • 제41권2호
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    • pp.199-207
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    • 2024
  • 이탄 토양으로부터 분리된 세균의 유기물 분해능, 항균 활성 및 식물생육촉진능을 확인하고 다기능성 유용 미생물을 확보하고자 하였다. 분리된 48 균주를 대상으로 β-glucosidase, cellulase, amylase, protease 생성능을 확인한 결과, SH-23, SH-26, SH-29 그리고 SH-33 균주가 우수 균주로 선발되었다. 선발된 유기물 분해 우수 세균 4 균주는 식물병원성곰팡이(Botrytis cinerea, Rhizoctonia solani, Fusarium oxysporum, Colletotrichum acutatum)에 대한 항진균 활성을 나타내었다. 유기물 분해능이 우수하고 식물병원성 곰팡이에 대해 항진균 활성을 나타낸 SH-26 균주를 최종 우수균주로 선발 하였다. 선발된 SH-26 균주의 16S rRNA 유전자 염기 서열을 결정한 결과, Pseudomonas knackmussii HG322950 B13T, Pseudomonas citronellolis BCZY01000096 NBRC 103043T, 그리고 Pseudomonas delhiensis jgi.1118306 RLD-1T와 100%의 상동성을 나타내었다. Pseudomonas sp. SH-26 균주는 siderophore 생성능, 질소고정능과 Indole-3-acetic acid 생성능이 있는 것으로 확인되었다.

Discovery of a Novel Cellobiose Dehydrogenase from Cellulomonas palmilytica EW123 and Its Sugar Acids Production

  • Ake-kavitch Siriatcharanon;Sawannee Sutheeworapong;Sirilak Baramee;Rattiya Waeonukul;Patthra Pason;Akihiko Kosugi;Ayaka Uke;Khanok Ratanakhanokchai;Chakrit Tachaapaikoon
    • Journal of Microbiology and Biotechnology
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    • 제34권2호
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    • pp.457-466
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    • 2024
  • Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 (CpCDH) was cloned and characterized. The CpCDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in CpCDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of CpCDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of CpCDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The CpCDH displayed excellent enzymatic activity at low temperatures (below 30℃), unlike other CDHs. Moreover, CpCDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of CpCDH for cellobiose and lactose were 2.05 × 105 and 9.06 × 104 (M-1 s-1), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of CpCDH. This study establishes CpCDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.

Porphyromonas endodontalis에 대한 Polyphosphate의 항균기전에 관한 연구 (MECHANISM IN ANTIBACTERIAL ACTIVITY OF POLYPHOSPHATES AGAINST PORPHYROMONAS ENDODONTALIS)

  • 최성백;박상진;최기운;최호영
    • Restorative Dentistry and Endodontics
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    • 제25권4호
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    • pp.561-574
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    • 2000
  • Poly-P has been used to prevent decomposition of foods and has been shown to have inhibitory effect on the growth of gram positive bacteria. The purpose of this study was to evaluate the effect of poly-P on the growth of Porphyromonas endodontalis, a gram negative obligate anaerobic rod, endodontopathic bacterium. P. endodontalis ATCC 35406 was in BHI broth containing hemin and vitamin K with or without poly-P. Inhibitory effect of each poly-P which was added at the beginning(lag phase) or during(exponential phase) the culture, MIC(minimum inhibitory concentration) was determined by measuring the optical density of the bacterial cell at 540nm. Viable cell counts were measured to determined whether poly-P has a bactericidal effect. Leakage of intracellular nucleotides from P. endodontalis was determined at 260nm and morphological change of P. endodontalis was observed under the TEM(transmission electron microscope). Binding of 32P-labeled poly-P to P. endodontalis was examined. SDS-polyacrylamide gel electrophoresis and zymography were performed to observe the changes in protein and enzyme profiles of P. endodontalis, respectively. The results from this study were as follows : 1. The minimal inhibitory concentration(MIC) of poly-P to P. endodontalis appeared to be 0.04~0.05%. 2. Poly-P added to the P. endodontalis culture during the exponential phase of P. endodontalis was as much effective as poly-P added at the begining of the culture, suggesting that the antibacterial effect of poly-P is not much dependent on the initial inoculum size of P. endodontalis. 3. Poly-P are bactericidal to P. endodontalis, demonstrating the decrease of the viable cell counts. 4. Intracellular nucleotide release from the P. endodontalis, was not increased in the presence of poly-P and was not reversed by the addition of divalent cations like $Ca^{2+}$ and $Mg^{2-}$. 5. Under the TEM, it was observed that fine electro-dense materials were prominent in the poly-P grown P. endodontalis, appearing locally in the cell, and the materials were more abundant and more dispersed in the cell as the incubation time with poly-P increased. In addition, highly electron dense granules accumulated in many poly-P grown cells, most of which were atypical in their shape. 6. Binding of 32P-labeled poly-P to P. endodontalis appeared to be 32.8 and 45.5 and 53.4% at 30 minutes, 1 hours and 2 hours, respectively. 7. In the presence of poly-P. the synthesis of proteins with apparent molecular masses of 25, 27, 35, 45 was lost or drastically decreased whereas expression of a protein with an apparent molecular mass of 75 was elevated. 8. Proteolytic activity of P. endodontalis was decreased by poly-P. The overall results suggest that use of poly-P may affect the growth of P. endodontalis, and the anti-bacterial activity of poly-P seems largely bactericidal. Changes in shape, protein expression, and proteolytic activity of P. endodontalis by poly-P may be directly and indirectly attributed to the antibacterial effect of poly-P. Further studies will be needed to confirm the effect of poly-P.

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남해안 갯벌 미생물의 세포외효소 활성 및 16S rDNA 분석에 의한 다양성 조사 (Microbial Population Diversity of the Mud Flat in Suncheon Bay Based on 16S rDNA Sequences and Extracellular Enzyme Activities)

  • 김유정;김성겸;권은주;백근식;김정호;김훈
    • Applied Biological Chemistry
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    • 제50권4호
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    • pp.268-275
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    • 2007
  • 순천만 갯벌에 존재하는 미생물의 다양성을 세포외 효소 활성과 16S rDNA 분석으로 조사하였다. 수초 주변과 갯벌에서 채취한 시료에서 얻은 배양 가능한 균 중에서 4개의 균주를 대상으로 CMCase, xylanase와 protease의 활성을 조사한 결과 2, 3, 27, 그리고 30번 4균주 모두 xylan 분해능이 가장 뛰어났다. 각각의 효소 활성의 최적 온도는 $50{\sim}70^{\circ}C$로 나타났고, 수초 주변과 갯벌 시료에 따른 차이는 거의 없었다. 갯벌 내 세균 군집을 조사하기 위해 메타게놈 DNA를 분리한 후 증폭된 16S rDNA를 갖는 약 2,000개의 클론을 얻어 그 다양성을 조사하였다. 제한효소 절단 양상으로부터 선별된 17개의 클론을 분석한 결과 클론들의 평균 유사도는 97.3%이었으며, 그 범위는$91.0{\sim}99.9%$를 보였고, 4번, 7번, 9번, 101번, 104번, 107번을 제외한 나머지 11개는 uncultured strain들로 확인되었다. 이들은 Proteobacteria, Bacteroidetes, Flavobacteria, Verrucomicrobia, Acidobacteria, Firmicutes, 그리고 Chloroflexi의 7개의 그룹으로 구분할 수 있었다. 9개 (52.9%)의 클론은 Proteobacteria, 3개(17.6%)의 클론은 Firmicutes에 속하였으며, 나머지 그룹에는 각각 1개씩의 클론이 해당되었다. Proteobacteria중에서는 황원소의 산화와 환원에 관여하는 gamma Proteobacteria의 비율이 높게 나타났다. 본 연구의 결과 순천만 갯벌은 미생물 다양성을 유지하고 있으며, 분석된 클론의 65% 가량은 배양되지 않은 미생물로 조사되었다. 이 갯벌로부터 메타게놈 라이브러리를 제작하여 보다 다양한 유전자원을 확보할 수 있을 것으로 판단된다.정에도 잘 활용될 수 있을 것으로 생각된다.생리 요인들 간에는 약한 상관이 존재했으나, 피험자들이 온열 쾌적을 유지하는 경우 착용한 의복 종류 및 노출 기온에 상관없이 불감체중손실량은 좁은 범위를 유지했다.것으로 판단된다.$4^{\circ}C$로 저장한 경우 치커리 고유의 초록색과 아삭한 조직감을 유지하고 있어 저온냉수 세척과 tray 포장이 세척 청경채의 선도 유지에 효과가 있는 것으로 나타났다.>$2.0{\sim}1459.4ppm(303.1{\pm}324.2)$으로 나타났다.다. 4. 이상의 결과를 바탕으로 위생교육 매뉴얼을 개발하였다. 위생교육 내용은 4가지 원칙 -개인위생확보, 교차오염방지, 시간온도관리, 냉장관리- 으로 구성하였고, 8영역으로 세분화하였다. 교육 설계방식은 강의식 교육을 위주로 하고, 주제별로, 실연식 교육, 시청각 교육, 토의식 교육을 위한 강의 자료를 마련하였다. 이상의 결과에서 보여 준 레스토랑에서 식중독 예방을 위한 위생관리 활동 내용으로 실천율이 낮은 활동을 규명하고 이 활동에 대한 지속적인 모니터링과 개선활동을 전개한다면 레스토랑의 식중독 예방에 크게 도움일 될 것으로 사료된다. 또한 본 연구에서 개발된 위생교육 자료를 활용한다면, 외식업체의 전반적인 위생수준을 향상시키고 식품의 안전성을 확보하는데 크게 기여할 것으로 기대된다. 최근에 외식업계에서 발생되고 있는 식중독 사고는 이상 기후의 영향이나 시설미비도 그 원인이겠지만, 무엇보다도 중요한 요인은 경영층의 위생에 관한 인식부족, 교육 프로그램의 부재로 사료된다. 그러므로 양적으로 과포화상태에 이르고 있는 외식업계의 질적인 향상을 위해서는 외식업체는 위생교육의 지속적인 실천과 모니터링을 강화하고, 그 결과를 인사고과시스템에 반영하는 통합적인 노력이 요구된다.야 하고, 검사관리를 통해 원재료와 최종제품의 안전성이 확보되어야 한다. 납품업체들의 검사관리 수행도를 높이기 위해서는 검사시설의