Filamentous Fungus Aspergillus oryzae의 유전공학적 응용

함영태 中央大學校 食糧資源硏究所 1993 食糧資源硏究所 論文集 Vol.5 No.1

A. oryzae, a filametous fungus classified in the group Aspergillaceae Ascomycetes, is an important microorganism for the industrial production of proteolytic and amylolytic enzymes and for the fermentation of rice and soy bean products. It secrets large quantities of proteins or enzymes into the culture medium which makes this organism appealing for the production of heterologous proteins. Secretion of proteins into the medium has several advantages when producing a product by fermentation which must be subsequently purified. the secreted protein is not degraded by intracellular proteases. It also does not affect the metabolism of the host cell by accumulating foreian proteins intracellularly. the recovery of a secreted product is simplified because it is already separated from cytoplamic proteins, Since A. oryzae has been used for a centuries to pro-duce fermented foods and enzymes, it may be a good choice for the production of useful products in the field of the medicine and the food industry.

곰팡이의 형질전환

함영태 中央大學校 遺傳工學硏究所 1993 遺傳工學硏究論集 Vol.6 No.1

The genetic transformation of fungi was first reported with an inositol-requiring mutant(inl) of Neurospora crassa. The fungi were cultured with DNA isolated from a wild type(inl+) in the presence of calcium and inositol-independent strains were obtained from the resulting conidia. Protoplast mediated transformation systems have been developed for Saccharomyces cerevisiae and other filamentous fungi. Several enzymes including helicase or flusulase and zymolyase have been used to generate fungal protoplast. The most common enzyme used to generate protoplast is Novozyme 234 isolated from the fungus Trichoderma viride. The protoplast should be stabilized by the addition of an osmotic stabilizer. Calcium ions and a high concentration of polyethylene glycol are generally required for most fungal transformation. Several types of nutritional selectable markers have been isolated and used to transform fungi including those selected by complementation of an auxotrophic defect. Some dominant selectable marker have been developed and used to the restricted fungal strains. The frequence of fungal transformants observed is generally low. In most reports on the transformation of fungus, stable transformation is a consequence of the integration of transforming DNA into the chromosome with multiple copies of the plasmid. Recently electroporation system have been used to fungal transformation to increase the transformation frequency.

중국산 야생 키위로부터 단백질 분해효소 유전자의 분리

이남근,함영태 中央大學校 遺傳工學硏究所 1999 遺傳工學硏究論集 Vol.12 No.1

A Kiwi fruit is originated from the Yangtze River Valley of Northern China and Zhejiang Province on the cost of Eastern China. It is called as the Chinese gooseberry. Around 1950, a large mass production began at New Zealand with an improved breeding. Kiwi fruits have been cultured n America, South Africa, Italy and Korea recently. The kiwi fruit includes vitamins and many proteases. Plant origin actinidin from kiwi fruit belongs to the papain family of cysteine proteinase, which in plants includes papain from papaya, bromelain from pineapple, C14 protease from tomato and aleurain from barley (Baker, 1980; Schaffer and Fischer, 1990). Actinidin is involved in the ripening-related gene family. In this study, protease gene of chinese wild kiwi fruit was isolated and characterized. 1.2kb PCR-amplified fragment was obtained from the total RNA using RT-PCR. pWACT-1 was obtained by subcloning of amplified fragment into pGEM-T Easy cloning vector and analyzed 35% percent of nucleotide sequence by DNA sequencing. In Result, high levels of homology between wild kiwi and cultured-kiwi was obtained.

지방산 결합단백질에 관한 연구 동향

권영아,노숙령,김혜경,함영태 中央大學校 食糧資源硏究所 1996 食糧資源硏究所 論文集 Vol.8 No.1

ABSTRACT Long chain free fatty acid (FFA) are the main energy source for many mammalian cell, and FFA metabolism is dependent upon the flux of FFA to sites of intra-cellular processing. Fatty Acid Binding Proteins (FABPs) are a family of abundant cytosolic proteins of low M.W.14-15 kDa. Although their precise physilogical role remains hypothetical, it has been proposed as a FFA transfer protein and as a binding protein responsible for controlling intracellular FFA concentration. Furthermore, significant correlations have been observed between the tissue content of FABP and the rate of FFA uptake or utilization in several different manipulations such as diet and hypolipidemic drugs. However, to date there has been no report which examines the role of FABP in physiological manipulations such as fatty liver, diabetes, obesity, hypercholesterolemia. Since lipid metabolism is abnormal and the liver is greatly affected in these disease, it is possible that FABP may be abnormal in these livers. Therefore, the realtionships of liver FABP and the disease with lipid abnormalities, especially fatty liver induced by alcohol and high cholesterol diet are examined in our lab. Where FABP is believed to be involved in FFA binding and transfer, cellular FABP amount as well as its binding activity are affected. If we further find the way to increase the expression of FABP in liver, it could be one means of settle for these kinds of diseases from lipid abnormality.

곰팡이의 원형질체를 이용한 항생제 저항성 연구

김기모,이경아,함영태 中央大學校 食糧資源硏究所 1994 食糧資源硏究所 論文集 Vol.6 No.1

A. oryzae와 T.inflatum의 wild-type host에 dominant selectable marker인 phleomycin resistancy를 가지는 E. coli Tn5의 ble gene과 hygromycin B에 대한 저항성을 나타내는 E. coli hph gene의 사용 가능성을 분석키 위하여 phleomycin과 hygromycin B를 농도별 (5㎍∼200㎍ of antibiotic/㎖ of media)로 곰팡이의 원형질체에 적용하여 그 저항성을 비교 분석하였다. Lysing enzyme과 β-glucuronidase를 germinated spore(??)에 처리하여 A. oryzae에서는 4.3∼4.8×10⁴protoplast를 얻은 반면, T.inflatum에서는 1.8∼2.5×10²protoplast만을 얻었다. A. oryzae의 hygromycin B 저항성을 농도 50∼200㎍/㎖ of media에서 조사하여 본 결과, hygromycin 200㎍에서도 강한 저항성을 보여 주었고, T.inflatum에서는 항생물질 농도 25㎍/㎖에서 60%의 세포수 감소를 보이며, 75㎍/㎖에서90% 이상의 세포수 감소를 보여주었다. Phleomycin 저항성에 대한 실험에서는 A.oryzae에서는 phleomycin 50㎍/㎖ of media에서 50% 정도의 저항성을 보여 주고 있으며 , 50㎍/㎖농도에서는 85% 정도의 세포수 감소가 관찰되었고, T.inflatum에서는 hleomycin 농도 5㎍/㎖에서도 강한 생장 억제효과가 나타나는 것을 보여 주었다. Antibiotic-resistancy on fungi, Aspergillus oryzae and Tolypocladium inflatum, were analyzed by using fungal protoplasts and antibiotics, hygromycin. Fungal protoplasts were prepared from the germinated spored treated with lysing enzyme and β-glucuronidase. 4.3∼4.8×10⁴protoplasts of A . oryzae per ㎖ were obtained from ?? spores whereas 1.8∼2.5×10²protoplasts of T. inflatum per ㎖ were harvester. A. oryzae protoplasts have strong resistancy against the hygromycin B, up to 20 ㎍/㎖ of media. Less than 10% of the initial population of T. inflatum protoplasts were survived on minimal media containing 75㎍/㎖ hygromycin B. In phleomycin-resistancy, the population of A. oryzae portoplasts were killed up to 50% of the initial population on minimal media containing 50㎍/㎖ antibiotic and up to 80% on the complex media with the same amount of antibiotic. No survived colonies of T. inflatum protoplasts were observed on the minimal media containing phleomycin, 5 to 20㎍/㎖.

Filamentous Fungi Aspergillus species에서 Phytase 유전자의 분리 및 동정에 관한 연구

이응석,함영태 中央大學校 遺傳工學硏究所 1998 遺傳工學硏究論集 Vol.11 No.1

Phytic acid는 인의 주형태를 이루는 유기화합물로 옥수수, 밀, 대두박, 종실류와 같은 기타 곡류로부터 만들어지는 부산물 사료나 식물성 식품에 다량으로 존재하는데, phytic acid가 protein, vitamin 그리고 mineral 등과 상호 작용하는 항 영양인자이기 때문에 사람이나 돼지 또는 가금류의 단위 동물에게는 효율적인 영양을 제공할 수가 없다. 흡수하지 못한 인 성분 이 토양이나 하천에 유기되어지면 부영양화로 인해서 용존 산소 결핍을 초래한다. 이는 토양이나 하천에 서식하는 호기성 미생물들을 사멸시키면서 토양이나 하천의 부패를 유발시키는 원인이 되기도 한다. 이러한 phytate를 제거하는 방법으로 분해효소인 phytase에 대한 연구가 시작되었고, 이러한 효소적 처리방법이 효율적인 방법임이 밝혀졌다. Phytase는 유기인산을 포함하는 물질로부터 무기인산을 유리화시키는 효소이다. 본 연구에서는 Aspergillus group중 phytase에 대해 가장 연구가 활발히 되어있는 A.ficuum와 이것을 토대로 A. oryzae에서 phytase gene을 분리하고자 두 Aspergillus species에서 total RNA 및 mRNA를 추출하였고, RT-PCR을 통해 cDNA를 합성하였다. 합성된 cDNA를 PCR을 사용하여 증폭시킨 뒤 phytase gene size와 유사한 1.2kb와 1.4kb DNA fragments를 분리하여 pT7 Blue-T vector에 subcloning하여 pPHY9과 pPHY13을 선별하였다. 선별된 두 plasmids를 DNA sequencing 하여 각각 약 250bp, 260bp의 염기서열을 확인하였으며, 이를 gene search하였다. 1.4kb의 5 prime의 250bp DNA fragment(pPHY9)는 A. ficuum의 phyA gene과 약 40%의 similarity를 보였으며, 이는 향후 phytase gene 분리를 위한 probe로 확보하였다. Phytases (myo-inositol hexakisphosphate phosphohydrolase; EC 3. 1. 3. 8) are enzymes which catalyze the hydrolysis of phytic acid or phytate into myo-inositol and inorganic phosphate. Phytases are found in plants and a variety of microorganisms. Phytase production of Aspergillus species, A. oryzae and A. ficuum, are influenced by nitrogen source, ammonium ions, especially ammonium phosphate, are markedly superior to nitrate for the phytase production. For the isolation of phytase gene, A. oryzae and A. ficuum were cultured on PSM media containing ammonium phosphate. After 4days, mycelia were harvested by filteration and isolated total RNA and cDNAs were synthesized by RT-PCR. The phytase gene was amplified with the primers designed from the conserved phytase sequence of A. ficuum and then amplified 1.2 and 1.4kb of DNA fragments by secondary PCR were subcloned into pT7 Blue-T vector. They were transformed into E. coli HB101 and selected two clones, pPHY9 and pPHY13 using LB/amp/IPTG/X-gal media. After DNA sequincing, the similarity analysis between the amplified fragments and A. ficuum phytase gene were carried out by using BLAST and DNASIS. 5'-250bp of pPHY9 fragment was 40% of similarity with phytase gene of A. ficuum

곰팡이의 형질전환을 위한 Dominant Selectable Markers

함영태,김종수 中央大學校 遺傳工學硏究所 1992 遺傳工學硏究論集 Vol.5 No.1

Expression of Mature Thaumatin in Escherichia coli

함영태,Batt, Carl A. 中央大學校 遺傳工學硏究所 1991 遺傳工學硏究論集 Vol.4 No.1

Thaumatin, a sweet-tasting plant protein, is initially produced as a precursor, preprothaumatin. The presignal sequence at aminoterminal end containing 22 mostly hydrophobic amino acid residues and the prosignal sequence at carboxy-terminal end carrying 6 mainly acidic amino acid residues of preprothaumatin II were removed by using a synthetic oligonucleotide and site-specific mutagenesis to produce mature thaumatin in E. coli. The presignal sequence was replaced by a 34mer oligonucleotide carrying a Shine-Dalgarno sequence and a translational initiation codon. The first amino acid of prosignal sequence, leu-230, was changed to an ochre stop codon TAA. The met-thaumatin gene in the plasmid pUTH-3 carrying two copies of the lac UV5 promoters was expressed in E. coli. A trace amount of an immunoreactive 22 kDa protein of a molecular weight similar to mature thaumatin was observed in E. coli SVS-3 [pUTH-3].

유전자재조합기술을 이용한 미생물 생체발광 유전자의 활용

함영태,이응석,전억한 中央大學校 遺傳工學硏究所 1996 遺傳工學硏究論集 Vol.9 No.1

지난 10여년간 해양미생물에서 분리한 생체발광 유전자의 활용에 대한 연구가 활발히 진행되고 있다. 초기에는 생체발광 기작에 대한 연구가 진행되었으며, 그 후 생체발광에 관여하는 효소와 그 유전자에 대한 연구가 진행되었다. 이러한 유전자는 일반세균에 형질전환시켜 생체발광 유전자 재조합 미생물을 얻으므로서 그 활용의 폭을 넓히고 있다. 특히 식품에서의 응용 연구가 많이 진척되었고, 환경분야에서는 특정 오염물질을 선택적으로 측정할 수 있는 biosensor개발에 활용이 시도되고 있다. 더우기 real-time assay, 즉 빠른 검사 결과가 요구되고 있는 HACCP system에서는 더욱더 필요시 되고 있다. 이와 같은 연구는 생체발광에 의한 매우 적은 양의 빛을 측정하는 기구의 개발도 동시에 이루어 지고 있다. Some microorganisms such as Vibrio, Photobacterium, Alteromonas and Xenorhabdus are capable of emitting light, called bioluminescent. They are found in marine, freshwater and terrestrial environments. Bacterial bioluminescence reaction is catalyzed by a enzyme, luciferase encoded in luxA and luxB genes with the oxidation of reduced reiboflavin phosphate and a long-chain aldehyde in the presence of a molecular oxygen. When the lux genes are genetically engineered and introduced into new microorganism which is originally not bioluminescent, the recombinant cell becomes light emitting. The light produced by cell be quantitatively detected by a instrument like luminometer. It has opened up a wide range of applications, especially in the fields of food industry and environment. Bioluminescent recombinant cell can be used as a reporter for the detection of a certain specific microorganism or pollutants, control of microbial contamination, etc. Bioluminescence assay is rapid, sensitive, and specific and provides a real-time noninvasive method. This review aims to provide the basic knowledge of recent development and applications for nature of bioluminescence.

흑생강 제조 공정 최적화 및 기능성 흑생강 음료 제조

반영주,백무열,함영태,김혜경,김병용 한국산업식품공학회 2010 산업 식품공학 Vol.14 No.2

생강이 가지고 있는 항산화력을 최대화 시키기 위해 다양한 제조 공정에서 흑생강을 제조하였다. 증숙 온도 및 시간에 따른 DPPH radical 소거능을 측정하였고, 이를 반응표면 분석법에 의해 최적화된 제조 공정을 선정하였다. DPPH radical 소거능을 최대화 할 수 있는 최적 점을 설정한 결과 93.2oC에서 6시간의 증숙 공정이 설정되었다. 제조한 흑생강을 음료로 개발하기 위해, 매실 농축액과 꿀을 혼합하여 기능성과 선호도가 높은 음료를 제조하였다. DPPH radical 소거능, 플라보노이드 함량, 관능평가의 canonical 계수를 이용하여 수적 최적화를 통하여 최적 배합비를 구한 결과, 흑생강 추출물 14.2%, 매실농축액 5%, 꿀 10.8%로 나타났고 desirability가 0.615로 설정되었다. 이때의 종속 변수값들은 DPPH radical 소거능 46.0 mg/L, 플라보노이드 함량 29.9 mg/L, 선호도 5.284로 예측되었다. Black ginger, obtained from steaming and drying process, provides the various functional properties. This study was performed to investigate the optimum processing conditions for black ginger with high content of biologically active substance such as anti-oxidations. Optimum processing conditions such as temperature and time for black ginger was determined by response surface methodology (RSM) with manufacturing process and functionality. The optimum steaming condition was determined 6 hours at 93.2oC, and 82.7 mg/L DPPH scavenging activities was obtained at this condition. The black ginger drink was made with black ginger extracts, Japanese apricot, and honey. Interaction effects of these ingredients were investigated by modified distance based on design and analyzed by linear, nonlinear regression model, and RSM. The optimization of mixture ratio was made by statistical modeling using DPPH scavenging activities and sensory properties which are the important target constraints in drink. Total flavonoids showed a linear canonical form, while preference and antiradical activity showed a nonlinear canonical form indicating the higher interaction among mixtures. The response trace plot revealed that antiradical activity, sensory properties and total flavonoids were quite sensitive to the drink blending. The optimum formulation of the drink was set at 14.2% of black ginger extracts, 5% of Japanese apricot, and 10.8% honey.