• Title/Summary/Keyword: fusion hybrids

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Construction of bifunctional xylanase-cellulase fusion protein from Bacillus licheniformis NBL420 and its expression in E. coli (Bacillus licheniformis NBL420 유래의 Xylanase-Cellulase 활성을 갖는 융합단백질 제작과 대장균에서의 발현)

  • Hong, In-Pyo;Choi, Shin-Geon
    • Journal of Industrial Technology
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    • v.29 no.A
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    • pp.161-167
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    • 2009
  • The bifunctional Xylanase-Cellulase hybrid protein was constructed by gene fusion. Two genes corresponding to endoxylanase gene (xylS) and endocellulase gene (celA) were amplified by PCR from Bacillus licleniformis NBL420. It was then linked through splicing by overlap extension (SOE) by PCR method. The two resulting fused hybrids, xyl/cel and cel/xyl, which differ by its orientation, were confirmed by its nucleotide sequencings. One of two fusion genes, xyl/cel was successfully expressed into pET22b(+) vector (pxyl/cel) with bifunctional xylanase-cellulase activity. On the contrary, the other cel/xyl fusion protein showed only cellulase activity with much decreased xylanase activity. Enzymatic properties of Xyl/Cel fusion protein were investigated regarding optimum pH, optimum temp, thermostability, and pH stability. It was revealed that Xyl/Cel fusion protein retained the bifunctional xylanase-cellulase activities eventhough two enzymes were connected with each other directly. These informations could be useful for construction of other hybrid proteins as well as increased range of substrate utilization.

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Engineered Recombinant PON1-OPH Fusion Hybrids: Potentially Effective Catalytic Bioscavengers against Organophosphorus Nerve Agent Analogs

  • Lee, Nari;Yun, Hyeongseok;Lee, Chan;Lee, Yikjae;Kim, Euna;Kim, Sumi;Jeon, Hyoeun;Yu, Chiho;Rho, Jaerang
    • Journal of Microbiology and Biotechnology
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    • v.31 no.1
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    • pp.144-153
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    • 2021
  • Organophosphorus nerve agents (OPNAs), including both G- and V-type nerve agents such as sarin, soman, tabun and VX, are extremely neurotoxic organophosphorus compounds. Catalytic bioscavengers capable of hydrolyzing OPNAs are under development because of the low protective effects and adverse side effects of chemical antidotes to OPNA poisoning. However, these bioscavengers have certain limitations for practical application, including low catalytic activity and narrow specificity. In this study, we generated a fusion-hybrid form of engineered recombinant human paraoxonase 1 (rePON1) and bacterial organophosphorus hydrolase (OPH), referred to as GV-hybrids, using a flexible linker to develop more promising catalytic bioscavengers against a broad range of OPNAs. These GV-hybrids were able to synergistically hydrolyze both G-type OPNA analogs (paraoxon: 1.7 ~ 193.7-fold, p-nitrophenyl diphenyl phosphate (PNPDPP): 2.3 ~ 33.0-fold and diisopropyl fluorophosphates (DFP): 1.4 ~ 22.8-fold) and V-type OPNA analogs (demeton-S-methyl (DSM): 1.9 ~ 34.6-fold and malathion: 1.1 ~ 4.2-fold above) better than their individual enzyme forms. Among the GV-hybrid clones, the GV7 clone showed remarkable improvements in the catalytic activity toward both G-type OPNA analogs (kcat/Km (106 M-1 min-1): 59.8 ± 0.06 (paraoxon), 5.2 ± 0.02 (PNPDPP) and 47.0 ± 6.0 (DFP)) and V-type OPNA analogs (kcat/Km (M-1 min-1): 504.3 ± 48.5 (DSM) and 1324.0 ± 47.5 (malathion)). In conclusion, we developed GV-hybrid forms of rePON1 and bacterial OPH mutants as effective and suitable catalytic bioscavengers to hydrolyze a broad range of OPNA analogs.

Ultrastructural Process of Protoplast Fusion Between Lentinula edodes and Coriolus versicolor

  • Kim, Chae-Kyun;Kim, Byong-Kak
    • Mycobiology
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    • v.29 no.1
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    • pp.15-18
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    • 2001
  • Protoplast fusion is a useful technique for establishing fungal hybrids to overcome the natural barriers. The ultrastructure of protoplast and its fusion process were observed using a scanning electron microscopy(SEM) and a transmission electron microscopy(TEM). The protoplasts were variable in size from $0.5{\sim}15{\mu}m$ in diameter, and the mean diameter was about $3{\sim}5{\mu}m$. It was impossible to discriminate protoplasts of Lentinula edodes from protoplasts of Coriolus versicolor by size and surface structure. Big aggregates of the dehydrated protoplasts were observed, after polyethylene glycol 4000 treatment. Nucleus, mitochondria, lipid granules and various vesicles having granules were scattered in the cytoplasm. The vesicles were heterogeneous in size and vary from one protoplast to another. The fused membrane layer of the two protoplasts was observed. Time protoplast membrane contact and reorganization of membrane components were essential condition for protoplast fusion. Transmission electron micrograph showed fused protoplasts and flattening of the cells in the area of the membrane contact. We hope that our electron microscopic observations provide some insights into the understanding of the fusion process of protoplast in fungi.

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Development of Lipase Hyper-producing Strain from Hybrids between Aspergillus niger and Penicillium chrysogenum by Nuclear Transfer (핵전이에 의한 Aspergillus niger와 Penicillium chrysogenum의 잡종에서의 Lipase 고생산균주의 개발)

  • 양영기;문명님;이윤희;임채영
    • Korean Journal of Microbiology
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    • v.33 no.1
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    • pp.31-37
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    • 1997
  • Intergeneric hybrids between Aspergillus niger and Perricillium ch~y.sop~um(Tyr ), hyperlipolytic enzyne-producing fungi, were obtained by nuclear transfer technique:. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts were obtainrd by 1% Novozym 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KC]. Frequencies of hybrid formation by nuclear transfer were $1.3{\times}$10^{-3}$$ $-3.8{\times}$10^{-3}$$. From the chervation of genetic stability, conidial size, DNA content, ;md nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed 1.4-2.2 fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.

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Genetic Analysis of the Life Cycle in Interspecific Hybrids of Pleurotus ostreatus and Pleurotus florida Following Protoplast Fusion (원형질 체융합에 의한 느타리버섯과 사철느타리버섯 체세포 잡종의 유전 분석)

  • Yoo, Young-Bok;You, Chang-Hyun;Park, Yong-Hwan;Peberdy, John F.
    • The Korean Journal of Mycology
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    • v.14 no.1
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    • pp.9-15
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    • 1986
  • Interspecific hybrids of Pleurotus ostreatus and Pleurotus florida were formed by using protoplasts of complementing auxotrophs. The genetic markers were shown to segregate and recombine in the first generation of monosporus isolates from basidiocarp of seven fusion products. The analysis provides proof of heterokaryosis and strong evidence for haploidy of vegetative nuclei, a sexual cycle consisting of nuclear fusion and meiosis. In all the crosses there was no evidence of linkage between the genetic markers. Clamp connections were formed in monosporus mycelia from basidiocarp of fusion products.

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The Use of Aureobasidin A Resistant Gene as the Dominant Selectable Marker for the Selection of Industrial Yeast Hybrid (산업용 효모 Hybrid의 선별을 위한 우성선별표지로서의 Aureobasidin A 내성유전자의 이용)

  • Jeon, Han-Taek;Park, Uhn-Mee;Kim, Keun
    • Microbiology and Biotechnology Letters
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    • v.39 no.2
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    • pp.111-118
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    • 2011
  • For the strain improvement of the industrial polyploid yeast strain through hybridization and protoplast fusion, a dominant selection marker other than a recessive marker such as the auxotrophic marker was required for the selection of the resulting hybrids. In the present investigation, the aureobasidin A resistant gene was tested in relation to whether it can be used as the dominant selectable marker for the isolation of hybrids of the yeast Saccharomyces. The plasmid pAUR112, carrying the gene responsible for resistance to aureobasidin A, was introduced into the haploid yeast strain K114/YIp. From the rare-mating between polyploid C6 and haploid K114/YIp carrying pAUR112, many hybrids were obtained from the agar medium containing 0.5 ${\mu}g$/ml of aureobasidin A. The hybrids exhibited characteristics derived from both of the parental strains; and the cell sizes of the hybrids were larger than those of the parental strains. These results showed that the aureobasidin A resistant gene could be successfully used as the dominant selectable marker for the isolation of yeast hybrids resulting from rare-mating.

Interspecific Hybridization between Pleurotus ostreatus and Pleurotus sajor-caju by Protoplast Fusion (원형질체(原形質體) 융합(融合)에 의한 느타리와 여름느타리버섯의 종간(種間) 교잡(交雜))

  • Yoo, Young-Bok;Lee, Haing-Sook
    • The Korean Journal of Mycology
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    • v.22 no.4
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    • pp.378-385
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    • 1994
  • Interspecific somatic hybrids were obtained by protoplast fusion between Pleurotus ostreatus and Pleurotus sajor-caju. The fusion products between incompatible strains did not form clamp connections. Fruiting body of the clampless fusants was induced by light-dark cycle on saw-dust-rice bran substrate in glass bottles. Out of them, seven somatic hybrids produced fruiting bodies of intermediate morphology of the two species. Light and low temperature were the initiating factors for the development of clamped hyphae from the clampless mycelial colonies. All of these basidiocarps had clamp connections. Eight fusants from the six crosses were analysed with the segregation of genetic characters by random spore isolates. In the three combinations, unexpected alleles were shown. Somatic hybrid between P188 (P. ostreatus 2-1 + P. sajor-caju 2-53) and P. florida 2-3 by triple cross produced fruiting bodies similar to those of fusant between P. ostreatus and P. florida. All the genetic charaters from the three strains were shown to segregate and recombine.

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Construction of Starch-Fermenting Yeast Using Protoplast Fusion Technique (원형질체 융합 방법을 이용한 전분 발효성 효모의 개발)

  • Chun, Soon-Bai;Bai, Suk;Lee, Kee-Young;Kim, Soo-Hyun;Lee, Jin-Jong;Kim, Sang-Moon
    • Korean Journal of Microbiology
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    • v.28 no.2
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    • pp.120-127
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    • 1990
  • Intraspecific fusion frequency between Filobasidium capsuligenum CBS6122-2 ade and met trp or met strains was $2.9\times 10^{-3}$ and $8.5\times 10^{-3}$, respectively. And intergeneric fusion frequency between Folobadidium capsuligenum CBS 4318 (cys his) and Saccharomyces cerevisiae 262-12-1 (hom3 thr1) or X2180-1A (ade thr) was $8.8\times 10^{-6}$ and $9.5\times 10^{-4}$, respectively. Nuclear fusion appears to occru in fusants between intraspecies and intergenera as strongly suggested by DNA content, nuclear staining, comparison of survival rate to UV light and mitotic segregation analysis. It was also found that $\alpha$-amylase and glucoamylase activity from intraspecific hybrids was 1.6-2.1 fold increased when compared with that from thier parents.

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Development of Lipase Hyper-producing Strain from Hybrids between Aspergillus niger and Penicillium notatum by Nuclear Transfer (핵전이에 의한 Aspergillus niger와 Penicillium notatum 잡종에서의 lipase 고생산 균주의 개발)

  • Yang, Young-Ki;Moon, Myeng-Nim;Lee, Yoon-Hee;Kang, Hee-Kyoung;Lee, Jung-Sup;Lim, Chae-Young;Kim, Jong-Se;Rhee, Young-Ha
    • The Korean Journal of Mycology
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    • v.25 no.2 s.81
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    • pp.143-151
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    • 1997
  • Interspecific hybrids between Aspergillus niger and Penicillium notatum (Tyr-), hyperlipolytic enzyme-producing fungi, were obtained by nuclear transfer technique. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts was obtained by 1% Novozyme 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KCl. Frequencies of hybrid formation by nuclear transfer were $3.8{\times}10^{-3}{\sim}1.3{\times}10^{-3}$. From the observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed $1.2{\sim}1.7$ fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.

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