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1.
Appl Biochem Biotechnol ; 170(6): 1533-45, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23700147

RESUMO

Genome shuffling is a powerful approach for efficiently engineering industrial microbial strains with interested phenotypes. Here we reported a high producer of nuclease P1, Penicillium citrinum G-16, that was bred by the classical physics-mutagenesis and genome shuffling process. The starting populations were generated by (60)Co γ-irradiation mutagenesis. The derived two protoplast fractions were inactivated by heat-treatment and ultraviolet radiation respectively, then mixed together and subjected to recursive protoplast fusion. Three recombinants, E-16, F-71, and G-16, were roughly obtained from six cycles of genome shuffling. The activity of nuclease P1 by recombinant G-16 was improved up to 1,980.22 U4/ml in a 5-l fermentor, which was 4.7-fold higher than that of the starting strain. The sporulation of recombinant G-16 was distinguished from the starting strain. Random amplified polymorphic DNA assay revealed genotypic differences between the shuffled strains and the wild type strain. The close similarity among the high producers suggested that the genetic basis of high-yield strains was achieved by genome shuffling.


Assuntos
Embaralhamento de DNA/métodos , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Melhoramento Genético/métodos , Genoma Fúngico/genética , Mutagênese/genética , Penicillium/fisiologia , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Proteínas Fúngicas/isolamento & purificação , Endonucleases Específicas para DNA e RNA de Cadeia Simples/isolamento & purificação
2.
Sheng Wu Gong Cheng Xue Bao ; 28(11): 1388-97, 2012 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-23457791

RESUMO

To establish a prokaryotic expression and purification protocol for nuclease P1 (NP1), we first obtained a synthetic NP1 by splicing 22 oligonucleotides with overlapping PCR. We constructed and transformed a secretory expression vector pMAL-p4X-NP1 into Escherichia coli host strains T7 Express and Origami B (DE3) separately. Then, the recombinant NP1 was purified by amylose affinity chromatography, and its activity, thermo-stability and metal-ion dependence were investigated systematically. The results indicated that the expressed fusion proteins MBP-NP1 (Maltose binding protein-NP1) existed mainly in soluble form both in host strains T7 Express and Origami B (DE3), but the specific activity of recombinant protein from Origami B(DE3) strain was higher than T7 Express strain (75.48 U/mg : 51.50 U/mg). When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased up to 258.1 U/mg and 139.2 U/mg. The thermal inactivation experiments demonstrated that the recombinant NP1 was quite stable, and it retained more than 90% of original activity after incubated for 30 min at 80 degrees C. Zn2+ (2.0 mmol/L) could increase enzyme activity (to 119.1%), on the contrary, the enzyme activity was reduced by 2.0 mmol/L Cu2+ (to 63.12%). This research realized the functional expression of NP1 in the prokaryotic system for the first time, and provided an alternative pathway for NP1 preparation.


Assuntos
Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Genes Sintéticos , Vetores Genéticos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
3.
J Control Release ; 107(3): 537-46, 2005 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16087268

RESUMO

Polyethylenimine (PEI) is one of the most potent non-viral vectors. We have developed a lactosylated PEI (Lac-PEI) to enhance cell-specific transfection and have shown that Lac-PEI is more efficient than unsubstituted PEI for gene transfer into immortalized cystic fibrosis airway epithelial SigmaCFTE29o-cells. As both intact PEI/plasmid and Lac-PEI/plasmid complexes are found in the cell nucleus, we have investigated the transcription efficiency of the plasmid complexed with PEI or Lac-PEI, according to the polymer nitrogen/DNA phosphate (N/P) ratio (from 0 to 20). The initiation of transgene transcription was analyzed in an acellular nuclease S1 transcription assay. For both PEI and Lac-PEI complexes, transcription efficiency varied with the N/P ratio of the complexes. Transcription inhibition was observed when plasmid DNA was either loosely (N/P<5) or tightly condensed (N/P>15). For an N/P ratio of 5 and up to 15, transcription of the complexed plasmid was as efficient as that of the free plasmid. Similar results were observed when gene expression was studied after nuclear microinjection of the complexes into SigmaCFTE29o-cells. Our study shows that condensation of DNA influences the accessibility of the plasmid to the transcription machinery. Interestingly, the charge ratios that allow the most efficient transcription are those usually known to be the most efficient for gene transfer in vitro and in vivo.


Assuntos
DNA/genética , Plasmídeos/genética , Polietilenoimina/química , Transcrição Genética , Fenômenos Químicos , Química Física , Eletroquímica , Eletroforese em Gel de Ágar , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lactose/química , Microinjeções , Microscopia Eletrônica , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
4.
Wei Sheng Wu Xue Bao ; 38(6): 449-53, 1998 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-12548924

RESUMO

Mycelia of Penicillium citrinum were adsorbed and immobilized efficiently within porous polyurethane. Nuclease P1 produced by the immobilized cells were studied in shaking flasks. The suitable glucose and peptone contents in medium were 10 g/L and 1 g/L, respectively. And the shaking speed was 180-200 r/min. After 48 h fermentation, the nuclease P1 activity reached 513.3 U/ml, the productivity was 3.6 times higher than that of the free cells. The production cost is obviously reduced. In repeated batch fermentation, the immobilized cells kept the capacity after 28 batches for 56 days with an average nuclease P1 activity of 507.4 U/ml.


Assuntos
Células Imobilizadas/metabolismo , Penicillium/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Meios de Cultura , Fermentação , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo
5.
Appl Microbiol Biotechnol ; 44(3-4): 425-31, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8597544

RESUMO

The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced. It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in length. The nucS gene had an open-reading frame of 963 base pairs and coded for a protein of 287 amino acid residues, comprising the signal peptide of 20 amino acids and a mature protein of 267 amino acids. The deduced amino acid sequence agreed well with the published amino acid sequence except for one substitution. Southern hybridization analysis showed that the nucS gene existed as a single copy in the A. oryzae chromosome. When the structural gene of nucS was fused with the promoter of the glaA gene and introduced into A. oryzae, the yield of secreted nuclease S1 increased about 100-fold compared with the recipient strain.


Assuntos
Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Sequência de Aminoácidos , Aspergillus oryzae/enzimologia , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Glucana 1,4-alfa-Glucosidase/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/isolamento & purificação
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