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1. The protective effect of organic media on protein conformation of covalent immobilization enzyme | |||
SU Jianhua,CHU Maoqing,ZHU Xiongjun,LIU Daling | |||
Biology 06 April 2013 | |||
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Abstract:In order to know the feasibility of protecting active conformation of enzyme during the covalent-immobilization, three lyophilized enzymes catalase (CAT), trypsin (TRY) and laccase (LAC) are immobilized under organic media and aqueous media, and kinetic constants are investigated. Specific activity of CAT immobilized in chloroform is 1.67 times larger with Kcat 2.43 times higher and Kmapp is 0.74 times smaller than that of CAT immobilized in aqueous phase. Specific activity of TRY immobilized in ethyl acetate is 5.33 times larger with Kcat 55.58 times higher and Kmapp is 0.40 times smaller than that of TRY immobilized in aqueous phase. Specific activity of LAC immobilized in ethyl acetate is 0.24 times larger with Kcat 0.29 times higher and Kmapp is 0.27 times smaller than that of LAC immobilized in aqueous phase.Results show that organic media has more advantages over aqueous media to remarkably improve activities and kinetic constant values. Organic media makes the enzymes under〝rigid〞state, and this "rigid" state helps enzymes maintain their original active conformation and prevent enzymes from conformation disorder during the modification reaction occurring. | |||
TO cite this article:SU Jianhua,CHU Maoqing,ZHU Xiongjun, et al. The protective effect of organic media on protein conformation of covalent immobilization enzyme[OL].[ 6 April 2013] http://en.paper.edu.cn/en_releasepaper/content/4535583 |
2. Identification, characterization and gene cloning of a phytase with potential industrial interest | |||
Ming-Lv Sun,Bo Zhou,Jian Sun,Dong-Min Zhao,Xiao-Yun Wang | |||
Biology 12 April 2007 | |||
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Abstract:A high phytase-producing strain of Aspergillus niger N-3 was identified by screening 94 microbial strains. The phytase displayed physicochemical characteristics of potential industrial interest with independent intellectual property. The enzyme activity in fermented broth achieved a maximum of 495 U/mL. The enzyme was purified by a combination of ammonium sulfate fractionation and gel filtration chromatography. The molecular weight of the enzyme as determined by SDS-PAGE was 60-80 kDa, with maximum activity at about 55 C (after incubation at 10 min). Dual optima pH (pH 2.0 and pH 5.5) was gained. Activity at pH 2.0, which is more suitable to the circumstance of monogastric animals’ stomach was about 30% higher than that at pH 5.5. The phytase retained about 45% of its enzymatic activity under heat treatment at 90 C for 5 min. It showed a greater affinity for sodium phytate (Km = 132.3 μM) than for pNPP (Km = 2.23 mM). The phyA gene was isolated and sequenced. The coding region without the introns and putative signal sequence was comprised of 1347 nucleotides. It encoded a polypeptide of 448 amino acids, exhibiting high amino acid sequence homologies (94.87%) with the typical phytase Aspergillus niger NRRL 3135. | |||
TO cite this article:Ming-Lv Sun,Bo Zhou,Jian Sun, et al. Identification, characterization and gene cloning of a phytase with potential industrial interest[OL].[12 April 2007] http://en.paper.edu.cn/en_releasepaper/content/12158 |
3. Screening, Cloning and Overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favorable characteristics | |||
Dong-Min Zhao,Min Wang,Xi-Jun Mu,Ming-Lv Sun,Xiao-Yun Wang | |||
Biology 09 April 2007 | |||
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Abstract:An Aspergillus niger spp. which produces extracellular phytase was isolated. The phyA gene was cloned and sequenced. The results show that the coding region comprises 1347 bp and the homology of nucleotide sequence and amino acid sequence with A. niger NRRL 3135 were about 96% and 93.8%, respectively. The coding sequence phyA fragment was cloned into Pichia secretive expression vector pPICZA and then transformed into chromosome of Pichia pastoris GS115 strain by electroporation. After stepwise screenings by antibiotics zeocin, yeast PCR and induction by methanol, one transformant showed high expression. The activity of fermented broth was 30000-fold of original Aspergillus niger spp. phytase (0.008 U•ml-1) and the specific activity was 503 U•mg-1 of protein. The Km value was 0.196 mmoll-1 for sodium phytate and 18.16 mmoll-1 for pNPP. It showed activity at pH range values of 2-6 with the optimum at 5.5. Studies on thermostability showed that the recombinant phytase remained 70% activity after exposure to 90℃ for 5 min and 65% for 30 min. Fluorescence analyses show that when the enzyme was treated at increasing temperatures, a little fluorescence red-shift was observed (4 nm) with an increase in emission intensity, which indicates that the conformation of the enzyme was very stable during the heating process. | |||
TO cite this article:Dong-Min Zhao,Min Wang,Xi-Jun Mu, et al. Screening, Cloning and Overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favorable characteristics[OL].[ 9 April 2007] http://en.paper.edu.cn/en_releasepaper/content/12053 |
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