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Xiong, Ai Sheng,Yao, Quan-Hong,Peng, Ri-He,Li, Xian,Fan, Hui-Qin,Guo, Mei-Jin,Zhang, Si-Liang Korean Society for Biochemistry and Molecular Biol 2004 Journal of biochemistry and molecular biology Vol.37 No.3
Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of $60^{\circ}C$.
Directed Evolution of Beta-galactosidase from Escherichia coli into Beta-glucuronidase
Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Liu, Jin-Ge,Xu, Fang,Cai, Bin,Guo, Zhao-Kui,Qiao, Yu-Shan,Chen, Jian-Min,Zhang, Zhen,Yao, Quan-Hong Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.3
In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli $\beta$-galactosidase and $\beta$-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more $\beta$-glucuronidase activity than wild-type $\beta$-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high $\beta$-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.
( Ai Sheng Xiong ),( Quan Hong Yao ),( Ri He Peng ),( Xian Li ),( Hui Qin Fan ),( Mei Jin Guo ),( Si Liang Zhang ) 생화학분자생물학회 2004 BMB Reports Vol.37 No.3
Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyII) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyII and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyII and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyIIs, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pustoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of I? pustoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that -4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and anoptimum temperature of 60℃.
Xue-Wei Cao,Hong-Mi Cui,Yuan Yao,Ai-Sheng Xiong,Xi-Lin Hou,Ying Li 한국식물학회 2017 Journal of Plant Biology Vol.60 No.4
Shoot branching (tillering) primarily determinesplant shoot architecture and has been studied in many plants. Shoot branching is an important trait in non-heading Chinesecabbage (Brassica rapa ssp. chinensis Makino). The B. rapassp. chinensis var. multiceps exhibits unique and multipleshoot branching characteristics. Here, we analyzed the variationin shoot branching between ‘Maertou,’ with multiple shootbranching, and ‘Suzhouqing,’ a common variety. The levelsof endogenous indole-3-acetic acid (IAA), zeatin ribosideand active gibberellins in the shoot meristem tissues of thetwo cultivars were quantified by enzyme-linked immunosorbentassay during the vegetative growth stage. High levels of IAAmaintained axillary bud dormancy and repressed axillary budoutgrowth allowing shoot branching to form in the vegetativestage in ‘Suzhouqing.’ In contrast, low levels of IAA did notinhibit axillary buds in ‘Maertou,’ while a high level of cytokininpromoted axillary bud growth and branch shoot development. Exogenous hormone (rac-GR24 and 6-benzylaminopurine)treatment showed that ‘Maertou’ was relatively sensitive tocytokinin, because the fold changes of cytokinin-responsivegenes in ‘Maertou’ were significantly more frequent than thosein ‘Suzhouqing’. Cytokinin was the direct regulator for axillarybud growth of ‘Maertou’. Compared with ‘Suzhouqing’,‘Maertou’ was sensitive to cytokinin and this weakened thestrigolactone–cytokinin branching pathway.