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�й����ﻯѧ���������ѧ�� 2005, Vol. 21 Issue (04 ):516-516     DOI:
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Site-directed Mutagenesis Improves the Thermostability and Specific-activity of Phytase Mutated by F43Y and I354M,L358F
CHEN Hui 1),WANG Hong-Ning 2) *,YANG Wan-Shen 1),ZHAO Hai-Xia 2), WU Qi 1), SHAN Zhi 1) ( 1)College of Life and Science, 2) College of Animal Science and Technology, Sichuan Agricultural University, Ya'an 625014, China)

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ժҪ �������ĸPPNPm8��ֲ��øphyAm�������PCR�鵼�Ķ���ͻ��,����ֲ��ø43λ�ı������滻Ϊ�Ұ���(F43Y),����354��358λ���������ᡢ������ֱ��滻Ϊ������ͱ�����(I354M,L358F),�õ���2��ͻ����PPNPm-1(F43Y)��PPNPm-2(I354M,L358F).��ͻ����������������pPIC9kphyAm-1,pPIC9kphyAm-2�ڱϳ��ĸGS115�б��,�Ա��������ø���Բⶨ�����ȶ��Լ��.������:ͻ����PPNPm-1���ʷ�Ӧ�¶ȱ�δͻ����PPNPm8������3��,75�洦��10min,���ȶ������15%,�Ȼ������11%;PPNPm-2���ʷ�Ӧ�¶�δ�ı�,���ȶ��Ա�PPNPm8�����3%,�Ȼ�������6.5%.��ͻ��ǰ���ֲ��ø�ռ�ṹ���бȽ�Ԥ��,����ͻ�䰱����Tyr43��ռ�λ�����ڵ�Asn416֮���γ����,��ǿ��ø�����ȶ���.
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Abstract�� The inability of currently available phytase to tolerate heat denaturation from feed processing remains one of the major practical concerns.To enhance the catalytic efficiency and thermostability of phytase expressed in Pichia pastoris,the mutants F43Y (PP-NP m-1) and I354M, L358F (PP-NP m-2) were constructed in vitro by site-directed mutagenesis with long-distance inverse PCR. The recombinants pPIC 9k-phyA m-1 and pPIC 9k-phyA m-2 were expressed in Pichia pastoris. The comparison experiments of mutant enzymes with natural phytase showed that the optimum temperature of PP-NP m-1 was increased by 3��,and its thermostability was increased by 15%,when it was exposed for 10 min at 75��. Its specific activity was increased by 11%. The optimum temperature of PP-NP m-2 was not increased, and its thermostability was only increased by 3%. However, the specific activity of PP-NP m-2 was decreased by 6.5%. By comparison between natural phytase and mutant phytase, it was found that hydrogen bond between Tyr43 and Asn416 promoted the thermostability.
Keywords�� phytase   phyA gene   site-directed mutagenesis   Pichia pastoris   thermostability      
Received 2004-11-04;
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�»�.F43Y��I354M,L358F����ͻ���ֲ��ø���ȶ��Լ�ø���Եĸ��� [J]  �й����ﻯѧ���������ѧ��, 2005,V21(04 ): 516-516
.Site-directed Mutagenesis Improves the Thermostability and Specific-activity of Phytase Mutated by F43Y and I354M,L358F [J]  Chinese Journal of Biochemistry and Molecular Biol, 2005,V21(04 ): 516-516
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