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Structure-based fragment shuffling of two fungal phytases for combination of desirable properties

文献类型： 外文期刊

作者： Bei, Jinlong 2 ; Chen, Zhuang 2 ; Fu, Jie 1 ; Jiang, Zongyong 2 ; Wang, Jinwen 1 ; Wang, Xunzhang 1 ;

作者机构： 1.Sun Yat Sen Zhongshan Univ, State Key Lab Biocontrol, Sch Life Sci, Guangzhou 510275, Guangdong, Peoples R China

2.Guangdong Acad Agr Sci, Inst Anim Sci, Guangzhou 510640, Peoples R China

关键词： 6-Phytase;Chimeras;Enzymes;Nucleotide 6-Phytase;Chimeras;Enzymes;Nucleotide sequence;Protein engineering;pH effects;Aspergillus fumigatus;Aspergillus niger;Pichia pastoris

期刊名称：JOURNAL OF BIOTECHNOLOGY （ 影响因子：3.307； 五年影响因子：3.778 ）

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收录情况： SCI

摘要： Aspergillus niger NRRL 3135 phytase (Anp) and Aspergillus fumigatus ATCC 13073 phytase (Afp) are quite different but mutually complementary in many properties. A semi-rational protein engineering strategy based on 3D structure and sequence alignmentwas used to take advantage of the desirable characteristics of both enzymes. Each phytase was divided into seven fragments, including regions I-VII (I, 1-47; II, 59-133; III, 139-172; IV, 178-237; V, 246-329; VI338-381; VII, 404-444). The equivalent regions were swapped to construct an array of chimeras. Among the functional chimeras expressed in the yeast Pichia pastoris, novel phytases with combinations of the most desirable properties, including heat-resistance, were obtained. Correlations of individual regions with detailed differences were established by systematic evaluation of the substitutions. Regions II and VI contributed to the difference in specific activity at pH 5.0. Regions IV and V of Anp fully accounted for its second pH optimum at pH2.5. Most influences of substitutions were additive, except those of regions V and VI. Exchanging both regions led to different impacts upon K sub(m) and activity at pi H 4.0 compared with the replacement of either.

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