Structure-based fragment shuffling of two fungal phytases for combination of desirable properties.
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 alignment was 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 pH 2.5. Most influences of substitutions were additive, except those of regions V and VI. Exchanging both regions led to different impacts upon K(m) and activity at approximately pH 4.0 compared with the replacement of either.
Bei
Jinlong
J
State Key Laboratory for Biocontrol, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China.
Chen
Zhuang
Z
Fu
Jie
J
Jiang
Zongyong
Z
Wang
Jinwen
J
Wang
Xunzhang
X
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003627:Data Interpretation, Statistical; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013696:Temperature
D005656:Fungal Proteins; D050506:Mutant Chimeric Proteins; D010832:6-Phytase