Immobilization of Starch Phosphorylase from Germinating Wheat Seeds

Starch phosphorylase is an industrially important enzyme used in the production of glucose-l-phosphate. Here, we extracted the enzyme from the germinating wheat seeds and partially purified using ammonium sulfate fractionation. The partially purified enzyme showed maximum enzyme activity at pH 6.2 and pH 7.2 in the polysaccharide and glucose-l-phosphate formation directions, respectively. The enzyme showed maximum enzyme activity at 37 ℃ temperature with 50% of the enzyme activity at 32 ℃ and 42 ℃. The desalted ammonium sulfate fractionated enzyme has been immobilized on brick dust with nearly 60% enzyme activity retention. The specific activity of the immobilized starch phosphorylase increased from 0.410 to 0.925. There was a slight alkaline shift in the optimum pH when assayed in both the directions. The immobilized starch phosphorylase also displayed increased optimum temperature and thermo-stability and could be reused many times. The desalted ammonium sulfate fractionated enzyme exhibited a half life of 4 h at 30 ℃ and 30 rain at 50 ℃ whereas brick dust immobilized enzyme exhibited a half life of 7 h at 30 ℃ and 45 min at 50 ℃. The immobilized enzyme may be exploited for glucose-l-phosphate production.

Starch phosphorylase 2 is essential for cellular carbohydrate partitioning in maize

Carbohydrate partitioning is essential for plant growth and development,and its hindrance will result in excess accumulation of carbohydrates in source tissues.Most of the related mutants in maize(Zea mays L.)display impaired whole-plant sucrose transport,but other mechanisms affecting carbohydrate partitioning have seldom been reported.Here,we characterized chlorotic leaf3(chl3),a recessive mutation causing leaf chlorosis with starch accumulation excessively in bundle sheath chloroplasts,suggesting that chl3 is defective in carbohydrate partitioning.Positional cloning revealed that the chl3 phenotype results from a frameshift mutation in ZmPHOH,which encodes starch phosphorylase 2.Two mutants in ZmPHOH exhibited the same phenotype as chl3,and both alleles failed to complement the chl3 mutant phenotype in an allelism test.Inactivation of Zm PHOH in chl3 leaves reduced the efficiency of transitory starch conversion,resulting in increased leaf starch contents and altered carbohydrate metabolism patterns.RNA-seq revealed the transcriptional downregulation of genes related to photosynthesis and carbohydrate metabolism in chl3 leaves compared to the wild type.Our results demonstrate that transitory starch remobilization is very important for cellular carbohydrate partitioning in maize,in which Zm PHOH plays an indispensable role.

Cloning, Analysis and Prokaryotic Expression of DsSP Gene from Dunaliella salina

[Objective] The purpose of this study was to clone a starch phosphorylase gene from Dunaliella salina and to preliminarily analyze its basic properties and protein expression. [Method] RT-PCR and RACE （rapid amplification of cDNA ends） method was used for gene cloning; basic properties of the gene were analyzed using bioinformatics method; prokaryotic expression vector PGS21a-DsSP was constructed and transformed into E. coil BL21; the fusion protein was purified and detected by GST-SefinoseTM Kit and Western Blot, respectively. [Result] A starch phos-phorylase gene （GenBank accession No. KF061044） named DsSP was successfully isolated from D. salina. Basic properties, subcellular localization, secondary structure and tertiary structure of the protein were analyzed and predicted. The fusion protein was found in the supernatant and inclusion bodies. The supernatant protein was successfully purified. Western Blot analysis showed that the fusion protein was successfully expressed in E. coil BL21. [Conclusion] This study laid experimental foun- dation for further clarifying the function and mechanism of DsSP.