Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, India.
J Biotechnol. 2010 Aug 20;149(1-2):8-15. doi: 10.1016/j.jbiotec.2010.06.017. Epub 2010 Jun 25.
The Pichia anomala gene PPHY, which codes for a cell-bound phytase, was isolated from genomic DNA by PCR, using oligonucleotide sequences derived from the N-terminal region of the purified phytase protein (Pphyp) and a degenerate primer derived from conserved sequences of yeast and fungal phytases as primers. The gene harbours an ORF of 1389bp, encoding a 462-amino-acid protein. The deduced amino acid sequence has similarity, to a varied extent, with those of phosphatases from Pichia stipitis (62%), Candida dubliniensis (51%), Candida albicans (51%), Arxula adeninivorans (35%) and phytases from Debaryomyces castellii (50%) and Pichia fabianii (39%). The sequence contains the phytase consensus heptapeptide motif (-Arg-His-Gly-X-Arg-X-Pro-) as well as two phosphohistidine signature motifs found in histidine acid phosphatases. After transformation of PPHY into the yeasts Saccharomyces cerevisiae, A. adeninivorans and Hansenula polymorpha, the last species was selected as the most suitable for synthesis of recombinant Pphyp. The cell-bound enzyme activities produced by wild-type P. anomala and transgenic H. polymorpha strains bearing the PPHY gene placed under the control of the inducible H. polymorpha-derived FMD promoter were characterized. In both cases, a molecular mass of approximately 380kDa was determined for the native enzyme (corresponding to a hexamer); the pH and temperature optima for the activity were 4.0 and 60 degrees C, respectively. The enzyme was active on phytic acid, p-nitrophenylphosphate, glucose-6-phosphate, ADP, sodium pyrophosphate, AMP, 1-naphthylphosphate and ATP. Based on the K(m)/K(cat) and further biochemical parameters, the enzyme was classified as a cell-bound phytase with acid phosphatase activity and not as acid phosphatase, despite its strong similarity to the latter class of enzymes. The yeast biomass containing phytase has been demonstrated to be useful as a feed additive in poultry and aquaculture, and dephytinization of foods and feeds.
从基因组 DNA 中通过 PCR 分离出毕赤酵母基因 PPHY，该基因编码一种细胞结合植酸酶，使用寡核苷酸序列从纯化植酸酶蛋白（Pphyp）的 N 末端区域和从酵母和真菌植酸酶的保守序列衍生的简并引物作为引物。该基因含有一个 1389bp 的 ORF，编码一个 462 个氨基酸的蛋白质。推导的氨基酸序列与酿酒酵母（62%）、杜布氏毕赤酵母（51%）、白色念珠菌（51%）、腺嘌呤营养型酵母（35%）和假丝酵母（50%）和毕赤酵母（39%）的植酸酶有不同程度的相似性。该序列包含植酸酶的保守七肽基序（-Arg-His-Gly-X-Arg-X-Pro-）以及在组氨酸酸性磷酸酶中发现的两个磷酸组氨酸特征基序。将 PPHY 转化为酿酒酵母、腺嘌呤营养型酵母和汉逊酵母后，选择后者作为合成重组 Pphyp 的最适酵母。对野生型毕赤酵母和携带受诱导汉逊酵母来源的 FMD 启动子控制的 PPHY 基因的转基因汉逊酵母菌株产生的细胞结合酶活性进行了表征。在这两种情况下，天然酶的分子量约为 380kDa（对应于六聚体）；活性的最佳 pH 和温度分别为 4.0 和 60°C。该酶对植酸、对硝基苯磷酸酯、葡萄糖-6-磷酸、ADP、焦磷酸钠、AMP、1-萘基磷酸酯和 ATP 均有活性。基于 K(m)/K(cat) 和进一步的生化参数，该酶被归类为具有酸性磷酸酶活性的细胞结合植酸酶，而不是酸性磷酸酶，尽管它与后一类酶具有很强的相似性。含有植酸酶的酵母生物质已被证明可作为家禽和水产养殖中的饲料添加剂，以及食品和饲料的脱植酸作用。
