| 摘要: |
| 【目的】从成熟的小麦籽粒中纯化小麦木聚糖酶抑制蛋白，并研究其对木聚糖酶的抑制作用，从而探明小麦中木聚糖酶抑制蛋白对外加木聚糖酶的影响。【方法】采用硫酸铵盐析、Macro-prep CM阳离子交换层析、Macro-prep DEAE阴离子交换层析以及Macro-prep 25S 阳离子交换层析等方法，从小偃22小麦中分离纯化木聚糖酶抑制蛋白，通过SDS-PAGE电泳检测木聚糖酶抑制蛋白的纯度和分子质量，并进一步研究了不同温度和反应时间对木聚糖酶抑制蛋白抑制活力的影响。【结果】小麦木聚糖酶抑制蛋白分子质量约为29 ku，N端氨基酸序列为SVSSVVS，经NCBI BLAST比对，该抑制蛋白N端序列与其他木聚糖酶抑制蛋白无同源性，但与大麦几丁质酶的N端序列高度同源，却无几丁质酶活性；该蛋白对来自黑曲霉的GH11家族木聚糖酶有强烈的抑制活性，但对GH10家族的木聚糖酶不敏感；该蛋白与木聚糖酶结合的最适温度为30 ℃，最佳时间为30 min；该抑制蛋白的半失活温度为74 ℃，具有较好的热稳定性。【结论】所获得的小麦木聚糖酶抑制蛋白可能是一种新型的XIP型木聚糖酶抑制蛋白。 |
| 关键词: 小麦 木聚糖酶 木聚糖酶抑制蛋白 抑制性质 |
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| 基金项目:NSFC-河南人才培养联合基金项目（U1404324） |
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| Purification and properties of xylanase inhibitor protein in wheat |
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LIU Xinyu,CUI Shixiu,LIU Yanfang,et al
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| Abstract: |
| 【Objective】This experiment was conducted to purify xylanase inhibitory protein from mature wheat seed and study its inhibition to xylanase,which clarified the effects of xylanase inhibitor proteins in wheat-based diet on added xylanase.【Method】The xylanase inhibitory protein from wheat variety Xiaoyan 22 was purified by salting out with ammonium sulfate,Macro-prep CM cation exchange chromatography,Macro-prep DEAE anion exchange chromatography and Macro-prep 25S cation exchange chromatography.The purity and molecular mass of xylanase inhibitory protein were checked by SDS-PAGE.Then,the effects of temperature and reaction time on inhibition of xylanase inhibitor protein were investigated.【Result】The obtained inhibitor had a molecular mass of 29 ku approximately.Its N-terminal amino acid sequence of SVSSVVS was not similar to those of other xylanase inbititors by NCBI BLAST program,while it had high similarity to chitinases in barley.The protein had no chitinase activity and strongly inhibited the activity of Aspergillus niger GH11 family xylanases,while insensitive to GH10 family xylanases.The optimum inhibition temperature and time for the inhibitor protein and xylanase were 30 ℃ and 30 min,respectively.The inhibitor had high thermal stability,with a temperature of remaining half inhibitory activity of 74 ℃.【Conclusion】The obtained inhibitor in this work was probablly a noval class of XIP-type xylanase inhibitor. |
| Key words: wheat xylanase xylanase inhibitor protein inhibition characteristics |
