In vitro pullulanase activity of wheat (Triticum aestivum L.) limit-dextrinase type starch debranching enzyme is modulated by redox conditions

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• 作者：Anne Repellin ; Monica Bå ; ga ; Ravindra N. Chibbar
• 关键词：cDNA ; Germination ; Kernel development ; Limit-dextrinase ; Starch debranching enzyme ; Wheat (Triticum aestivum L.)
• 刊名：Journal of Cereal Science
• 出版年：2008
• 出版时间：March 2008
• 年：2008
• 卷：47
• 期：2
• 页码：302-309
• 全文大小：290 K

文摘

Expression of a limit-dextrinase (LD) type starch debranching enzyme (EC 3.2.1.41) was observed in developing wheat (Triticum aestivum L.) endosperm and germinating grains, indicating a role for the enzyme in both biosynthesis and degradation of starch. A full-length cDNA, TaLD1, encoding LD in wheat developing kernels was isolated and predicted to encode a 98.6 kDa mature protein active in amyloplasts. Isolated cDNA was expressed in Escherichia coli to produce a recombinant His-tagged LD, which mainly accumulated in inclusion bodies as an inactive enzyme. Extraction of His-tagged LD from the inclusion bodies followed by dialysis under thiol/disulphide redox conditions allowed partial refolding of the protein and detection of pullulanase specific activities by zymogram analysis and enzyme assays. Several active conformations were demonstrated by the recombinant TaLD1 and pullulanase activity could be modulated by redox conditions in vitro. The results suggest that cellular redox conditions may regulate the level of LD activity in wheat tissues.