Cloning,Expression and Characterization of a Hyper-thermophilic Pullulanase and Its Application in Vermicelli Processing

In order to develop a hyper-thermophilc pullulanase suitable for vermicelli processing,the pullulanase gene was heterologously expressed and the application properties of recombinant enzyme were studied. Pullulanase encoding gene TtP5331 was cloned from genome DNA of Thermus thermophilus XQ5331 by PCR and over-expressed in Escherichia coli. The recombinant enzyme TtP was preliminarily purified by ion exchange chromatography. The results of enzymatic properties analysis showed that,the optimum temperature and pH of the TtP were 75℃ and 6.5,respectively. Under optimal conditions,the specific activity of TtP was 17 U/mg. After treatment at 90℃ for 10 min,more than 60% of the enzyme's activity was preserved,and the enzyme was completely inactivated after 5 min in a boiling water bath. Based on the traditional vermicelli-making process,TtP was used as a substitute of alum,which has been used as a treatment for starch paste. The enzyme was added before gelatinization at an optimal dosage of 1 U TtP for 1 g of paste starch. Under optimal conditions,the breaking rate of the vermicelli was 4.5%,which was comparable to that of vermicelli made by adding alum. The above results showed that in the process of enzymatic production of vermicelli,TtP can be added to starch suspension before gelatinization,thus,TtP is a low-dose,easy-to-use alum substitute.