一种高耐热普鲁兰酶的克隆表达、酶学性质及其在粉丝制作中的应用

为了获得一种适合在粉丝制作中使用的高耐热普鲁兰酶,对Thermus thermophilus的普鲁兰酶基因进行了异源表达及应用性质研究。以T.thermophilus XQ5331基因组DNA为模板,通过PCR方法扩增获得普鲁兰酶编码基因TtP5331,在大肠杆菌中实现高效表达。重组酶TtP通过离子交换层析获得初步纯化。酶学性质研究结果显示,重组酶最适作用温度为75℃,最适pH6.5,在最适条件下比酶活为17 U/mg。重组酶TtP在90℃下保温10 min仍能保留约60%的酶活,沸水浴5 min可以完全灭活。在传统粉丝制作工艺基础上,采用先加酶后制芡糊的方式制作粉丝,按照每克芡糊淀粉1 U的量添加TtP处理芡糊,制作的粉丝的断条率为4.5%,与添加明矾获得的粉丝基本一致。以上结果说明,在酶法粉丝制作工艺中,TtP适合在芡糊制作前在淀粉悬液中添加,是一种用量小、使用简便的明矾替代物。

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.