Kim Jong-Hyun, Sunako Michihiro, Ono Hisayo, Murooka Yoshikatsu, Fukusaki Eiichiro, Yamashita Mitsuo
Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.
J Biosci Bioeng. 2009 Feb;107(2):124-9. doi: 10.1016/j.jbiosc.2008.10.019.
A gene (apuA) encoding amylopullulanase from a starch-hydrolyzing lactic acid bacterium, Lactobacillus plantarum L137, which had been isolated from traditional fermented food made from fish and rice in the Philippines, was found to contain two unique amino acid repeating units in the N- and C-terminal region. The former is a six amino acid sequence (Asp-Ala/Thr-Ala-Asn-Ser-Thr) repeated 39 times, and the latter is a three amino acid sequence (Gln-Pro-Thr) repeated 50 times. To clarify the role of these repeating units, a truncated apuA in the C-terminal region was constructed and expressed in L. plantarum NCL21, which is the ApuA- derivative of strain L137. The recombinant truncated amylopullulanase (ApuADelta), which lacks the 24 kDa of the C-terminal repeat region, was purified and characterized, and compared with wild-type amylopullulanase (ApuA). The enzyme production and specific activity of ApuADelta were higher than those of ApuA. The two enzymes, ApuA and ApuADelta, showed similar pH (4.0-4.5) and temperature (40-45 degrees C) optima. However, the activity of ApuADelta was more stable in the pH and temperature than that of ApuA. The catalytic efficiencies of ApuADelta toward soluble starch, pullulan and amylose were higher than those of ApuA, although their substrate specificities towards saccharides were similar. From these results, we conclude that the C-terminal repeating region of ApuA is negatively involved in the stability of amylopullulanase and binding of substrates. Thus, the truncated amylopullulanase is more useful in processing of amylose and pullulan.
从菲律宾鱼米制成的传统发酵食品中分离出的植物乳杆菌L137是一种淀粉水解乳酸菌，编码支链淀粉酶的基因（apuA）在其N端和C端区域含有两个独特的氨基酸重复单元。前者是一个六氨基酸序列（天冬氨酸-丙氨酸/苏氨酸-丙氨酸-天冬酰胺-丝氨酸-苏氨酸），重复39次，后者是一个三氨基酸序列（谷氨酰胺-脯氨酸-苏氨酸），重复50次。为了阐明这些重复单元的作用，构建了C端区域截短的apuA，并在植物乳杆菌NCL21中表达，NCL21是L137菌株的ApuA衍生物。纯化并表征了缺少24 kDa C端重复区域的重组截短支链淀粉酶（ApuADelta），并与野生型支链淀粉酶（ApuA）进行比较。ApuADelta的酶产量和比活性高于ApuA。ApuA和ApuADelta这两种酶的最适pH（4.0 - 4.5）和温度（40 - 45摄氏度）相似。然而，ApuADelta在pH和温度方面的活性比ApuA更稳定。尽管ApuADelta和ApuA对糖类的底物特异性相似，但ApuADelta对可溶性淀粉、支链淀粉和直链淀粉的催化效率高于ApuA。从这些结果中，我们得出结论，ApuA的C端重复区域对支链淀粉酶的稳定性和底物结合有负面影响。因此，截短的支链淀粉酶在直链淀粉和支链淀粉的加工中更有用。
