耐热普鲁兰酶CBM68结构域中关键位点对其酶学性质的影响  

Key amino acid sites in the CBM68 structure of thermostable pullulanase and their effects on the enzymatic properties

作　　者：申莹莹[1,2,3] 郑宏臣[2,3] 李树芳[2,3] 付晓平[2,3] 徐健勇[2,3] 宋诙[2,3] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,工业酶国家工程实验室,天津300308 [3]中国科学院天津工业生物技术研究所,天津工业生物系统与过程重点实验室,天津300308

出　　处：《食品与发酵工业》2016年第3期12-17,共6页Food and Fermentation Industries

基　　金：国家863计划(2012AA022205);天津市科学基金(13JCYBJC39500)

摘　　要：通过对Anoxybacillus sp.LM18-11来源的耐热普鲁兰酶(Pul A)与麦芽三糖共结晶的晶体结构分析,在新命名的底物结合域CBM68中预测出4个与底物结合有关的氨基酸位点(Y14、D16、K62和R96),分别将4个氨基酸位点突变为丙氨酸,得到突变体Pul A^(Y14A)、Pul A^(D16A)、Pul A^(K62A)和Pul A^(R96A)。其中,突变酶Pul A^(Y14A)和Pul A^(R96A)的底物结合力及催化效率与野生酶Pul A相比均有明显降低,其K_m值分别比Pul A提高了4.2倍和2.5倍,k_(cat)/K_m分别为Pul A的29%和37%。同时,Pul A^(Y14A)和Pul A^(R96A)的最适作用温度均降低了10℃,Pul A^(R96A)的最适作用p H也降低了0.5个单位。说明Y14和R96是CBM68结构域中的关键氨基酸位点,这两个位点的发现为进一步探究CBM68结构域的作用机制奠定了基础。Based on the analysis of the crystalline structure of pullulananse（ Pul A）-maltotetraose complex,four key amino acid sites（ Y14,D16,K62 and R96） in a new carbohydrate-binding module CBM68 closely related to substrate binding were discovered. The thermostable pullulanase（ Pul A） was derived from Anoxybacillus sp. LM18-11. Four mutants of Pul A^Y14A,Pul A^D16A,Pul A^K62 Aand Pul A^R96Awere obtained respectively by site-directed mutation.Among them,Pul A^Y14Aand Pul A^R96Ashowed lower affinity for substrate and lower catalytic efficiency compared with the wild-type pullulanase（ Pul A）. The K_mvalues of Pul A^Y14Aand Pul A^R96Awere 4. 2 times and 2. 5 times higher than that of Pul A,respectively. The k_（cat）/ K_mvalues of Pul A^Y14Aand Pul A^R96Aonly remained 29% and 37% of the original k_（cat）/ K_mvalue（ Pul A）,respectively. Moreover,the optimum temperatures for both Pul A^Y14Aand Pul A^R96Awere 10 ℃lower than that for the wild-type pullulanase（ Pul A）. And Pul A^R96Aalso showed decreasing of the optimum p H by 0. 5units. All the results in this work have proved the significance of the key amino acid sites Y14 and R96 on the structure CBM68. Further study on the function of the two key amino acid sites could enhance the understanding of the functional mechanism of CBM68.

关 键 词：耐热普鲁兰酶 CBM68 定点突变 底物亲和力 催化效率 

分 类 号：TS201.25[轻工技术与工程—食品科学]