, 张东晨1
, 吴学凤2, 邓胜松21. 安徽理工大学材料科学与工程学院, 淮南 232001;
2. 合肥工业大学食品与生物工程学院, 合肥 230009
收稿日期: 2021-02-19; 修回日期: 2021-04-25; 录用日期: 2021-04-25
基金项目: 国家自然科学基金（No.51274012）
作者简介: 王方略(1989—), 男, E-mail: wangfanglue66@163.com
通讯作者（责任作者）: 张东晨, E-mail: dchzhang202101@163.com
摘要：为探究漆酶降解聚丙烯酸酯（PAA）/阴离子型聚丙烯酰胺（HPAM）的微观机理，采用对接模拟了其结构模型与枯草芽孢杆菌漆酶（B. subtilis laccase）的结合.根据-CDOCKER_Energy score打分最高的原则，对获得的最佳结合构象进行分析.然后基于亲和力虚拟氨基酸突变进行丙氨酸（ALA）扫描和饱和突变.结合模式分析表明，HPAM比PAA可更深地埋入活性口袋，B. subtilis laccase对HPAM的亲和力和结合能皆高于PAA.相互作用分析表明，疏水相互作用可能对B. subtilis laccase与底物的结合起到促进作用，而氢键作用会阻碍该酶与底物的结合.通过ALA扫描进一步得知，ARG487、GLY486和TYR133是B. subtilis laccase降解HPAM的关键氨基酸残基，而ASP113和TYR133是B. subtilis laccase降解PAA的关键氨基酸残基.通过饱和突变表明，ASP113>ARG可以提高B. subtilis laccase降解PAA的活性，这些数据为理性设计增强活性的B. subtilis laccase突变体提供了理论参考.
关键词：漆酶阴离子型聚丙烯酰胺(HPAM)聚丙烯酸酯(PAA)对接虚拟氨基酸突变
Rational design of degradation activity of anionic polyacrylamide/polyacrylate by laccase
WANG Fanglüe1
, ZHANG Dongchen1, WU Xuefeng2, DENG Shengsong21. College of Material Science and Engineering, Anhui University of Science and Technology, Huainan 232001;
2. College of Food and Bioengineering, Hefei University of Technology, Hefei 230009
Received 19 February 2021; received in revised from 25 April 2021; accepted 25 April 2021
Abstract: In order to explore the degradation mechanism of anionic polyacrylamide (HPAM)/polyacrylate (PAA) by laccase, the docking was employed to investigate the binding modes of their structural model to Bacillus subtilis laccase (B. subtilis laccase). The optimal conformation was obtained and analyzed according to the highest principle of-CDOCKER_Energy score. Then, the alanine (ALA) scanning and saturated mutation were performed by the virtual amino acid mutation based on affinity. The analysis results of binding model showed that HPAM was buried deeper in the active pocket than PAA, and B. Subtilis laccase had higher affinity and binding energy with HPAM than PAA. The interaction analysis showed that the hydrophobic interaction may promote the binding of B. subtilis laccase to substrate, while the hydrogen bond could hinder their combination. It was further informed by the ALA scanning that the ARG487, GLY486 and TYR133 were the key amino acid residues of B. subtilis laccase degrading HPAM, while the ASP113 and TYR133 were the key amino acid residues in PAA degradation. The saturated mutation showed that the ASP113>ARG could enhance the activity of B. subtilis laccase degrading PAA. These data provided theoretical reference for rational design of B. subtilis laccase mutants with enhanced activity.
Keywords: laccaseanionic polyacrylamide(HPAM)polyacrylate (PAA)dockingvirtual amino acid mutation
1 引言(Introduction)阴离子型聚丙烯酰胺(HPAM)作为絮凝剂, 通常用于细煤的浮选加工过程(Zou et al., 2016).其中, HPAM的碳主链与细煤颗粒形成疏水相互作用, 而HPAM的阴离子与电负性的煤颗粒通过静电相互作用结合(Zou et al., 2018).现代选煤厂中工艺水一般是闭路循环回浮选过程, 但随着絮凝剂的大量使用, HPAM在选煤循环水中的大量累积不利于煤炭的浮选(Castro et al., 2015).此外, 含HPAM的选煤污水的排放对生态环境也会造成潜在威胁(Hu et al., 2018).因此, 急需对煤泥水中的HPAM进行脱除.
微生物酶法是一种降解HPAM的绿色、高效的方法, 其优势在于成本低、条件温和、无二次污染等(Zhao et al., 2020).Song等(2018)探究了细菌分泌的漆酶活性与HPAM浓度的关系, 试验结果表明, HPAM浓度的变化对细菌漆酶的活性不敏感, 这是因为细菌漆酶不仅能提高污染物的氧化速率, 还能扩大氧化底物的范围.在好氧环境下, 细菌漆酶可以从HPAM分子上得到4个电子, 将氧化剂分子氧还原成水分子(Tayssir et al., 2017).Bao等(2010)通过HPLC检测到, 聚丙烯酸酯(PAA)是HPAM降解的中间产物.HPAM经脱氨基后残留的碳骨架PAA作为碳源, 可供微生物生长(Nyyss?l? et al., 2019).PAA分子链是由加氧酶或氧化酶催化发生氧化而断裂, 这类似于脂肪酸上的α-C氧化(Joshi et al., 2017).但仅通过实验无法得到漆酶与底物相互作用的信息, 而且从土壤或水样中分离得到的酶活性一般不高, 且周期较长(Zhang et al., 2013).漆酶与HPAM或PAA的微观作用信息不仅可用于设计更高活性的漆酶突变体, 还有助于深入理解酶催化机理.
漆酶是一种多酚类的氧化酶, 用于催化氧化各种聚合物、酚类化合物的降解和芳香化合物的开环等(Thurston, 1994), 其主要来源于真核细胞, 如真菌、植物、昆虫等(Mayer et al., 2002).目前研究比较多的是白腐真菌分泌的漆酶, 常用于木质素的降解、合成染料的脱色和纺织废水的处理.但该酶存在要求低的pH、水力停留时间过长等缺点(Singh, et al., 2014; Chen et al., 2015).而细菌漆酶可以弥补真菌漆酶的缺点, 具有更广泛的工业和生物技术的应用价值, 但目前关于细菌漆酶降解环境污染物的报道还不多.
分子对接是一种优化受体蛋白与配体结合构象的分子模拟手段, 通常用于药物设计, 但也可以用来探究环境有机污染物与酶活性口袋的结合模式(Liu et al., 2018).目前, 细菌漆酶的三维晶体结构已经通过实验解析出来, 这为进一步在分子水平上探究漆酶与PAA或HPAM的相互作用提供了可能性(Singh et al., 2014). Nyyss?l?等(2019)通过实验在HPAM降解过程中仅能检测到漆酶及中间产物PAA的存在, 据此提出降解途径的假设.但关于HPAM或PAA与漆酶相互作用的报道还很少.基于此, 本文采用Discovery Studio 2019软件的CDOCKER半柔性对接程序分别进行枯草芽孢杆菌漆酶(B.subtilis laccase)与PAA和HPAM的分子对接, 然后根据亲和力的虚拟氨基酸突变进行丙氨酸(ALA)扫描和饱和突变, 该研究的数据用于理性设计高活性的B.subtilis laccase突变体, 以期对HPAM及其中间产物PAA进行彻底降解.
2 材料与方法(Materials and methods)2.1 细菌漆酶结构的获取细菌漆酶的三维晶体结构(Enguita et al., 2003)下载于蛋白质数据库(RCSB), PDB编号为1GSK, 分辨率为0.170 nm, A链.该酶来源于枯草芽孢杆菌(简称为B.subtilis laccase).采用Discovery Studio 2019软件的Prepare Protein工具对其进行去水加氢、删除全部配体、修补缺失的氨基酸残基等处理.图 1所示为预处理后的B.subtilis laccase晶体结构, 其中红色球体代表活性位点的位置.
图 1(Fig. 1)
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| 图 1 预处理后的枯草芽孢杆菌漆酶的晶体结构 Fig. 1Prepared crystal structure of B.subtilis laccase |
2.2 底物结构的获取酶促反应本质上是官能团之间发生的化学反应, 而高分子聚合物的二聚体包含其所有的反应官能团.因此, 选择HPAM和PAA的二聚体作为结构模型用于研究酶降解底物的作用机理.HPAM与PAA结构模型采用Discovery Studio 2019软件的Sketching工具绘制, 分子链两端采用甲基进行封端(图 2).采用分子力学方法对结构进行能量最小化, 力场选CHARMm(Tu et al., 2018).
图 2(Fig. 2)
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| 图 2 最小化的棍状HPAM(a)和PAA(b)结构模型 (元素包括碳(灰色)、氢(白色)、氧(红色)、氮(蓝色)) Fig. 2Minimized stick structural models of HPAM(a) and PAA(b)(Elements: carbon(gray), hydrogen(white), oxygen(red), nitrogen(blue)) |
2.3 分子对接选用Discovery Studio 2019软件的CDOCKER程序进行B.subtilis laccase与PAA或HPAM的对接.该程序采用半柔性对接, 首先, 高温动力学产生配体构象集, 然后, 采用模拟退火对配体构象集依次在受体的活性位点进行结构优化.默认参数设置是保存最佳的前10种配体构象, 采用-CDOCKER_Energy score进行打分排序, 选择打分最高的配体进行分析.该程序已经成功地用于血管紧张素转换酶的短肽抑制机理研究, 其中, 短肽来源于酪蛋白水解物(Tu et al., 2018).
2.4 基于亲和力的虚拟氨基酸突变为进一步探究B.subtilis laccase与HPAM或PAA对接中起关键作用的氨基酸残基, 采用Discovery Studio 2019软件的Calculate Mutation Energy(Binding)程序对酶与底物复合物中配体周围0.40 nm范围内的氨基酸残基进行ALA扫描, 然后选择突变能量变化较大的氨基酸残基进行饱和突变, 此数据可以帮助试验人员优化突变位点, 进而理性设计酶蛋白(Zong et al., 2015; Ren et al., 2019).
3 结果与讨论(Results and discussion)3.1 结合模式分析本研究选择HPAM和PAA的结构模型, 用于探究B.subtilis laccase的微观降解机理.目前还没有从分子水平上揭示B.subtilis laccase与PAA或HPAM之间的相互作用, 本文首次采用Discovery Studio 2019软件的CDOCKER程序探究其结合模式.配体的结合构象直接影响其与活性位点的结合情况, 最终影响降解效果.从图 3可以看出, HPAM和PAA都结合在B.subtilis laccase的活性位点上, 但HPAM的结合位置比PAA更接近于活性位点中的3个铜离子, 说明HPAM可以更好地结合在B.subtilis laccase活性中心上.
图 3(Fig. 3)
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| 图 3 B.subtilis laccase与PAA或HPAM的结合模式 (碳原子为绿色代表PAA, 而碳原子为粉色代表HPAM) Fig. 3The binding model of B.subtilis laccase with PAA or HPAM (Carbon of PAA is represented by green, while carbon of HPAM is represented by pink) |
从表 1的对接结果中的-CDOCKER_Energy score值可以看出, B.subtilis laccase对HPAM的亲和强度较PAA好(Tu et al., 2018).酶与底物结合能越大, 结合越稳定, 说明该酶的活性越高.通过对比-CDOCKER_Interaction_Energy score得知, B.subtilis laccase与HPAM结合更稳定, 说明该酶对HPAM有较高的酶活；B.subtilis laccase与PAA之间的结合能越小, 结合就越不紧密, 说明该复合物就越不稳定存在.从亲和力的角度判断, B.subtilis laccase难以PAA作为生长所需的碳源.Li等(2015)通过试验发现, 与利用HPAM的碳链作为碳源相比, 微生物更倾向于将HPAM的酰胺基水解成羧基, —NH2释放入溶液中, 以其作为营养物质(氮源).
表 1(Table 1)
| 表 1 通过CDOCKER工具获得的对接结果 Table 1 Docking results obtained from CDOCKER protocol | |||||||||
表 1 通过CDOCKER工具获得的对接结果 Table 1 Docking results obtained from CDOCKER protocol
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采用View interactions工具对酶的受体活性表面按氢键性质进行可视化, 结果如图 4所示.可以发现, B.subtilis laccase活性口袋入口很宽很大, 而且口袋底部是空的.PAA的大部分位于结合口袋外面, 仅有一少部分镶嵌在活性口袋里面.而与PAA相比, HPAM嵌入活性口袋更深, 说明HPAM与活性口袋结合更紧密.同时也发现, PAA和HPAM上的高分子链两端都与口袋处的氨基酸残基发生疏水性相互作用.HPAM侧链上的酰胺基和羧基都可以与口袋处的氨基酸残基发生氢键作用, 而PAA侧链上两个羧基都可以与口袋周围的氨基酸残基发生氢键作用, 具体相互作用细节将在后续探讨.
图 4(Fig. 4)
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| 图 4 B.subtilis laccase与HPAM或PAA的结合口袋形貌图 Fig. 4Morphology of binding pocket between B.subtilis laccase and PAA or HPAM |
3.2 相互作用细节分析通过Discovery Studio 2019软件对最稳定的酶与底物复合物进行可视化, 其相互作用关系如图 5所示.从图 5可以看出, HPAM的分子链两端与ARG487形成两个疏水相互作用(粉色虚线表示), 同时还与TYR118形成一个疏水相互作用, 而PAA的分子链两端与ARG487形成两个疏水相互作用, 说明疏水相互作用促进了B.subtilis laccase与底物的结合, 该酶对HPAM的活性高于PAA.HPAM羧基上的羰基氧原子与LYS135上的氮氢原子形成一个氢键(绿色虚线表示), 而HPAM酰胺基上的氨基氢原子与TYR133上的羰基氧原子形成一个氢键.LYS135上的氮氢原子同时与PAA羧基上的羰基氧原子和羟基氧原子形成两个氢键, 而PAA羧基上的羟基氢原子与TYR133上的羰基氧原子形成一个氢键.虽然氢键可以稳定酶与底物复合物, 但氢键对B.subtilis laccase与底物的结合不利.同时, 该酶与HPAM相互作用有4个氨基酸残基参与, 比PAA多了一个氨基酸残基, 这进一步说明该酶对HPAM的活性偏高.研究表明, 基于淀粉-碘化镉和总有机碳量(TOC)的结果的显著差异, 细菌更倾向于利用HPAM上的酰胺侧链, 以其作为生长的氮源, 而难以利用HPAM的碳链骨架作为生长的碳源, 即细菌很难利用其脱氨产物PAA作为营养物质(Li et al., 2015).因此, 分子对接的结果与试验结果相互验证.将上述相互作用分析的信息总结于表 2和表 3, 包含相互作用的残基、距离及类型等.
图 5(Fig. 5)
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| 图 5 HPAM-B.subtilis laccase(a)和PAA-B.subtilis laccase(b)三维相互作用图 Fig. 5The 3D interaction of HPAM-B.subtilis laccase (a) and PAA-B.subtilis laccase (b) |
表 2(Table 2)
| 表 2 B.subtilis laccase与HPAM最稳定的复合物相互作用分析 Table 2 Interaction analysis of the most stable B.subtilis laccase-HPAM complex | ||||||||||||||||||
表 2 B.subtilis laccase与HPAM最稳定的复合物相互作用分析 Table 2 Interaction analysis of the most stable B.subtilis laccase-HPAM complex
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表 3(Table 3)
| 表 3 B.subtilis laccase与PAA最稳定的复合物相互作用分析 Table 3 Interaction analysis of the most stable B.subtilis laccase-PAA complex | ||||||||||||||||||
表 3 B.subtilis laccase与PAA最稳定的复合物相互作用分析 Table 3 Interaction analysis of the most stable B.subtilis laccase-PAA complex
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3.3 ALA扫描为了确定酶与底物相互作用的关键氨基酸残基, 采用Calculate Mutation Energy(Binding)程序对配体周围0.4 nm范围内的氨基酸残基进行ALA扫描, 表 4和表 5皆显示了ALA扫描后突变蛋白与配体亲和力的能量变化.对于B.subtilis laccase-HPAM复合物(表 4), 可以发现ARG487、GLY486和TYR133突变为ALA, 突变能大于0.5 kcal·mol-1, 效果为不稳定, 即这种突变导致酶与底物的亲和力降低, 由此判断这3种氨基酸残基是B.subtilis laccase与HPAM结合的关键氨基酸残基.对于B.subtilis laccase-PAA复合物(表 5), 可以发现ASP113和TYR133突变为ALA, 效果为不稳定, 即这种突变会导致亲和力下降, 可以推测这2种氨基酸残基是B.subtilis laccase与PAA结合的关键氨基酸残基.通过比较发现, B.subtilis laccase-HPAM比B.subtilis laccase-PAA多了一个关键氨基酸残基, 进一步说明B.subtilis laccase-HPAM结合更紧密, 即该酶对HPAM的活性高于PAA.
表 4(Table 4)
| 表 4 B.subtilis laccase-HPAM的ALA扫描结果 Table 4 ALA scanning results of B.subtilis laccase-HPAM | ||||||||||||||||||||||||||||||||||||||||
表 4 B.subtilis laccase-HPAM的ALA扫描结果 Table 4 ALA scanning results of B.subtilis laccase-HPAM
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表 5(Table 5)
| 表 5 B.subtilis laccase-PAA的ALA扫描结果 Table 5 ALA scanning results of B.subtilis laccase-PAA | ||||||||||||||||||||||||||||||||||||||||||||
表 5 B.subtilis laccase-PAA的ALA扫描结果 Table 5 ALA scanning results of B.subtilis laccase-PAA
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3.4 饱和突变对于B.subtilis laccase-HPAM, 对ARG487、GLY486和TYR133等关键氨基酸残基进行饱和突变, 分别突变为除ALA外的其余19种标准氨基酸残基, 饱和突变结果列于表 6.当TYR133>THR、GLY486>LEU和ARG487>PRO时, 突变能变化最大, 分别为1.43、2.07和2.42 kcal·mol-1, 突变影响均为不稳定.这些突变会使该酶对HPAM的亲和力下降, 相互作用减弱, 最终导致对HPAM的活性降低.
表 6(Table 6)
| 表 6 B.subtilis laccase-HPAM饱和突变的结果 Table 6 Results of B.subtilis laccase-HPAM saturation mutations | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
表 6 B.subtilis laccase-HPAM饱和突变的结果 Table 6 Results of B.subtilis laccase-HPAM saturation mutations
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对于B.subtilis laccase-PAA, 对ASP113和TYR133等关键氨基酸残基进行除ALA外其余19种标准氨基酸残基的饱和突变, 结果如表 7所示.当ASP113>THR和TYR133>GLY时, 突变能变化最大, 分别为2.75 kcal·mol-1和1.22 kcal·mol-1, 突变影响均为不稳定, 这些突变会使受体与配体相互作用减弱.同时发现, 当ASP113>ARG时, 突变能变化最小, 为-0.63 kcal·mol-1, 影响为稳定, 这表明该点突变可以用于指导试验进行合理的氨基酸突变, 从而提高酶的活力.
表 7(Table 7)
| 表 7 B.subtilis laccase-PAA饱和突变的结果 Table 7 Results of B.subtilis laccase-PAA saturation mutations | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
表 7 B.subtilis laccase-PAA饱和突变的结果 Table 7 Results of B.subtilis laccase-PAA saturation mutations
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4 结论(Conclusions)1) 从HPAM和PAA叠合的结合构象来看, HPAM比PAA更接近于酶催化活性中心, 而且HPAM比PAA更深地嵌入到活性口袋中, 说明HPAM与B.subtilis laccase结合更紧密.无论是基于-CDOCKER_Energy score还是-CDOCKER_Interaction_Energy score, HPAM-B.subtilis laccase都高于PAA-B.subtilis laccase, 进一步说明该酶对HPAM的亲和力高于PAA.
2) 通过相互作用细节的对比得知, PAA与LYS135形成两个氢键, 而HPAM与LYS135只形成一个氢键, 可以推测氢键对B.subtilis laccase与底物结合不利.同时没有发现TYR118与PAA存在疏水相互作用, 而HPAM与TYR118形成一个疏水相互作用, 可以判断疏水相互作用促进了B.subtilis laccase与底物结合.
3) 通过丙氨酸扫描得到, ARG487、GLY486和TYR133是B.subtilis laccase降解HPAM的关键氨基酸残基, 而ASP113和TYR133是B.subtilis laccase降解PAA的关键氨基酸残基.
4) 通过饱和突变得到, 当TYR133>THR、GLY486>LEU和ARG487>PRO时, B.subtilis laccase降解HPAM的活性皆减小.当ASP113>THR和TYR133>GLY时, B.subtilis laccase降解PAA的活性皆较小, 而ASP113>ARG时, 该酶降解PAA的活性将增强, 这些数据将用于对B.subtilis laccase进行理性设计.
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