微生物学报  2017, Vol. 57 Issue (7): 1014-1025
http://dx.doi.org/10.13343/j.cnki.wsxb.20160386
中国科学院微生物研究所,中国微生物学会,中国菌物学会
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文章信息

刘艳如, 赵丙春, 董盼盼, 邱黎清, 黄建忠, 朱晓兰, 王作镇, 舒正玉. 2017
Yanru Liu, Bingchun Zhao, Panpan Dong, Liqing Qiu, Jianzhong Huang, Xiaolan Zhu, Zuozhen Wang, Zhengyu Shu. 2017
理性设计盐桥构建伯克霍尔德菌脂肪酶热稳定突变体
Computer-aid screening of thermostable lipase LipA from Burkholderia sp. ZYB002
微生物学报, 57(7): 1014-1025
Acta Microbiologica Sinica, 57(7): 1014-1025

文章历史

收稿日期:2016-10-01
修回日期:2016-11-11
网络出版日期:2016-12-02
理性设计盐桥构建伯克霍尔德菌脂肪酶热稳定突变体
刘艳如, 赵丙春, 董盼盼, 邱黎清, 黄建忠, 朱晓兰, 王作镇, 舒正玉     
福建师范大学生命科学学院, 工业微生物发酵技术国家地方联合工程研究中心, 教育部工业微生物工程中心, 福建 福州 350108
摘要[目的] 借助蛋白质工程技术提高伯克霍尔德菌ZYB002脂肪酶LipA的热稳定性,以期更好地将其应用于工业生产中。 [方法] 利用YASARA、FoldX、Rosetta、Gromacs等生物信息学软件,构建1个脂肪酶LipA的热稳定性提高的微型突变体电子文库;通过对突变体的结构信息和自由能变化进行评估,筛选出潜在的有价值的突变体。继而利用基因定点突变技术,构建上述候选突变体的突变基因文库,通过实验筛选出热稳定性提高的脂肪酶LipA突变体。 [结果] 利用上述方法,从构建的20个脂肪酶LipA突变体中,筛选到热稳定性有显著提高的3个突变体LipA-Asn125Asp、LipA-Asn125Glu和LipA-Gln262Glu。经55℃处理12 min后,上述3个突变体的T5012值较野生型分别提高4.0℃、5.5℃和4.4℃;在55℃下的半衰期较野生型分别提高了2.2倍、3.8倍和2.6倍。 [结论] 利用生物信息学软件,构建脂肪酶LipA突变体电子文库,结合蛋白质的结构信息,可以快速筛选到热稳定性提高的脂肪酶LipA突变体。
关键词: 伯克霍尔德菌     脂肪酶A     电子设计与筛选     盐桥     热稳定性突变体    
Computer-aid screening of thermostable lipase LipA from Burkholderia sp. ZYB002
Yanru Liu, Bingchun Zhao, Panpan Dong, Liqing Qiu, Jianzhong Huang, Xiaolan Zhu, Zuozhen Wang, Zhengyu Shu     
National & Local United Engineering Research Center of Industrial Microbiology and Fermentation Technology, Engineering Research Center of Industrial Microbiology, Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou 350108, Fujian Province, China
Received 1 October 2016; Revised 11 November 2016; Published online 2 December 2016
*Corresponding author: Yanru Liu, Tel/Fax:+86-591-22868212, E-mail:yrliu@fjnu.edu.cn
Zhengyu Shu, Tel/Fax:+86-591-22868212, E-mail:shuzhengyu@fjnu.edu.cn
Supported by the National Natural Science Foundation of China (31370802, 30870545), by the Key Project from Science and Technology Bureau of Fujian Province (2013H0021) and by the Natural Science Foundation for Distinguished Young Scholar of Fujian Province (2009J06013)
Abstract: [Objective] We improved the thermostability of lipase LipA from Burkholderia cecapia ZYB002 using protein engineering technology. [Methods] Lipase LipA mutant library was designed and screened using the following software, YASARA, FoldX, Rosetta, and Gromacs. We screened 27 thermostable lipase LipA mutants displaying the salt bridge effect among the resulting library of 341 variants, and then further screened using the site-directed mutagenesis technology. [Results] Three mutants LipA-Asn125Asp, LipA-Asn125Glu and LipA-Gln262Glu displayed improved thermostability. The T5012 value of LipA-Asn125Asp, LipA-Asn125Glu and LipA-Gln262Glu increased by 4.0℃, 5.5℃ and 4.4℃, respectively. The half-life of LipA-Asn125Asp, LipA-Asn125Glu and LipA-Gln262Glu at 55℃ increased by 2.23-fold, 3.8-fold and 2.6-fold, respectively. [Conclusion] It is feasible to screen thermostable mutant from the computationally designed library.
Key words: Burkholderia sp.ZYB002     lipase LipA     in silico design and screening     salt bridge     thermostable mutant    

微生物脂肪酶(Triacylglycerol acylhydrolase,EC 3.1.1.3) 又名三酰甘油酯水解酶,是一类能催化长链脂肪酸甘油酯水解为甘油和脂肪酸的生物催化剂。作为一种非水相酶,脂肪酶已被广泛应用到食品加工、精细化工、生物能源及制药等诸多领域,具有重要的经济价值[1]。大量微生物脂肪酶资源陆续被挖掘、开发,以Candida antarctica脂肪酶B、Burkholderia cepacia脂肪酶A和Rhizopus oryzae脂肪酶为代表的系列脂肪酶均已实现了商业化生产[2]。到目前为止,已开发的脂肪酶多为中温脂肪酶,欠佳的热稳定性一定程度上制约了脂肪酶的规模化工业应用。虽然近些年来陆续有嗜脂肪酶或耐热脂肪酶资源的零星报道[3-4],但极端脂肪酶到目前为止,尚未实现工业化生产及大规模工业化应用。

快速获得具有良好热稳定性的突变体酶分子,一直是生物催化剂领域诸多专家(包括学界和业界)的追求目标。近些年来,基于蛋白质工程技术实现快速筛选或构建热稳定的酶分子,得到迅猛发展,诞生了系列新方法新技术。以Reetz教授为代表的科学家基于酶分子氨基酸残基B-factor值的大小作为筛选影响酶分子热稳定性的潜在氨基酸残基突变位点的标准,继而利用迭代饱和突变技术(Iterative saturation mutagenesis),极大地缩小了突变文库容量,可快速筛选出理想的热稳定性突变体[5-6]。随着越来越多蛋白质晶体结构的解析及生物信息学的迅猛发展,通过构建电子文库,并以ΔΔGFold数值高低及突变体结构模型作为筛选标准,能进一步缩小突变文库的数量。Floor等利用此方法,从仅含150个突变体的突变库中快速筛选到表观熔点温度(Apparent melting temperature)提高了23 ℃,60 ℃下半衰期延长了200倍的卤代烷烃脱卤酶(Haloalkane dehalogenase)热稳定性突变体[7]。在酶分子3D结构信息指导下,还可直接理性设计出热稳定性突变体。如Zhou等利用来自于嗜热水解酶的结构模体(motiy)置换中温脂肪酶的相应模体获得的脂肪酶突变体,50 ℃下的温度稳定性提高了100倍[8]

本课题组自主分离筛选到1株脂肪酶高产菌株,经鉴定并命名为伯克霍尔德菌ZYB002 (Burkholderia sp.)[9]。在前期研究中,该菌株分泌的胞外脂肪酶LipA,在TMP纸浆造纸工艺中,可有效分解纸浆中的树脂成分,对控制树脂障碍具有一定的潜在价值[10]。为进一步提高该脂肪酶的应用效果,有必要利用蛋白质工程技术提高其热稳定性(机械浆温度高达80 ℃)。本文报道利用生物信息学软件设计、筛选潜在的Burkholderia sp. ZYB002脂肪酶热稳定性突变体;通过基因定点突变技术,获得重组突变体蛋白,筛选到性能优良的热稳定性突变体,实验验证了该技术的可行性和有效性。

1 材料和方法 1.1 材料

1.1.1 菌株与载体: Burkholderia sp. ZYB002菌株由本实验室从油污土壤中分离鉴定并保存[9];脂肪酶编码基因lipA及其对应的伴侣蛋白编码基因lipB由本实验室克隆并提交NCBI核酸数据库(登录号为:EU768869)。表达载体pACYC-lipA/lipB及重组表达菌株E. coli BL21(DE3)-pACYC-lipA/lipB由本实验室构建并保存[11]

1.1.2 工具酶、引物及试剂: PrimeSTAR HS DNA Polymerase、QuickCut Dpn I限制性内切酶及标准分子量的DNA Marker均购于宝生物工程(大连)有限公司;Wizard SV Gel and PCR Clean-Up System试剂盒购于普洛麦格(北京)生物技术有限公司;4-硝基苯月桂酸酯(p-Nitrophenyl laurate,pNPL)购于Sigma (美国)公司;氯霉素购于鼎国生物技术有限公司(北京);其他试剂均为市售分析纯。本实验使用的引物对如表 1
表 1. 本实验使用的PCR引物对 Table 1. Pairs of the primers used in this study
Primers Mutation sites Oligonucleotide sequence (5'→3’)* Tm /℃ Annealing temperature/℃
BF1F Q34K CGGCATCAAAGAGGACCTGCAACAG 65.3 60
BF1R GTCCTCTTTGATGCCGTACCAATACTCGAG 66.1
BF2F Q39K GGACCTGCAAAAAAACGGTGCGACC 65.3 60
BF2R CGCACCGTTTTTTTGCAGGTCCTCC 65.3
BF3F N40R CTGCAACAGCGTGGTGCGACCGTCT 68.5 63
BF3R TCGCACCACGCTGTTGCAGGTCCTC 68.5
BF4F T79E GCGACGGGGGCGGAAAAGGTGAATC 68.5 62
BF4F GCCGACGAGATTCACCTTTTCCGCC 66.9
BF5F A97K GCTATGTTAAAGCCGTCGCGCCCGATCTCG 70.2 60
BF5R GACGGCTTTAACATAGCGCGACGAGAGGCC 70.2
BF6F A100K CCGTCAAACCCGATCTCGTTGCGTC 66.9 62
BF6R CGGGTTTGACGGCAGCAACATAGCG 66.9
BF7F D102E GTCGCGCCCGAACTCGTTGCGTCGG 71.8 62
BF7R CAACGAGTTCGGGCGCGACGGCAGC 71.8
BF8F N125E GCCGACTTCGTGCAGGAAGTGCTGG 68.5 63
BF8R CGCCAGCACTTCCTGCACGAAGTCG 68.5
BF9F N125D AGGATGTGCTGGCGTACGATCCGAC 66.9 62
BF9R ATCGTACGCCAGCACATCCTGCACG 66.9
BF10F S135K CGGGCTTCGTTCATCGGTGATCGCC 68.5 63
BF10R CCGATGAACGAAGCCCGGTCGGATC 68.5
BF11F A140R GGTGATCCGTGCGTTCGTCAATGTG 65.3 60
BF11R ACGAACGCACGGATCACCGATGAAG 65.3
BF12F N144K GATCGCCGCGTTCGTCAAAGTGTTC 65.3 62
BF12R GATTCCGAACACTTTGACGAACGCG 63.6
BF13F S152K ACGAGCAAAAGCCACAACACCAACC 63.6 58
BF13R TTGTGGCTTTTGCTCGTCAGGATTC 62.0
BF14F T166K CTGCAGAAACTGACCACCGCACGGG 68.5 63
BF14R TGGTCAGTTTCTGCAGTGCGGCGAG 66.9
BF15F Y175D CCGCCACGGATAACCAGAACTATCC 65.3 60
BF15R ATAGTTCTGGTTATCCGTGGCGGCC 65.3
BF16F N178R TACAACCAGCGTTATCCGAGCGCGG 66.9 62
BF16R CGGATAACGCTGGTTGTACGTGGCG 66.9
BF17F V199K GACCGAAACCAAAGGCGGCAACACG 66.9 62
BF17R GTGCGTGTTGCCGCCTTTGGTTTCG 66.9
BF18F Q262E CTCCGGAGAAAACGACGGGCTCGTG 68.5 63
BF18R GTCGTTTTCTCCGGAGCCGCGGTTG 68.5
BF19F L278E GTACGGCAAGGTGGAAAGCACGAGC 66.9 62
BF19R TTGTAGCTCGTGCTTTCCACCTTGC 63.6
BF20F S281D CAAGGTGCTGAGCACGGATTACAAG 63.6 58
BF20R GGTTCCACTTGTAATCCGTGCTCAG 63.6
BF21F N285R CTACAAGTGGCGTCACCTCGACGAG 66.9 62
BF21R GAGGTGACGCCACTTGTAGCTCGTG 66.9
BF22F V305R GTATGCTGAAGATCCGCGTGCGGTG 66.9 60
BF22R ATCACCGCACGCGGATCTTCAGCAT 65.3
BF23F A306E GATCCGGTCGAAGTGATCCGCACGC 68.5 62
BF23R GCATGCGTGCGGATCACTTCGACCG 68.5
*Bold nucleotides: the mutated sites.

1.2 影响脂肪酶LipA热稳定性的潜在突变位点的筛选

影响脂肪酶LipA热稳定性的潜在突变位点筛选方法如下:(1) 脂肪酶LipA三级结构模型的模拟、优化及评估,具体方法参照刘艳如等[12]。(2) 利用YASARA软件[13],确定距离脂肪酶LipA活性中心氨基酸残基Ser87(组成活性中心催化三联体...Ser87...His286...Asp264...的氨基酸残基之一) 9 Å范围以内的所有氨基酸残基。(3) 在排除上述活性中心及其临近氨基酸残基的基础上,利用FoldX软件和Rosetta软件对脂肪酶LipA其他位点的氨基酸残基进行虚拟饱和突变,并计算出每个突变体的自由能变化值[14-15]。(4) 以ΔΔG<-0.5 kcal/mol (FoldX软件分析)和ΔΔG<0 kcal/mol (Rosetta软件分析)作为热稳定性提高的突变体的筛选标准,合并上述2个突变电子文库。(5) 利用YASARA软件对上述突变电子文库中的每个突变体的三级结构进行分析,删除结构明显不合理的突变体[7];(6) 剩余的突变体,进一步利用YASARA软件进行分析,筛选出突变位点的氨基酸残基与周围氨基酸残基之间能形成盐桥的突变体,作为本实验的研究对象。

1.3 脂肪酶lipA基因突变体的构建

以质粒pACYC-lipA/lipB为模板,利用表 1中的引物对,PCR扩增全长质粒。PCR扩增程序为:95 ℃ 5 min;95 ℃ 30 s,退火30 s (退火温度参考表 1),72 ℃ 6 min,25个循环;72 ℃ 6 min。PCR扩增产物经电泳鉴定后,加入0.5 μL QuickCut Dpn I限制性内切酶(25 μL PCR反应体系) 37 ℃酶切4 h后,电泳、纯化目的产物,并转化E. coli BL21 (DE3) 感受态细胞。重组转化子经培养后,抽提质粒并送交测序公司进行序列分析。

1.4 脂肪酶lipA基因及其突变体的诱导表达

将测序验证正确的重组转化子转接到5 mL LB培养基(含34 μg/mL的氯霉素)中,37 ℃、220 r/min振荡培养12 h。按2% (V/V)的接种率转接到20 mL LB培养基中(含34 μg/mL的氯霉素,250 mL三角瓶),37 ℃、220 r/min培养至OD600达0.6-0.9时,加入IPTG至终浓度为1 mmol/L。30 ℃、220 r/min培养16 h后,离心收集菌体。用pH 7.4 20 mmol/L Na2HPO4-NaH2PO4缓冲溶液洗涤菌体1次,并重新用8 mL相同的缓冲溶液悬浮菌体。悬浮菌体利用超声破碎仪进行裂解,裂解条件为:处理时间6 min,功率35%,工作2 s,间歇2 s。4 ℃、12000 r/min,离心10 min,收集上清液,作为粗酶液,进行后续分析。

1.5 脂肪酶酶活的测定

脂肪酶酶活的测定采用比色法[15]。具体酶学反应体系及测定方法参照刘艳如等[12]

1.6 热稳定性提高的脂肪酶突变体的筛选

脂肪酶热稳定性的测定方法参照刘艳如等[12]。比较突变体脂肪酶与野生型脂肪酶在55 ℃处理12 min后的残余酶活,筛选出残余酶活大于野生型脂肪酶的突变体,进行复筛。

1.7 脂肪酶及其突变体T5012t1/2的测定

T5012t1/2的测定方法参照Zhao和Arnold[16]。具体实验方法、酶活测定体系等参照刘艳如等[12]

1.8 分子动力学模拟

利用Gromacs 4.5.6软件在55 ℃条件下对野生型脂肪酶LipA和3个阳性突变体进行分子动力模拟分析,分析其RMSD变化趋势。分子动力学模拟使用的力场为CHARMM27,溶质原子距离盒子边缘的距离设置为0.9 nm。同时,向水溶剂中添加合适的抗衡离子(Na+/Cl-),使模拟系统成电中性。系统能量优化采取最陡下降法(Steepest descents)。经NVT平衡和NPT平衡后,在系统达到温度328 K、压力1.05 bar、密度998.3 kg/m3时,对系统进行时长为1 ns的MD分析[17]

2 结果和分析 2.1 热稳定性增强且存在盐桥效应的LipA突变体电子文库的构建

利用YASARA软件,对模拟的LipA 3D结构进行分析,以距离Ser87 9 Å为标准,可将Ser87、His286、Asp264、Leu17和Gln88 (前3个氨基酸残基为组成催化三联体的氨基酸残基,后2个氨基酸残基为构成阴离子氧洞的氨基酸残基)等40个氨基酸残基排除在突变范围之外。以ΔΔG<-0.5 kcal/mol为标准,利用FoldX构建的虚拟饱和突变文库含511个突变体;而以ΔΔG<0 kcal/mol为标准,利用Rosetta构建的虚拟饱和突变文库含322个突变体。合并上述2个突变体文库后,构建出1个覆盖率更高、包含711个突变体的电子文库。利用YASARA软件,逐一评估上述711个突变体的3D结构,删除空间结构冲突或空间结构不合理的突变体后,最终获得1个包含341个突变体的微型电子文库(表 2)。对上述341个突变体进一步分析,发现其中27个突变体存在盐桥效应(图 1)。

表 2. 热稳定性脂肪酶LipA突变体电子文库及其对应的自由能变化值 Table 2. The thermostable lipase LipA mutant library designed by computer aid and the corresponding ΔΔG value of every mutant
Mutation site △△G/(kcal/mol) Mutation site △△G/(kcal/mol) Mutation site △△G/(kcal/mol) Mutation site △△G/(kcal/mol)
G3R -0.7785 A128S -0.6913 A186T -0.5100 V240D -0.178
G3K -0.5344 A128D -0.4540 A186V -0.5580 V240Q -0.246
T7V -0.8362 V129K -0.5161 A186K -0.6990 V240K -0.321
R8K -0.1200 A128K -0.7085 T192L -0.5124 L243T -0.051
D21Q -0.1930 Y129S -0.8340 T192N -0.5048 L243S -0.240
D21E -0.3920 V129L -0.7060 T192H -0.5344 T245K -0.921
A24C -0.5588 V129R -0.9818 T192P -1.4413 T245N -0.147
A24E -0.6514 V129E -0.2590 A194L -0.5913 L246R -1.205
A24H -0.8405 Y129A -0.3040 A194R -0.6576 A247L -0.570
A24G -1.3081 Y129T -0.4570 A194K -0.1120 A247R -0.871
A24L -1.3944 Y129N -0.4930 A194N -0.1400 A247D -0.008
A24R -2.7022 Y129Q -0.5260 A194S -0.2260 A247V -0.115
A24N -1.3826 L134R -0.8425 A194Q 0.2890 A247E -0.201
A24S -0.0110 L134D -0.5611 T198R -0.8394 A247Q -0.223
A24D -0.1850 S135T -0.5618 T198H -0.5816 A247I -0.399
A24K -1.3030 S135Q -0.6608 T198D -1.0785 F249R -0.517
A24Q -05320 S135R -1.8750 V199I -0.9693 G250N -0.450
L27I -0.1160 S135K -1.4986 V199H -0.6688 G250A -0.576
E28P -2.1582 S136Q -1.2996 V199L -1.0340 G250K -0.767
Q34R -0.9208 S136P -2.4084 V199R -2.1772 G250I -0.716
Q34K -0.5994 S136R -1.3254 V199K -1.3822 G250T -0.347
Q38R -0.8354 S136K -1.6446 H204K -0.6588 G250E -0.507
Q39R -0.9360 S136E -1.1263 H204I -0.8610 M255L -0.832
N40K -0.0860 V138L -0.9007 H204L -1.2558 Q262E -1.464
N40R -0.3750 V138I -0.5829 H204M -0.5532 K269E -1.712
T43R -0.9446 S138K -0.6372 L206F -0.7061 K276D -0.087
T43L -1.1647 A140L -0.9693 L206Y -0.8394 L278E -0.112
T43K -1.1706 A140K -0.5391 A210C -0.5386 S279D -0.533
T43E -0.0350 A140D -1.0960 A210L -0.6448 S281E -0.659
T43I -0.3740 V143L -0.5951 A210V -0.5816 S281D -0.109
V44I -0.6242 V143I -0.7605 A210T -0.2900 S281N -0.124
A47V -0.9243 N144R -0.7610 L218K -1.0785 N285R -3.381
A67K -0.2760 V145L -0.5312 L218N -0.0050 N285K -1.936
A67R -0.6817 V145I -0.5654 L218S -0.1150 L294K -0.645
A67N -0.0150 V145R -0.5241 L218T -0.1760 L294R -0.517
A67E -0.0820 T150R -0.6940 S219E -0.4060 L294N -0.378
K70L -0.6738 T150C -0.7912 S219T -0.3770 V296L -0.715
T71A -0.5249 T150K -0.9236 S219Q -0.9693 V296I -0.655
T71L -0.9591 S152K -0.6748 V220I -2.1772 V296K -0.967
T71K -0.9708 S153T -0.4030 V220T -0.7750 A299L -0.927
T71E -0.6674 S153R -0.6373 F221D -1.3700 A299V -0.622
T71Q -0.8760 S153K -0.5605 T224V -1.0490 A299I -0.736
T71I -1.0157 S153E -0.6592 T224I -1.328 A299R -0.734
T71R -1.1490 S153Q -0.6412 T224R -1.308 A299C -0.893
T71D -0.0460 S153D -0.6851 T224K -1.789 A299Q -0.599
V72I -1.0980 S153N -0.0520 G225A -0.571 A299T -0.047
A74N -0.0620 S153V -0.2740 G225L -0.745 A299K -1.059
A75Q -0.0850 N155D -0.5002 G225K -0.772 E302P -0.971
A75I -0.1030 T156G -0.7054 G225D -0.876 E302Q -0.238
A75K -0.1090 T156A -0.7947 G225R -1.012 V305R -0.586
A75L -0.3200 T156R -0.9517 T227K -0.563 V305Q -0.038
A75E -0.5730 T156S -0.6136 D228K -1.126 A306L -0.555
A78R -0.9365 T156C -0.8422 T229S -0.558 A306S -0.651
A78S -0.6248 T156D -0.7406 T229E -0.919 A306Q -0.504
A78K -0.9667 T156N -0.8129 T229Q -0.875 A306E -0.067
T79P -0.9351 T156L -1.0726 T229R -2.095 A306K -0.157
T79R -0.9866 T156P -1.0719 T229K -1.584 I308M -0.642
T79E -0.7094 T156K -1.2328 T231Q -0.783 T310E -1.226
T79K -1.0755 T156E -1.0154 T231R -1.979 T310I -1.043
K80R -0.8256 T156Q -1.0131 L232Q -0.171 T310R -0.556
A97R -0.8281 N157D -0.7379 L232E -0.296 T310K -0.843
A97K -1.5113 L161E -0.5420 L232K -0.458 T310Q -0.888
A98I -0.6848 Q165K -0.5024 L234A -0.895 T310N -0.636
A98L -1.6157 T166Q -1.7472 L234G -1.366 T310S -0.331
A98R -2.2462 T166N -1.5465 L234R -1.456 A312L -0.534
A98K -1.4528 A170Q -0.5121 L234D -1.209 A312M -3.206
A100L -1.4349 A170D -0.5624 L234N -0.214 N313A -0.632
A100R -1.3800 A170E -0.0770 L234D -0.220 N313K -0.903
A100K -1.4495 A170T -0.0830 L234K -0.012 N313Q -0.656
D102R -0.7704 A173E -0.7490 L234K -0.309 N313H -0.544
D102E -0.6733 T174R -0.9199 V235G -0.045 N313L -1.870
D102Q -0.7028 T174N -0.0700 V235A -0.169 N313R -0.136
D102N -0.5314 Q177N -0.0650 V235E -0.304 R314I -0.043
V104I -0.7344 Q177K -0.3540 V235T -0.431 R314N -0.249
S117M -1.3885 N178Q -0.9495 V235S -0.523 R314E -0.322
N125K -0.0370 N178L -1.0417 V235D -0.563 R314A -0.358
N125T -0.1110 N178R -1.0936 V235N -0.631 R314Q -0.389
N125L -0.1610 N178K -1.1880 V235L -0.811 R314K -0.422
N125D -0.1960 P180R -0.8366 V235I -0.575 R314K -0.518
N125I -0.2750 A186E -0.6911 V235R -0.793 K316R -0.543
N125Q -0.7590 A186S -0.0020 V235K -0.773 L317R -0.949
N125E -1.3540 A186D -0.1090 V235Q -0.786 L317K -0.564
V126L -0.7786 A186L -0.0510 N239R -1.611 L317N -0.077
V126I -0.8724 A186C -0.2560 N239K -2.227
A128N -0.6060 A186H -0.2580 V240E -0.170
A128L -0.5365 A1861 -0.2640 V240N -0.141

图 1 在LipA突变体电子文库中存在盐桥效应的突变体 Figure 1 The corresponding mutation sites to form salt bridge in the LipA molecular model.

2.2 突变体的构建及突变效应

上述27个突变体中,LipA-Asn40Arg和LipA-Asn40Lys、LipA-Ala100Arg和LipA-Ala100Lys、LipA-Asn125Asp和LipA-Asn125Glu、LipA-Asn178Arg和LipA-Asn178Lys、LipA-Val199Arg和LipA-Val199Lys为同一位点突变成性质相似的不同氨基酸残基。在第1轮筛选实验中,仅构建了突变体LipA-Asn40Arg、LipA-Ala100Lys、LipA-Asn125Asp、LipA-Asn178Arg和LipA-Val199Lys。上述突变体经验证后,若具有正效应,再构建对应的另外1个突变体(如LipA-Asn125Glu),因此实际仅对23个突变体进行了基因突变的工作(表 1)。最终经测序验证,成功获得20个突变体的重组蛋白。另外3个突变体(Tyr175Asp、Asn178Arg和Asn285Arg),多次实验结果均显示,虽然在突变位点发生了正确的基因突变,但同时在其他位置均存在插入突变。插入突变的引入与上述突变位置附近的核苷酸序列结构特点(如GC含量、回文序列等)有关。后续实验中,PCR扩增体系及条件还有待进一步优化。不同突变体的突变效应如表 3。3个突变体LipA-Asn125Asp、LipA-Asn125Glu和LipA-Gln262Glu较野生型脂肪酶LipA,热稳定性有显著性的提高。55 ℃处理12 min后,突变体LipA-Asn125Asp、LipA-Gln262Glu和LipA-Asn125Glu的残余酶活较野生型LipA分别提高了40.99%、52.3%和57.69%。

表 3. 经热处理后,脂肪酶突变体与野生型脂肪酶残余酶活的比较 Table 3. The difference comparision of the residual activity between LipA mutants and the wild-type LipA
LipA mutants Increase rate of residual activity/% LipA mutants Increase rate of residual activity/%
LipA-Gln34Lys 3.52±1.14 LipA-Ala140Arg 1.25±0.51
LipA-Gln39Lys -8.899±0.600 LipA-Asn144Lys -1.070±1.067
LipA-Asn40Arg 2.56±0.65 LipA-Ser152Lys -8.35±2.15
LipA-Thr79Glu -1.55±1.22 LipA-Thr166Lys -9.12±1.56
LipA-Ala97Lys -7.83±1.25 LipA-Val199Lys -9.60±0.26
LipA-Ala100Lys -4.23±0.26 LipA-Gln262Glu 52.30±2.13
LipA-Asp102Glu -6.92±1.53 LipA-Leu278Glu 0.85±1.53
LipA-Asn125Glu 57.69±1.43 LipA-Ser281Asp -9.80±0.23
LipA-Asn125Asp 40.99±1.14 LipA-Val305Arg -1.210±1.413
LipA-Ser135Lys -6.39±1.75 LipA-Ala306Glu -7.63±2.56

2.3 野生型脂肪酶及正效应脂肪酶突变体T5012的测定

野生型脂肪酶与正效应脂肪酶突变体的T5012测定结果如图 2。野生型脂肪酶LipA的T5012为51 ℃,3个正效应的脂肪酶突变体较野生型脂肪酶的T5012均有一定程度的提高,分别为:55 ℃ (LipA-Asn125Asp)、56.5 ℃ (LipA-Asn125Glu)和55.4 ℃ (LipA-Gln262Glu)。

图 2 野生型脂肪酶及其突变体T5012的测定 Figure 2 The difference in T5012 value between the wild-type LipA and LipA mutants. The T5012 value at each temperature was the average value of the three independent assays and the errors of each T5012 value were presented as the standard deviation.

2.4 野生型脂肪酶及正效应脂肪酶突变体t1/2的测定

野生型脂肪酶与正效应脂肪酶突变体的t1/2测定结果如图 3。野生型脂肪酶LipA的t1/2为5.20 min,突变体LipA-Asn125Asp的t1/2为11.6 min,LipA-Asn125Glu为19.8 min,LipA-Gln262Glu为13.5 min。

图 3 野生型脂肪酶及其突变体t1/2的测定 Figure 3 The difference of the t1/2 graph between the wild-type LipA and LipA mutants. The residual activity at each temperature was the average value of the three independent assays and the errors of each ln(% residual activity) value were presented as the standard deviation.

2.5 脂肪酶突变体稳定性提高的分子机制分析

3个正效应脂肪酶突变体在突变位点与周围临近的氨基酸残基之间形成盐桥结构示意图(图 4-A-C)。突变体LipA-Asn125Asp (图 4-A)和突变体LipA-Asn125Glu (图 4-B)的Asp125、Glu125分别与Arg258氨基酸残基之间形成了盐桥。突变体LipA-Gln262Glu的Glu262与Lys269氨基酸残基之间形成了盐桥(图 4-C)。图 4-D显示,3个脂肪酶突变体的RMSD值的波动范围较野生型脂肪酶都有一定的降低,其中LipA-Asn125Glu的RMSD值下降最为明显,分子动力学模拟的结果与前述实验结果基本一致。

图 4 脂肪酶突变体热稳定性提高的分子机制 Figure 4 Molecular mechanism for thermostability improvement of three lipase mutants. The schematic model of the salt bridge position in the 3D structural model of LipA mutants was shown in the (A), (B) and (C), respectively. D displayed the overall RMSD values of wied-type LipA and LipA mutants.

3 讨论

热耐受性生物催化剂较中温催化剂而言,可以在较高温度下催化反应,提高催化效率;降低产物被污染的概率;同时,酶制剂具有较好的稳定性,可以提高重复使用批次,从而有效降低生产成本。近些年来,围绕热稳定催化剂资源的挖掘[18]、热稳定蛋白耐热的分子机制[3, 19]、热稳定催化剂突变体的构建[20]及热稳定蛋白突变体的高通量筛选[21]等方面的研究,取得了长足的进展,诞生了一系列新技术新方法。

同定向进化技术中构建突变基因文库一样,电子突变文库的质量直接决定了阳性突变率的高低。Floor等仅从150个突变体的电子文库中筛选到半衰期延长了200多倍的阳性突变体[7];Wijma等从包含64个突变体的电子文库中,筛选到21个热稳定突变体。其中最优热稳定性突变体,表观熔点温度从50 ℃提高到85 ℃,半衰期提高了250多倍[15]。本实验中,虽未对产生的341个突变体的电子文库全部进行实验验证,但验证存在盐桥效应的27个突变体的实验结果表明,仅3个脂肪酶突变体的热稳定性有显著性提高,阳性突变率依然较低。较低的阳性突变率与突变氨基酸的选定范围、筛选突变体自由能ΔΔG的划分标准存在一定的联系。本试验中,我们以9 Å作为活性中心范围的标准。事实上,LipA分子的3D结构具有较深的funnel-like状底物结合部位[22],其活性中心的范围远大于9 Å (如位于活性中心的Ser87的Cα原子与Leu127的Cα原子之间的距离为22.178 Å)。因此,通过扩大活性中心范围的氨基酸残基(本试验中,活性中心的氨基酸残基排除在潜在突变位点之外),可以有效降低突变体文库的容量,一定程度上有助于提高阳性突变率。另外,精确计算突变体自由能,并划定出合理的数值区间,作为筛选、评估突变体的突变效应的标准,也有助于提高阳性突变率。Morloy和Kazlauskas统计了目前报道的各类阳性突变体的ΔΔG自由能变化,发现-ΔΔG值在高于2-3 kcal/mol时,具有良好的阳性突变效应[23]。Christensen和Kepp通过优化FoldX程序并精准计算出ΔΔG的数值,有效提高了预测及评估潜在突变位点的能力[14]

突变体LipA-Gln262Glu在—Glu262......Lys269—之间形成盐桥,Glu262和Lys269均位于loop环上。通过提高蛋白质柔性区域(flexible region)的刚性(rigidity),实现增强蛋白质的稳定性,已成为构建热稳定蛋白质的常规策略[20, 24-25]。突变体LipA-Asn125Asp和LipA-Asn125Glu形成的盐桥位于LipA分子的α-螺旋α4与α9之间,而α4和α9是构成LipA分子2个盖子结构域(Lid1/Lid2) 的重要二级结构成分(Lid1由α4和α5及其两侧的loop区组成;Lid2由α8、α9、β3和β4及其临近的loop区组成)。脂肪酶的盖子结构域具有高度的柔性和灵活性,在油水界面,盖子结构由闭合状态转变为打开状态,暴露出脂肪酶分子的活性中心,完成脂肪酶的界面激活(Interfacial activation)功能[26-27]

本实验仅测定了突变体电子文库中存在盐桥效应突变体的突变效应,初步实验结果证实利用生物信息学构建的突变体电子文库具备筛选热稳定性突变体的潜力和价值。进一步深入分析该电子文库中各类突变体提高热稳定性的分子机制,可发现存在多种效应,如二硫键效应(如Ala24Cys/Thr150Cys间可形成二硫键)、脯氨酸效应(如Ser136Pro、Thr156Pro等)、疏水效应、β-转角构象优化等。初步试验结果表明:不同效应叠加产生的突变体,具备更好的热稳定性。如本实验筛选出的具有盐桥效应的突变体继续叠加脯氨酸效应突变产生的叠加突变体,其T5012较野生型LipA提高了10.2 ℃;t1/2延长了18倍,更详细深入的调查仍在进行之中。

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