李坤鹏, 张荣珍
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江南大学生物工程学院, 教育部工业生物技术重点实验室, 江苏 无锡 214122
收稿日期:2016-12-29;修回日期:2017-03-15;网络出版日期:2017-04-05
基金项目:国家自然科学基金(31370100,31670070);江苏省六大人才高峰高层次人才项目(2015-SWYY-010);中央高校基本科研业务费专项资金(JUSRP51409B);教育部新世纪优秀人才(NCET-13-0833);高等学校学科创新引智计划(111-2-06)
*通信作者:张荣珍, Tel:+86-510-85197760;Fax:+86-510-85864112;E-mail:rzzhang@jiangnan.edu.cn
摘要:[目的]以金黄色葡萄球菌(Staphylococcus aureus)的sortase A为“分子订书机”,用于(S)-羰基还原酶Ⅱ分子之间的连接,获得催化功能与稳定性增强的氧化还原酶寡聚体,高效催化2-羟基苯乙酮,合成(S)-苯基乙二醇。[方法]从S. aureus基因组中克隆sortase A基因,在大肠杆菌中表达,通过镍柱和凝胶层析纯化重组酶,获得纯酶sortase A。通过基因工程手段在(S)-羰基还原酶Ⅱ的C末端添加GGGGSLPETGG序列,蛋白纯化获得(S)-羰基还原酶Ⅱ-GGGGSLPETGG,摸索了sortase A催化(S)-羰基还原酶Ⅱ-GGGGSLPETGG的分子连接,形成(S)-羰基还原酶Ⅱ寡聚体的最佳条件,并研究了寡聚体酶学性质及生物转化(S)-苯基乙二醇的效率。[结果](S)-羰基还原酶Ⅱ寡聚体比酶活力为38.5 U/mg,比原始型(S)-羰基还原酶Ⅱ提高了6倍,最适反应温度为50 ℃,最适pH为6.0,在50 ℃放置1 h后酶活仍旧保持90%以上;蛋白质变性实验结果显示,(S)-羰基还原酶Ⅱ寡聚体的变性温度为60.1 ℃,比原始酶提高了10 ℃;生物转化结果显示(S)-羰基还原酶Ⅱ寡聚体在3 h内完全转化5 g/L 2-羟基苯乙酮,产生光学纯度为100%的(S)-苯基乙二醇,相比于重组大肠杆菌(S)-羰基还原酶Ⅱ全细胞催化时间缩短了16倍。[结论]本研究首次将sortase A应用于氧化还原酶的分子连接,显著提高了酶的催化效率和热稳定性,表明sortase在手性催化中有很大的潜在应用价值。
关键词: Sortase A 分子连接 寡聚体 氧化还原酶 手性催化
Sortase A-mediated oligomers of (S)-carbonyl reductase Ⅱ suitable for biotransformation of (S)-phenyl-1, 2-ethanediol
Kunpeng Li, Rongzhen Zhang
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Key Laboratory of Industrial Biological, Ministry of Education, School of Bioengineering, Jiangnan University, Wuxi 214122, Jiangsu Province, China
Received 29 December 2016; Revised 15 March 2017; Published online 5 April 2017
*Corresponding author: Rongzhen Zhang, Tel:+86-510-85197760;Fax:+86-510-85864112;E-mail:rzzhang@jiangnan.edu.cn
Supported by the National Natural Science Foundation of China (31370100, 31670070), by the Program for Advanced Talents within Six Industries of Jiangsu Province (2015-SWYY-010), by the Fundamental Research Funds for the Central Universities (JUSRP51409B), by the Program for New Century Excellent Talents in Universities (NCET-13-0833) and by the Program of Introducing Talents of Discipline to Universities (111-2-06)
Abstract: [Objective]To obtain oligomers of (S)-carbonyl reductase Ⅱ with strong activity and stability, we used sortase A from Staphylococcus aureus as molecular "stapler" to conjugate (S)-carbonyl reductase Ⅱ. The (S)-carbonyl reductase Ⅱ oligomers efficiently catalyzed the biotransformation of 2-hydroxyacetophenone to (S)-1-phenyl-1, 2-ethanediol.[Methods]We cloned sortase A gene from S. aureus genome and expressed it in Escherichia coli. The recombinant enzyme was purified through Ni-affinity and gel filtration chromatography. Meanwhile, we added GGGGSLPETGG to C terminus of (S)-carbonyl reductase Ⅱ using genetic techniques, and purified recombinant SCRII-GGGGSLPETGG to homogeneity. We determined the optimal reaction conditions of sortase A-mediated ligation of (S)-carbonyl reductase Ⅱ to oligomers. The enzyme characteristics of the generated oligomers were studied. And the oligomers-catalyzed biotransformation efficiency of (S)-1-phenyl-1, 2-ethanediol was further detected.[Results]Oligomers showed a specific activity of 38.5 U/mg, 6-fold increase compared to (S)-carbonyl reductase Ⅱ. The optimal temperature and pH of ligation reaction by oligomers were 35 ℃ and 6.0 respectively. The relative activity was maintained over 90% at 50 ℃ for 1 hour. Denaturation test showed that the denaturation temperature of oligomers was 60.1 ℃, 10 ℃ higher than that of (S)-carbonyl reductase Ⅱ. Biotransformation results indicated that oligomers completely transformed 5 g/L 2-hydroxyacetophenone within 3 hours to generate (S)-1-phenyl-1, 2-ethanediol with an optical purity of 100%. With oligomers, we reduced transformation duration for 16 folds compared to that of recombinant E. coli-(S)-carbonyl reductase Ⅱ cells.[Conclusion]This work first described sortase A-mediated the ligation of oxidoreductase and significantly improved catalytic efficiency and thermal stability of enzyme, suggesting sortase had great potential application in chiral synthesis.
Key words: Sortase A molecular ligation oligomer oxidoreductase chiral catalysis
光学纯手性醇作为一类重要的中间体,在药物、农用化学品、精细化学品以及一些特殊材料的合成中被广泛应用。羰基还原酶能够将内源性和外源性羰基化合物还原为手性醇,该催化反应具有高度立体选择性的特点[1-3],相比于传统化学方法,生物催化具有安全、绿色的优势,因此羰基还原酶已越来越多地应用于化学和制药产业中[4-6]。来源于近平滑假丝酵母(Candida parapsilosis) CCTCC M203011的(S)-羰基还原酶Ⅱ(SCRII)能够催化2-羟基苯乙酮(2-HAP)生成(S)-苯基乙二醇((S)-PED),该手性化合物是许多药物和液晶材料合成必不可少的中间体[7]。然而,重组菌株Escherichia coli-SCRII催化2-HAP的活性较低,催化转化5 g/L 2-HAP为(S)-PED需要48 h。最近SCRII在C. parapsilosis CCTCC M203011中进行原位表达[8],重组菌株C. parapsilosis-SCRII催化2-HAP的酶活力提高了2倍,不对称转化(S)-PED的光学纯度和产率均高达99%以上,然而,整个生物转化过程需要24 h,限制了其在手性催化中的应用。
sortase是一类存在于革兰氏阳性菌中的转肽酶,其功能与细菌菌毛蛋白组装和表面蛋白锚定有关[9]。来源于金黄色葡萄球菌(Staphylococcus aureus)的sortase A (SrtA)能识别带有LPXTG(X可以为任何氨基酸)的序列,并且特异性地切开苏氨酸与甘氨酸之间的酰胺键形成一个硫酯中间体,然后由位于细胞壁上的五聚甘氨酸来进攻这个硫酯中间体,从而将LPXT序列与五聚甘氨酸相连,实现了将蛋白质固定于细胞壁上[10-12]。目前sortase介导的连接反应已经广泛应用于蛋白质标记、环化和固定化等[13-15]。然而sortase应用于蛋白质与蛋白质分子之间的连接鲜有报道。Mao等成功地将sortase用于绿色荧光蛋白之间的连接[16],认为LPXT序列与一个甘氨酸与五聚甘氨酸的连接效果相同。迄今为止,还未有sortase介导的蛋白质互连用于连接氧化还原酶的手性催化。
本研究尝试将sortase A应用于SCRII自身分子连接(寡聚化),通过基因工程在SCRII的C端添加GGGGSLPETGG序列(简称为SCRII-mtf),以其N端存在的天然甘氨酸作为亲核试剂,在sortase A的介导下进行连接反应,本研究对其连接产物的组成进行了分析,考察了寡聚体的酶学性质,获得了催化功能加强和稳定性显著提高的寡聚体,高效催化(S)-PED的不对称转化。
1 材料和方法 1.1 材料
1.1.1 菌种与质粒: 本研究中使用的菌株、质粒及引物如表 1所示。
表 1. 实验菌株,质粒和引物 Table 1. Strains, plasmids and primers in this work
Strains and plasmids | Characteristics | Source |
Strains | ||
Staphylococcus aureus | Source of srtA | This lab |
E. coli/T-scrⅡ-mtf | E. coli JM109 harboring T-scrⅡ-mtf | This study |
E. coli/T-srtA | E. coli JM109 harboring T-srtA | This sutdy |
E. coli/pET28a-scrⅡ | E. coli BL21 harboring pET28a-scrⅡ | This study |
E. coli/pET28a-scrⅡ-mtf | E. coli BL21 harboring pET28a-scrⅡ-mtf | This study |
E. coli/pET21a-srtA | E. coli BL21 harboring pET21a-srtA | This sutdy |
Plasmids | ||
pMD19-T | Ampr, 2692 bp | TaKaRa |
T-scrⅡ-mtf | pMD19-T harboring 0.87 kb scrⅡ-mtf gene, 3.5 kb | This sutdy |
T-srtA | pMD19-T harboring 0.4 kb srtA gene, 3.1 kb | This sutdy |
pET28a-scrⅡ-mtf | pET28a harboring 0.87 kb scrⅡ-mtf gene, 6.3 kb | This study |
pET28a-scrⅡ | pET28a harboring 0.84 kb scrⅡ gene, 6.2 kb | This lab [7] |
pET21a-srtA | pET21a harboring 0.4 kb srtA gene, 5.8 kb | This study |
Primers | Sequence (5'→3') | |
srtA_F | CGCCATATGCAAGCTAAACCTCAAATTC (Nde Ⅰ) | |
srtA_R | CCGCTCGAGTTTGACTTCTGTAGCTAC (Xho Ⅰ) | |
scrⅡ-mtf_F | CCCATGGGCGAAATCGAATC (Nco Ⅰ) | |
scrⅡ-mtf_R | CCCTCGAGGCCGCCGGTTTCCGGAAGGC (Xho Ⅰ) TGCCACCGCCACCTGGACAAGTGTAACCACCATC | |
The restriction endonuclease sites are underlined. |
表选项
1.1.2 主要试剂: Taq DNA polymerase、T4 DNA ligase、DNA marker、限制性内切酶、IPTG、基因组DNA提取试剂盒购于TaKaRa生物有限公司,质粒提取和DNA回收试剂盒购于OMEGA BIO-TEK。引物由上海生工生物工程有限公司合成。2-羟基苯乙酮、辅酶NADPH和(S)-苯基乙二醇购于Sigma-Aldrich。其余试剂均为分析纯。
1.2 菌体培养 LB培养基(g/L):胰蛋白胨10,酵母提取物5,NaCl 10,pH 7.0,固体培养基添加1.5%琼脂粉。将S. aureus和E. coli/pET28a-scrⅡ[7]菌种分别接种于装有5 mL LB液体培养基(无抗)和5 mL LB液体培养基(50 μg/mL卡那霉素)的试管中,37 ℃、200 r/min振荡培养10 h。
1.3 基因组DNA和质粒的提取 参照试剂盒说明书提取S. aureus的基因组DNA以及E. coli/pET28a-scrⅡ的质粒。
1.4 Sortase A和SCRII-mtf基因的获得 分别以srtA和scrⅡ基因序列为模板,通过DNAMAN软件设计引物如表 1中所示(下划线为酶切位点)。以S. aureus基因组为模板,PCR扩增srtA基因,条件为:98 ℃ 1 min;98 ℃ 30 s,55 ℃ 30 s,72 ℃ 30 s,30次循环;72 ℃ 10 min。以pET28a-scrⅡ质粒为模板,PCR扩增scrⅡ-mtf基因,条件为:98 ℃ 1 min;98 ℃ 30 s,55 ℃ 30 s,72 ℃ 45 s,30次循环;72 ℃ 10 min。PCR产物经胶回收纯化后,连接至pMD19-T上,连接产物转化E. coli JM 109感受态细胞,涂布于LB固体培养基(含100 μg/mL氨苄青霉素),获得阳性克隆后提取质粒。
1.5 表达载体的构建 使用限制性内切酶Xho Ⅰ和Nde Ⅰ分别对载体pET-21a和质粒T-srtA进行双酶切,酶切后回收DNA片段通过粘性末端连接获得带有srtA的重组表达质粒pET21a-srtA。同时,用Xho Ⅰ和Nco Ⅰ分别对载体pET-28a和T-scrⅡ-mtf进行双酶切,获得的DNA片段连接后获得带有SCRII-mtf的表达质粒pET28a-scrⅡ-mtf。重组质粒由上海生工生物工程有限公司进行测序。
1.6 重组菌株的构建 将重组质粒pET21a-srtA和pET28a-scrⅡ-mtf转化感受态细胞E. coli (DE3)菌株,从氨苄青霉素抗性平板上筛选出重组菌株E. coli/pET21a-srtA,从卡那霉素抗性平板上筛选出E. coli/pET28a-scrⅡ-mtf阳性克隆。
1.7 蛋白纯化 从平板上挑取E. coli/pET21a-srtA、E. coli/pET28a-scrⅡ-mtf、E. coli/pET28a-scrⅡ单菌落分别接种于含对应抗性的100 mL LB液体培养基中,当菌体培养的OD600值达到0.6–0.8时,加入终浓度为1 mmol/L诱导剂IPTG,30 ℃诱导表达10 h。将诱导表达后的菌液离心,收集菌体,用生理盐水洗涤3次后重悬于20 mmol/L Tris-HCl缓冲液(pH 8.0),超声破碎。12000 r/min,4 ℃离心收集上清液,经0.22 μm滤膜过滤后的滤液即为粗酶液。利用镍离子亲和层析柱和Superdex 200凝胶柱两个纯化步骤,对SrtA、SCRII和SCRII-mtf进行纯化,具体参考张荣珍等方法[17]。
1.8 SrtA介导SCRII-mtf寡聚化 向连接体系(50 mmol/L Tris-HCl,150 mmol/L NaCl,10 mmol/L CaCl2,pH 7.5)中加入SrtA (25 μmol/L)和SCRII-mtf (30 μmol/L)分别在10、15、20、25、30和35 ℃的条件下进行反应,同时,在相同的体系中加入同等量的SrtA和SCRII作为对照,反应时间为8 h。连接产物经SDS-PAGE分析后确定最佳连接温度。在最佳温度下将反应进行12、24、36 h,观察连接产物产量的变化,确定连接时间。连接反应结束后,将连接体系超滤浓缩至500 μL,用50 mmol/L Tris-HCl,150 mmol/L NaCl,pH 8.0的缓冲平衡凝胶柱Superdex 200,接下来将浓缩后的样品过柱,根据分子筛效应分离得到SCRII寡聚体,SDS-PAGE鉴定连接产物的纯度,连接产物条带切下后用于MALDI-TOF-MS分析,获得的质谱数据利用Mascot搜索引擎(www.matrixscience.com)进行鉴定,以确定连接产物的成分。
1.9 酶活力测定 辅酶NADPH被还原后,会引起340 nm的吸光度的降低。因此可通过测定反应过程中340 nm处吸光度的变化来衡量氧化还原酶的活力。酶活测定体系:100 μL体系中含有100 mmol/L磷酸钾缓冲液(pH 6.0),0.5 mmol/L NADPH,5 mmol/L 2-HAP,35 ℃恒温3 min,加入适量酶液混匀后,酶标仪扫描340 nm处的吸光值变化。酶活测定实验重复3次,取平均值。蛋白含量采用Bradford法测定[18],以BSA为标准蛋白。定义1个酶活单位(U)为每分钟催化氧化1 μmol辅酶NADPH的酶量(公式1、2)。
![]() | 公式(1) |
![]() | 公式(2) |
1.10 酶学性质测定 对分别在不同温度梯度(20–70 ℃)和pH梯度(4.0–9.0)下测定SCRII、SCRII-mtf及SCRII寡聚体的酶活,确定三者的最适pH和最适温度。将3种酶置于50 ℃下并在不同时间间隔取样进行酶活的测定,评估温度稳定性。在4 ℃条件下将3种酶放置在不同pH梯度(4.0–9.0)的缓冲溶液中,24 h后测定酶活,确定三者的pH耐受性。
1.11 不对称转化 用SCRII、SCRII-mtf、SCRII寡聚体分别转化2-HAP,对比转化结果。反应体系:100 mmol/L磷酸钾缓冲液(pH 6.0),5 g/L 2-HAP或10 g/L 2-HAP和等摩尔的NADPH,以及适量的纯酶液,总体积2 mL。在35 ℃、200 r/min条件下反应10 h,每隔0.5 h取1次样,乙酸乙酯萃取后取液相部分检测转化结果。每个实验重复3次取平均值。
1.12 圆二色谱分析 三种纯化蛋白用10 mmol/L磷酸盐缓冲液(pH 6.0)稀释至0.1 mg/mL,应用Jasco J720光谱仪在25 ℃条件下进行二级结构测定,光谱值范围为190–250 nm,扫描速度50 nm/min,带宽2 nm,反应时间1 s,读数30次,取信号平均值并基线校正,将所测定的CD值转换成摩尔椭圆度(molar ellipticity) θ (deg×cm2×dmol–1),用计算机专用软件(J-700 for Windows Secondary Structure Estimaton,Version 1.10.00)分析各蛋白二级结构组成。取上述制备好的样品,分别加入1 mm比色皿中,以1 ℃/min的速度将蛋白溶液从20 ℃加热至80 ℃,波长209 nm,用圆二色谱仪记录摩尔椭圆度随温度的变化,考察三种蛋白随温度升高解螺旋情况,确定蛋白质变性温度。
2 结果和分析 2.1 重组质粒pET21a-srtA和pET28a-scrⅡ-mtf的构建 以S. aureus基因组为模板,以srtA_F和srtA_R为引物进行PCR扩增,获得目的基因srtA,经过1%琼脂糖凝胶电泳分析,其条带大小为0.6 kb。将srtA片段连入pMD19T,经Xho Ⅰ和Nde Ⅰ双酶切后连入pET21a载体。以scrⅡ-mtf_F和scrⅡ-mtf_R为引物,从pET28a-scrⅡ质粒上扩增出scrⅡ-mtf基因,电泳分析结果表明基因片段大小接近0.9 kb。将scrⅡ-mtf片段连入pMD19-T,经Xho Ⅰ和Nco Ⅰ双酶切后连入pET28a载体。两种重组质粒均经双酶切鉴定后转入E. coli BL21 (DE3),挑取阳性克隆提取质粒并送去测序,测序结果显示两种基因均已克隆到相应表达载体上,获得重组质粒pET21a-srtA和pET28a-scrⅡ-mtf。
2.2 SrtA、SCRII和SCRII-mtf的表达及纯化 挑取E. coli BL21/pET21a-srtA、E. coli/pET28a-scrⅡ、E. coli BL21/pET28a-scrⅡ-mtf的单菌落至含相应抗性的LB液体培养基中培养,经IPTG诱导后收集菌体,洗涤后重悬于Tris-HCl缓冲液中,超声破碎、离心、过膜得到粗酶液。然后将粗酶液用Ni柱和Superdex 200两个步骤进行分离纯化,经SDS-PAGE分析,3种重组蛋白纯化后获得的目的蛋白条带均一,大小分别约为23 kDa (SrtA)、32 kDa (SCRII)、33 kDa (SCRII-mtf),与理论分子量大小一致(图 1和图 2)。纯化蛋白可用于后续的连接反应及酶学性质分析。
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图 1 SrtA的表达与纯化 Figure 1 Expression and purification of SrtA. M: protein molecular weight marker (Low); Lane1: the cell-free extracts of the recombinant E. coli BL21/pET-srtA; lane2: the purified SrtA. |
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图 2 SCRII和SCRII-mtf的纯化 Figure 2 Purification of SCRII and SCRII-mtf. M: protein molecular weight marker (Low); lane 1: the purified SCRII-mtf; lane 2: the purified SCRII. |
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2.3 SrtA介导SCRII分子连接的最佳条件的确定 在Ca2+存在的情况下,向Sortase介导的连接反应体系中加入SrtA和SCRII-mtf,分别在不同温度下反应8 h,同时设置SrtA+SCRII为对照组,SDS-PAGE检测结果如图 3所示,不同温度下均有连接产物生成,肽指纹图谱显示与连接产物匹配度最高的是来自于Candida parapsilosis的stereospecific carbonyl reductase 2,即本研究中的SCRII,根据大小可以判断连接产物的成分主要是二聚体(约66 kDa)和三聚体(约99 kDa),还有少量多聚体生成。此外,在SCRII-mtf条带的下方还有两条带生成,根据大小以及文献报道[19],我们推测是切去末尾甘氨酸的SCRII-GGGGSLPET和环化的SCRII-GGGGSLPET。随着反应温度的升高,连接产物的产量逐渐增加,但当温度高于25 ℃时产物有所减少,因此确定25 ℃为最适连接温度。在25 ℃下进一步进行反应条件优化,在12、24和36 h分别用SDS-PAGE鉴定连接产物的变化,结果发现随着时间的延长,连接产物的量逐渐增加,SCRII-mtf的量逐渐减少,直到36 h时几乎所有的SCRII-mtf被消耗,转化为连接产物,同时SCRII-GGGGSLPET和环化的SCRII-GGGGSLPET也几乎被消耗完,此时连接产物的产量达到最多,因此确定连接时间为36 h。作为对照,在同等条件下连接36 h的SCRII经蛋白电泳检测无任何连接产物生成。SCRII寡聚体经凝胶柱Superdex 200去除SrtA蛋白和残余的少量SCRII-mtf蛋白,得到的寡聚体混合物用于酶学性质的测定。
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图 3 SrtA介导的SCRII寡聚体的形成 Figure 3 SrtA-mediated oligomerization of SCRII. M: protein molecular weight marker (Low); lane 1: SrtA+SCRII for 8 h at 25 ℃; lane 2–7: SrtA+SCRII-mtf for 8 h at 10, 15, 20, 25, 30 and 35 ℃, respectively; lane 8: SrtA+SCRII-mtf for 36 h at 25 ℃; lane 9: the purified SCRII oligomers. |
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2.4 SCRII寡聚体的温度和pH稳定性显著提高 SCRII、SCRII-mtf和SCRII寡聚体的活性在同一条件下进行测定,SCRII对2-HAP的比活为6.3 U/mg,与之前文献当中报道的结果基本一致[7];而在C端添加了GGGGSLPETGG序列的SCRII-mtf的比活较之有所增加,为8.7 U/mg;SCRII寡聚体对2-HAP的活性比较高,为38.5 U/mg,与SCRII相比提高了6倍。
在20–70 ℃范围内,SCRII和SCRII-mtf随着温度的升高,活性逐渐增加,在35 ℃时酶活最高,此后随温度升高,活性迅速降低(图 4-A);在20–50 ℃范围内,SCRII寡聚体随着温度升高,相对酶活增加,在50 ℃时相对酶活最高,温度高于60 ℃时活性迅速降低。SCRII和SCRII-mtf在pH 6.0时酶活最高,而SCRII寡聚体在pH 6.0和pH 6.5均具有最高活性(图 4-B)。
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图 4 SCRII、SCRII-mtf和SCRII寡聚体的酶学性质 Figure 4 The enzyme characteristics of SCRII, SCRII-mtf and SCRII oligomers. A: the optimal temperature of SCRII, SCRII-mtf and SCRII oligomers towards 2-HAP reduction (pH 6.0); B: the optimal pH value of SCRII, SCRII-mtf and SCRII oligomers towards 2-HAP reduction (35 ℃); C: the thermostability of SCRII, SCRII-mtf and SCRII oligomers (pH 6.0); D: the pH stability of SCRII, SCRII-mtf and SCRII oligomers (4 ℃). |
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温度稳定性测定结果如图 4-C示,SCRII和SCRII-mtf在50 ℃放置10 min后,能够保留原始酶活的80%,SCRII寡聚体在50 ℃放置1 h,活性仍旧维持在90%以上。在pH 5.0–9.0的范围内放置24 h后,3种酶的酶活性都能保持75%以上,但在同等条件下,SCRII寡聚体的pH稳定性明显高于其他两种酶(图 4-D)。
2.5 蛋白变性实验进一步证实SCRII寡聚体热稳定性显著提高 随着波长的变化,SCRII、SCRII-mtf、SCRII寡聚体的摩尔椭圆度显示出相似的变化趋势,并且分别在190和209 nm处出现波峰和波谷,说明三种蛋白具有非常相近的二级结构,识别序列的添加以及寡聚化并没有改变SCRII的蛋白结构。蛋白变性温度是蛋白二级结构解螺旋达到最大速率时所对应的温度,即变性曲线最大斜率处所对应的温度。随着温度的升高,SCRII和SCRII-mtf在45 ℃处开始解螺旋,通过计算曲线的最大斜率,得出二者的Tm值为49.6 ℃和50.3 ℃。SCRII寡聚体在55 ℃左右开始丧失二级结构,经计算Tm值为60.1 ℃ (图 5),说明SCRII经寡聚化后,变性温度提高了近10 ℃,蛋白变性实验进一步证实SCRII寡聚体热稳定性显著提高。
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图 5 圆二色谱分析SCRII、SCRII-mtf和SCRII寡聚体的二级结构含量(A)和变性温度(B) Figure 5 Circular dichroism analysis of secondary structures (A) and denaturation temperature (B) for SCRII, SCRII-mtf and SCRII oligomers. |
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2.6 SCRII寡聚体显著提高不对称转化(S)-PED效率 以2-HAP为底物,NADPH为辅酶,SCRII、SCRII-mtf和SCRII寡聚体作为催化剂分别进行不对称转化反应。在SCRII寡聚体最适反应条件35 ℃和pH 6.0下,在反应的不同时间段对转化产物进行取样分析。结果如图 6所示,SCRII寡聚体在2 h内的产率即达到了90%以上,在3 h左右能够将5 g/L的2-HAP完全转化,产物(S)-PED的光学纯度高达100%,而原始酶SCRII和SCRII-mtf均在8 h左右才将底物完全转化。SCRII寡聚体转化时间比SCRII和SCRII-mtf缩短了2.7倍。另外,我们尝试了三种酶催化转化10 g/L 2-HAP,结果如表 2中所示,SCRII寡聚体能够在8 h左右将2-HAP几乎完全转化,(S)-PED的产率和光学纯度均为100%,而SCRII、SCRII-mtf转化产率低于50%。
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图 6 SCRII、SCRII-mtf和SCRII寡聚体不对称转化(S)-PED Figure 6 Asymmetric transformation of (S)-PED by SCRII, SCRII-mtf and SCRII oligomers. The data represented three independent experiments and standard errors were not more than 5%. |
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表 2. SCRII、SCRII-mtf和SCRII寡聚体催化转化(S)-PED Table 2. The biotransformation of (S)-PED by SCRII, SCR-mtf and SCRII oligomers
Enzymes | Optical purity/(% e.e.) | Yield/% |
SCRII | 100.00±0.00 | 41.20±1.15 |
SCRII-mtf | 100.00±0.00 | 46.10±1.79 |
SCRII oligomers | 100.00±0.00 | 98.30±0.83 |
2-HAP concentration and reaction time is 10 g/L and 8 h, respectively. The data represented three independent experiments and standard errors were not more than 5%. |
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3 讨论 羰基还原酶是一种具有高度化学、区域和立体选择性的生物催化剂,可以用于催化合成许多不同种类的手性化合物[20-21],这些化合物可作为中间体可用于制备农药、医药和功能材料等[22-24]。本研究中的SCRII催化2-HAP生成的(S)-PED是液晶材料中不可缺少的手性添加剂[7]。前期研究发现重组SCRII催化2-羟基苯乙酮的活性较低,及时更换蛋白表达的宿主细胞,如在酿酒酵母、毕赤酵母中异源表达[25]和近平滑假丝酵母中原位表达[8],酶催化转化(S)-PED的周期均较长,至少24 h以上。因此,本研究从蛋白质工程角度出发,对SCRII进行改造,以此来提高酶的稳定性和催化效率。目前常用的蛋白质改造的方法主要有两种,即定向进化和理性设计。定向进化的完成需要建立在大量随机突变和高通量筛选方法的基础上,如Zhao等通过易错PCR获得了羰基还原酶ChKRED20的突变文库,通过在高温下孵育后检测酶活,最终成功筛选得到了热稳定性提高的突变酶[26];理性设计是基于对某一蛋白质的结构与功能之间的具体关系,实施定点突变或饱和突变来获得性能提高的突变酶,如Luo等采用同源建模和分子对接锁定了羰基还原酶KIAKR的Tyr295和Trp296,通过实施点饱和突变,最终成功获得了功能强化的突变酶[27]。然而氧化还原酶的高通量筛选方法是定向进化技术应用的瓶颈,而对于缺乏足够蛋白结构信息的氧化还原酶来讲,理性设计的工作量较大且成功率不高。因此,我们借助于sortase的转肽作用来构建寡聚酶,在不改变SCRII结构的基础上,构建新型共价聚合的寡聚酶提高酶的催化活性和热稳定性。
sortase由于其识别序列较短,不容易导致蛋白质活性的丧失,广泛应用于蛋白质的修饰[13-14]。但迄今为止,sortase很少用于蛋白质与蛋白质之间的分子连接,分析其主要原因可能是由于蛋白质的分子量较大,连接成功率比较低等因素限制了它在蛋白质连接研究中的发展。通过对SCRII的序列进行分析,发现在其N端存在一个天然甘氨酸,因此只需在其C端添加SrtA的识别序列,即LPXTG(X为任意氨基酸)序列,随后依靠SrtA的转肽作用便能够介导寡聚反应的发生。对于识别序列的添加,一方面,为了排除蛋白质的位阻效应,使该识别序列充分暴露出来能够被SrtA顺利切开,另一方面,也为了避免引入的LPETG序列对SCRII的活性造成影响,因此,有必要在该识别序列前面添加GGGGS柔性多肽,并在其后面再添加了一个G,使整个识别序列可以暴露在蛋白质表面并且不影响SCRII的正确折叠。值得一提的是,由于C端距离SCRII酶的活性区域较远,GGGGS柔性序列添加至LPETGG识别序列的前段,使得SCRII酶活性未受影响,同时增加了酶蛋白的柔韧性,从而导致添加GGGGSLPETGG的SCRII-mtf酶活比原始酶更高。此外,SCRII寡聚体的高度聚合必然离不开sortase介导反应条件的优化,主要包括两个方面,即连接温度和反应时间。首先,在不同温度下sortase介导反应8 h,通过观察连接产物的量,确定了25 ℃时连接率最佳,随后,在25 ℃下进行连接反应时间的摸索,发现在36 h时几乎所有的单体全部转化为SCRII寡聚体,主要为二聚体和三聚体。寡聚体催化2-HAP酶活性比原始酶有显著提高,说明以共价键连接的多聚体的催化效率比单体酶要高;与原始酶相比,寡聚体的温度稳定性显著提高,圆二色谱分析结果进一步证实SCRII寡聚体变性温度比SCRII提高了10 ℃左右。不对称转化实验结果表明寡聚体不对称催化2-HAP转化(S)-PED的时间大大缩短,在3 h内完全转化5 g/L的2-HAP,相比重组大肠杆菌(S)-羰基还原酶Ⅱ全细胞催化时间缩短了16倍[6],并且在8 h内几乎完全转化10 g/L 2-HAP,表明寡聚体能够耐受更高的底物浓度。综上所述,本研究首次将sortase介导氧化还原酶分子连接及其手性催化反应,成功构建了SCRII寡聚体,以共价键连接的寡聚体具有更好的热稳定性和催化活性,sortase介导的蛋白质连接也为羰基还原酶的改造提供了一个全新的思路。
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