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1. 江南大学 食品学院,江苏 无锡 214122;
2. 江南大学 无锡医学院,江苏 无锡 214122;
3. 江南大学 生物工程学院,江苏 无锡 214122
收稿日期:2019-08-26;接收日期:2019-11-21;网络出版时间:2019-12-11
基金项目:中国博士后基金项目(No. 2018M630522),江苏省青年基金项目(No. BK20180622)资助
通讯作者:Minchen Wu. Tel: +86-510-85327662; E-mail: biowmc@126.com.
摘要:为提高L-苯乳酸(L-phenyllactic acid,L-PLA)的生产效率,以干酪乳杆菌Lactobacillus casei L-乳酸脱氢酶突变体L-LcLDH1Q88A/I229A为研究对象,实现其在毕赤酵母Pichia pastoris GS115中的分泌表达,并与葡萄糖脱氢酶SyGDH偶联,构建并优化体外辅酶循环体系,不对称还原苯丙酮酸(Phenylpyruvate,PPA)制备L-PLA。结果显示,毕赤酵母重组酶reLcLDH1Q88A/I229A的表观分子量为36.8 kDa,比活力为270.5 U/mg,是原酶的42.9倍。在40 ℃,初始pH为5.0,底物PPA、辅酶NAD+和葡萄糖浓度分别为100、2和120 mmol/L,SyGDH和reLcLDH1Q88A/I229A添加量分别为1和10 U/mL的最优条件下,L-PLA的产率可达99.8%,对映体过量(ee)值> 99.9%,时空产率和平均转化率分别高达9.5 g/(L·h)和257.0 g/(g·h)。结果表明,reLcLDH1Q88A/I229A在不对称还原PPA制备L-PLA中生产效率高,具有工业应用的潜力。
关键词:乳酸脱氢酶毕赤酵母葡萄糖脱氢酶辅酶循环体系L
Expression of a Lactobacillus casei L-lactate dehydrogenase mutant in Pichia pastoris for asymmetric reduction of phenylpyruvate
Ting Zhang1, Jianfang Li1, Die Hu1,2, Chuang Li3, Bochun Hu3, Minchen Wu2
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1. School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China;
2. Wuxi School of Medicine, Jiangnan University, Wuxi 214122, Jiangsu, China;
3. School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
Received: August 26, 2019; Accepted: November 21, 2019; Published: December 11, 2019
Supported by: China Postdoctoral Science Foundation (No. 2018M630522), Natural Science Foundation of Jiangsu Province for Youth of China (No. BK20180622)
Abstract: To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1Q88A/I229A, a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1Q88A/I229A with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1Q88A/I229A was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 ℃ and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1Q88A/I229A, 1 U/mL SyGDH, 2 mmol/L NAD+ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1Q88A/I229A in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.
Keywords: L-lactate dehydrogenasePichia pastorisglucose 1-dehydrogenasecoenzyme regeneration systemL-phenyllactic acid
苯乳酸(Phenyllactic acid,PLA)是一种高附加值的天然有机酸,具有广谱抑菌性,可代替防腐剂应用于食品和饲料中[1]。L-PLA和D-PLA是苯乳酸的两种对映异构体[2],它们具有不同的应用价值,D-PLA可用于合成降血糖药物恩格列酮和驱肠虫药PF1022A,而L-PLA可用于合成非蛋白氨基酸Statine和新型生物可降解材料聚苯乳酸[3-5]。生物酶法合成光学纯PLA具有反应条件温和、光学纯度高及环境友好等优点而备受青睐[6]。
乳酸脱氢酶(Lactate dehydrogenase,LDH)分为L-LDH (EC. 1.1.1.27)和D-LDH (EC. 1.1.1.28),是以辅酶NADH或NAD+作为氢传递[7],在生物体内催化丙酮酸与乳酸之间的还原与氧化反应[8]。LDH能够不对称还原潜手性α-酮酸生成α-羟基酸,具有较广的底物谱,对天然底物丙酮酸具有较高酶活力,而对非天然底物苯丙酮酸(Phenylpyruvate,PPA)的酶活力较低。如来源于植物乳杆菌Lactobacillus plantarum的L-LpLDH[9]、L-LpLDH1和L-LpLDH2[10]、巨大芽孢杆菌Bacillus megaterium的L-BmLDH[11]、嗜热脂肪土芽孢杆菌Geobacillus stearothermophilus的L-BsLDH[12]和凝结芽孢杆菌B. coagulans的L-BcLDH[6, 13]催化PPA的比活力分别为28.1、71.1、0.1、3.4、7.4和72.6 U/mg;来源于L. plantarum的D-LpLDH[10]、菊糖芽孢乳杆菌Sporolactobacillus inulinus的D-SiLDH[14]、乳酸片球菌Pediococcus acidilactici的D-PaLDH[15]和戊糖片球菌P. pentosaceus的D-PpLDH[16]催化PPA的比活力分别为215.8、4.3、140.0和116.0 U/mg。
目前,采用微生物发酵法制备非光学纯PLA的PPA转化率及PLA产率均较低[17]。多种来源L-LDH和D-LDH在大肠杆菌Escherichia coli表达,并分别应用于制备光学纯L-PLA和D-PLA。Wang等[14]利用表达D-SiLDHM307L的E. coli全细胞,采用体内辅酶循环体系催化158.2 mmol/L PPA制备D-PLA (eep > 99.7%)的产率为82.3%;Zhu等[9]将L-LpLDH与葡萄糖脱氢酶(Glucose 1-dehydrogenase,GDH)在E. coli中共表达,全细胞催化186.2 mmol/L PPA的转化率为89.2%,L-PLA (eep=99.7%)产率仅为55.7%。Zhu等[9, 18]研究表明,E. coli全细胞自身代谢酶在催化PPA过程中产生副产物,如图 1所示,转氨酶可将PPA转化为苯丙氨酸(Phe),且葡萄糖参与细胞新陈代谢生成乳酸,造成PLA产率低且分离困难。巴斯德毕赤酵母Pichia pastoris本身不分泌内源蛋白,外源蛋白可通过信号序列进行胞外分泌表达。近年,来源于肠系膜明串珠菌Leuconostoc mesenteroides的D-LmLDH[19]和L. plantarum D-LpLDHY52L突变体[20]在P. pastoris胞内表达,分别利用重组P. pastoris全细胞生成D-乳酸和(R)-2-羟基-4-苯基丁酸。
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图 1 LDH和GDH双酶共表达大肠杆菌全细胞不对称还原PPA制备PLA[9, 18] Fig. 1 Asymmetric reduction of PPA to PLA by co-expressing LDH and GDH in E. coli cells[9, 18]. |
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本研究室前期从干酪乳杆菌Lactobacillus casei CICIM B1192基因组中克隆出一种编码L-LDH的基因lcldh1 (GenBank登录号CAP07851),并实现其在E. coli中表达[21],通过定点和迭代饱和突变构建L-LcLDH1Q88A/I229A突变体,显著提高其催化PPA的酶活力。为避免副产物形成,提高L-PLA产率,本研究利用P. pastoris GS115分泌表达L-LcLDH1Q88A/I229A突变体,并与嗜酸热源体Thermoplasma aciddophilium葡萄糖脱氢酶SyGDH构建体外辅酶NADH循环体系,通过进一步优化reLcLDH01Q88A/I229A和SyGDH双酶偶联不对称还原PPA的催化条件,实现高效制备L-PLA。
1 材料与方法1.1 材料与试剂重组菌E. coli/pET-22b-lcldh1、E. coli/pET-22b-lcldh1Q88A/I229A、E. coli/pET-22b和E. coli/pET-28a-sygdh,表达宿主E. coli JM109和P. pastoris GS115和表达质粒pPIC9K均由实验室保藏;克隆载体pUCm-T购自生工生物工程(上海)股份有限公司;T4 DNA连接酶、内切酶EcoRⅠ、NotⅠ和SalⅠ,购自大连TaKaRa公司;质粒提取试剂盒和ES Taq Master Mixture购自江苏康为世纪公司;苯丙酮酸钠、D-/L-苯乳酸和苯丙氨酸均购于美国Sigma-Aldrich公司;还原型辅酶ⅠNADH和氧化型辅酶ⅠNAD+购自上海阿拉丁公司。
1.2 方法1.2.1 重组表达质粒的构建根据乳酸脱氢酶L-LcLDH1基因及酵母表达载体pPIC9K多克隆位点,设计上游和下游引物(L9K-F:5′-CTCGAGAAAAGAGTGGCAAGTATT ACGG-3′;L9K-R:5′-GCGGCCGCCTAGTGGTGG TGGTGGTGGTGCTGACGAGTTTCGATG-3′),酶切位点为EcoRⅠ和NotⅠ(下划线表示),并委托生工生物工程(上海)股份有限公司合成。
以重组质粒pET-22b-lcldh1或pET-28a-lcldh1Q88A/I229A为模板,L9K-F和L9K-R为引物,通过PCR扩增出目的基因(lcldh1和lcldh1Q88A/I229A),与pUCm-T载体连接构建克隆质粒pUCm-T-lcldh1和pUCm-T-lcldh1Q88A/I229A,经EcoRⅠ和NotⅠ双酶切的克隆质粒与经同样双酶切的pPIC9K连接后,转化E. coli JM109,构建重组菌JM109/ pPIC9K-lcldh1和JM109/pPIC9K-lcldh1Q88A/I229A。
1.2.2 重组毕赤酵母转化子的筛选和表达提取pPIC9K、pPIC9K-lcldh1和pPIC9K-lcldh1Q88A/I229A质粒,经SalⅠ线性化后,电转化P. pastoris GS115感受态细胞,构建重组P. pastoris菌GS115/lcldh1和GS115/lcldh1Q88A/I229A。参照Multi-Copy Pichia Expression Kit操作手册,补料添加1% (V/V)的甲醇在30 ℃诱导培养72 h,收集发酵液,以经同样条件诱导的GS115/pPIC9K作为阴性对照,SDS-PAGE分析重组酶表达。
1.2.3 酶活力的测定及检测方法LDH酶活力测定:1 mL醋酸-醋酸钠缓冲液(50 mmol/L,pH 5.5),加入10 mmol/L PPA,2 mmol/L NADH,混匀后35 ℃保温5 min,加入适量酶液反应5 min,加入2 mL甲醇终止反应,采用反相HPLC色谱分析PLA生成量。在上述条件下,每分钟生成1 μmol的PLA定义为1个LDH活力单位(U)。GDH酶活力测定参照文献报道[22],反应温度和pH修改为35 ℃和5.5。
反相HPLC分析方法:采用HPLC为Waters e2695系统,二极管阵列(PDA)检测器和ProntoSIL C18 AQ (4.6 mm×250 mm,5 μm)色谱柱进行样品分析。检测条件为:流动相为水/甲醇(62︰38,V/V,含0.05%三氟乙酸),流速1.0 mL/min,进样量为10 μL,色谱柱温度为30 ℃,检测波长210 nm。Phe、PPA和PLA的保留时间分别是4.52、9.23和10.9 min。
正相HPLC分析方法:采用Daicel OD-H (4.6 mm×250 mm,5 μm)色谱柱分析PLA的光学纯度。检测条件为:流动相为正己烷/异丙醇(98︰2,V/V,含0.05%三氟乙酸),其他条件与反相HPLC相同。D-PLA和L-PLA的保留时间分别是25.7和28.1 min。L-PLA的对映体过量值(eep)计算公式为:
$e{e_\mathit{\boldsymbol{p}}}(\% ) = \frac{{{\mathit{\boldsymbol{A}}_\mathit{\boldsymbol{L}}} -{\mathit{\boldsymbol{A}}_\mathit{\boldsymbol{D}}}}}{{{\mathit{\boldsymbol{A}}_\mathit{\boldsymbol{L}}} + {\mathit{\boldsymbol{A}}_\mathit{\boldsymbol{D}}}}} \times 100$ | (1) |
1.2.4 辅酶NADH循环体系的构建和验证重组葡萄糖脱氢酶SyGDH (GenBank登录号AL445065)的诱导表达及纯化参考文献报道[22],纯化SyGDH后经冷冻干燥制得SyGDH酶粉。构建不同辅酶NADH循环体系(表 1),催化体系中参数分别为50 mmol/L醋酸钠缓冲液(pH 5.5),10 mmol/L PPA,2 mmol/L NADH或NAD+,15 mmol/L葡萄糖,0.2 U/mL reLcLDH1Q88A/I229A和0.2 U/mL SyGDH;以E. coli/lcldh1Q88A/I229A全细胞催化作对照,反应体系中包含50 mmol/L醋酸钠缓冲液(pH 6.0),10 mmol/L PPA,15 mmol/L葡萄糖和50 mg/mL E. coli/LcLDH1Q88A/I229A湿菌体,反应条件均为35 ℃、220 r/min反应2 h,按照1.2.3反相HPLC色谱测定L-PLA的产量。
1.2.5 双酶偶联不对称还原苯丙酮酸催化条件的优化温度和pH对催化反应的影响:构建包含10 mmol/L PPA、0.5 mmol/L NAD+、20 mmol/L葡萄糖、0.2 U/mL reLcLDH1Q88A/I229A和0.2 U/mL SyGDH的催化体系,分别在不同反应温度(20–60 ℃)和pH 5.5缓冲液或者不同pH (3.5–7.0)缓冲液和40 ℃条件下进行催化反应,反应20 min后测定L-PLA的产量。
PPA浓度对催化反应的影响:构建包含不同底物浓度PPA (20–300 mmol/L)、葡萄糖浓度为PPA 1.5倍、2.5 mmol/L NAD+、15 U/mL reLcLDH1Q88A/I229A和1 U/mL SyGDH的催化体系,在40 ℃和pH 5.0条件下反应不同时间取样,测定L-PLA的产量。
酶添加量对催化反应的影响:构建包含100 mmol/L PPA、150 mmol/L葡萄糖、2.5 mmol/L的NAD+、不同酶量(0.5–15 U/mL) reLcLDH1Q88A/I229A和1 U/mL SyGDH的催化体系,在40 ℃和pH 5.0条件下反应2 h后,分别测定L-PLA的产量。
NAD+浓度对催化反应的影响:构建包含100 mmol/L PPA、150 mmol/L葡萄糖、不同浓度(0.2–2.5 mmol/L)的NAD+,10 U/mL的reLcLDH1Q88A/I229A和1 U/mL SyGDH的催化体系,在40 ℃和pH 5.0条件下反应2 h后,分别测定L-PLA的产量。
葡萄糖浓度对催化反应的影响:构建包含100 mmol/L PPA,不同浓度(80–200 mmol/L)葡萄糖,2.0 mmol/L NAD+,10 U/mL reLcLDH1Q88A/I229A和1 U/mL SyGDH的催化体系,在40 ℃和pH 5.0条件下反应2 h后,分别测定L-PLA的产量。
1.2.6 双酶偶联不对称还原苯丙酮酸的反应进程在25 mL双酶偶联催化体系,包含50 mmol/L醋酸-醋酸钠(pH 5.0)、100 mmol/L PPA、120 mmol/L葡萄糖,2.0 mmol/L NAD+、10 U/mL reLcLDH1Q88A/I229A和1 U/mL SyGDH的催化体系,在40 ℃、220 r/min条件下反应不同时间取样,按照1.2.3反相HPLC测定PPA的转化率和L-PLA的产量,正相HPLC检测L-PLA的对映体过量值,并计算出时空产率(Space time yield,STY)和平均转化率(Average turnover frequency,aTOF),其计算公式为:
$\mathit{\boldsymbol{STY}}({\rm{g}}/(\mathit{\boldsymbol{L}} \cdot \mathit{\boldsymbol{h}})) = \frac{{{\mathit{\boldsymbol{C}}_\mathit{\boldsymbol{P}}}}}{t}$ | (2) |
式中:cp为L-PLA的产量(g/L),t为反应时间(h),ce为酶蛋白添加量(g/L)。
2 结果与分析2.1 重组毕赤酵母的构建及表达重组酵母GS115/lcldh1和GS115/lcldh1Q88A/I229A经甲醇诱导收集发酵液,SDS-PAGE分析表明reLcLDH1和reLcLDH1Q88A/I229均在约36.8 kDa处有明显特异性条带,与E. coli/lcldh1Q88A/I229A表达reLcLDH1Q88A/I229的目的条带大小一致(图 2)。以PPA为底物,reLcLDH1和reLcLDH1Q88A/I229A的比活力分别为6.3 U/mg和270.5 U/mg,reLcLDH1Q88A/I229比活力明显高于其他来源天然L-LDH[6, 9-12]。Aslan等[12]通过半理性设计获得最优突变酶L-BsLDHN101D/Q102Y催化PPA比活力仅从7.4 U/mg提高至51.3 U/mg。
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图 2 重组酶LcLDH1 and LcLDH1Q88A/I229A的SDS-PAGE分析 Fig. 2 SDS-PAGE analysis of the recombinant LcLDH1 and LcLDH1Q88A/I229A. M: protein marker; 1: the cultured supernatant of GS115/pPIC9K; 2: the cultured supernatant of GS115/lcldh1; 3: the cultured supernatant of GS115/lcldh1Q88A/I229A; 4: the whole cells of E. coli/ pET-22b; 5: the whole cells of E. coli/lcldh1Q88A/I229A. |
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2.2 辅酶NADH循环体系的构建及验证重组葡萄糖脱氢酶SyGDH具有较强的耐酸和适酸性,以及热稳定性[22],通过构建reLcLDH1Q88A/I229A和SyGDH不同的双酶偶联体系,验证辅酶NADH循环再生的可行性。如表 1所示,催化体系1、2、3和4说明SyGDH在以葡萄糖作为底物实现了NADH辅酶循环再生,在未同时添加SyGDH和葡萄糖的体系2和4中,PLA产率决于NADH的添加量;催化体系4和6表明添加NAD+与NADH均能实现辅酶循环,产物L-PLA产率高达99.9% (图 3A,3C),其eep > 99.9% (图 3D–E),故选择价格较低的NAD+作为辅酶。对比7和8表明,E. coli/lcldh1Q88A/I229A全细胞作为催化剂时,不需要额外添加辅酶,但PLA产率仅为89.1%,其副产物的出峰时间与苯丙氨酸一致(图 3B)。全细胞含有各种酶系,无额外添加辅酶也可完成辅酶NADH的循环再生,但往往再生效率较低。与此同时,E. coli代谢途径转氨反应中的氨基酸转移酶,可将一部分PPA进行转氨形成苯丙氨酸[23]。Zhu等[9]也在研究中提到,其用E. coli/pETduet-lpldh催化时有将近30%的PPA被用于其他代谢途径,这导致PPA的转化效率和PLA的产率均不高。
表 1 不同催化体系验证体外辅酶NADH循环的构建Table 1 Verification of NADH regeneration in vitro in the different reaction systems
Catalytic system | Conversion (%) | Yield (%) |
1: reLcLDH1Q88A/I229A+PPA | ND a | ND |
2: reLcLDH1Q88A/I229A+PPA+NADH | 20.0±0.6 | 19.8±0.6 |
3: reLcLDH1Q88A/I229A+SyGDH+PPA+NADH | 19.9±0.5 | 19.7±0.5 |
4: reLcLDH1Q88A/I229A+SyGDH+PPA+NADH+glucose | 100±2.5 | 99.9±2.4 |
5: reLcLDH1Q88A/I229A+SyGDH+PPA+NAD+ | ND | ND |
6: reLcLDH1Q88A/I229A+SyGDH+PPA+NAD++glucose | 100±2.4 | 99.9±2.5 |
7: reLcLDH1Q88A/I229A+SyGDH+PPA+glucose | ND | ND |
8: E. coli/lcldh1Q88A/I229A+PPA+glucose | 100±2.1 | 89.1±1.6 |
a ND means not detected. |
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图 3 HPLC分析重组L-LcLDH1Q88A/I229A催化不对称还原PPA Fig. 3 HPLC analysis of the asymmetric reduction of PPA catalyzed by recombinant L-LcLDH1Q88A/I229A. (A) Phe, PPA and PLA standard samples. (B) E. coli whole cells catalyzed PPA. (C, E) reLcLDH1Q88A/I229A catalyzed PPA coupling with SyGDH. (D) Racemic PLA standard samples. |
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2.3 双酶偶联不对称还原苯丙酮酸的催化条件优化2.3.1 温度和pH对催化反应的影响酶促反应效率与温度和pH有关。如图 4A所示,L-PLA的产量随反应温度的升高而升高,且在40 ℃时达到最高值。如图 4B所示,L-PLA产量在pH为5.0时达到最高,当pH > 5.0后,随着pH的增高,产量明显降低,pH到达7.0时,产量为零,表明温度和pH对催化反应具有重要影响,双酶偶联催化反应的最适温度和pH为40 ℃和5.0,与reLcLDH1Q88A/I229A的最适反应温度和pH一致。由于SyGDH具有良好的温度和pH稳定性,而温度和pH对reLcLDH1Q88A/I229A的影响较大,故双酶偶联体系中催化生成L-PLA依赖于reLcLDH1Q88A/I229A。
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图 4 不同温度、pH值下双酶偶联体系催化不对称还原PPA Fig. 4 Asymmetric reduction of PPA at different temperatures (A) and pH values (B) in double-enzyme system. |
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2.3.2 PPA浓度对催化反应的影响为提高PPA的生产效率,构建reLcLDH1Q88A/I229A和SyGDH体外双酶偶联体系催化不同浓度的PPA。如表 2所示,10、50和100 mmol/L的PPA在2 h内完全水解,L-PLA的产率均大于99%,时空产率(STY)从1.64 g/(L·h)提高至8.2 g/(L·h);当PPA浓度为150 mmol/L时,L-PLA的产量为104.1 mmol/L,而产率仅为69.4%,由于随着L-PLA浓度增加,SyGDH催化葡萄糖产生等量葡萄糖酸,测定催化体系中pH值从5.0降至3.0,导致reLcLDH1Q88A/I229A和SyGDH酶活力显著降低,从而不能继续转化PPA。此外,当PPA浓度大于150 mmol/L时,L-PLA的产量随着PPA浓度的增加而降低,当底物浓度为300 mmol/L时,L-PLA的产量仅为9.6 mmol/L,表明存在底物抑制作用。据文献报道,由于高浓度PPA对LDH的催化具有抑制作用,采用补料添加PPA的手段可以显著增加PLA的产量[9, 24-25]。Zheng等[6]研究表明,L-BcLDH催化PPA抑制浓度为90 mmol/L,Zhu等[9]研究中指出L-LpLDH催化80 mmol/L PPA的转化效率最佳。
表 2 双酶偶联体系不对称还原不同浓度的PPATable 2 Asymmetric reduction of PPA at different concentrations in double-enzyme system
Substrate (mmol/L) | Time (h) | Conversion (%) | Production (mmol/L) | Yield (%) | STY (g/(L·h)) |
10 | 2 | 100±2.5 | 9.9±0.1 | 99.9 | 1.64 |
50 | 2 | 100±3.0 | 49.9±1.3 | 99.8 | 4.2 |
100 | 2 | 100±2.2 | 99.6±2.5 | 99.6 | 8.2 |
150 | 4 | 69.5±1.2 | 104.1±3.1 | 69.4 | 4.3 |
200 | 5 | 38.4±1.0 | 76.2±2.1 | 38.1 | 3.2 |
250 | 5 | 11.3±0.2 | 28.0±0.6 | 11.2 | 0.9 |
300 | 5 | 3.5±0.1 | 9.6±0.3 | 3.2 | 0.3 |
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2.3.3 酶添加量、NAD+浓度和葡萄糖浓度对催化反应的影响为进一步提高其生产效率,对酶添加量、NAD+浓度和葡萄糖浓度进行优化。如图 5所示,随着reLcLDH1Q88A/I229A添加量的增大,L-PLA的产率逐渐增加,当达到10 U/mL时,L-PLA产率可达99.9%;当NAD+添加量为2.0 mmol/L时,L-PLA产量达到最高;葡萄糖浓度超过120 mmol/L后L-PLA产量稍微降低。与初始反应体系相比,reLcLDH1Q88A/I229A酶添加量、NAD+浓度和葡萄糖浓度分别降低至10 U/mL、2.0和120 mmol/L以提高其利用率。
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图 5 双酶偶联不对称还原PPA催化条件的优化 Fig. 5 Optimization of the catalytic conditions for the asymmetric reduction of PPA in double-enzyme system. |
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2.4 双酶偶联不对称还原苯丙酮酸以优化的催化条件下,扩大双酶偶联反应体系至25 mL,检测不对称还原100 mmol/L PPA制备L-PLA的反应进程。如图 6所示,反应105 min后,PPA的转化率 > 99.9%,L-PLA的产率可达到99.8%,eep > 99.9%,平均转化率aTOF为257.0 g/(g·h),时空产率为9.5 g/(L·h)。与其他不同来源的LDH不对称还原PPA制备PLA的比较如表 3所示,尽管L-LpLDH与GDH双酶共表达E. coli全细胞催化PPA生成L-PLA具有较高时空产率,但其L-PLA的产率仅为55.8%;Xu等[26]利用来源于乳杆菌Lactobacillus sp. CGMCC 9967的苯丙酮酸还原酶LaPPR与GDH双酶共表达E. coli全细胞(20 g干细胞)催化PPA生成D-PLA浓度高达550.0 mmol/L,产率为91.3%,由于较低的催化活力,其aTOF仅为0.5 g/(g·h)。与已有报道相比,reLcLDH1Q88A/I229A和SyGDH体外双酶偶联催化不对称还原PPA具有更高的产率和生产效率。
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图 6 reLcLDH1Q88A/I229A不对称还原PPA产L-PLA的进程曲线 Fig. 6 Time course of the production of L-PLA from PPA by reLcLDH1Q88A/I229A. |
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表 3 比较不同来源LDH不对称还原PPA制备PLATable 3 Comparison of asymmetric reduction of PPA to prepare PLA by LDH
Enzyme | Catalyst | Activity (U/mg) | PPA feeding (times) | PPA (mmol/L) | PLA (mmol/L) | Time (h) | c (%) | Yield (%) | STY (g/(L·h)) | aTOF (g/(g·h)) | eep(%) | Refernce |
L-LpLDH a | E. coli cells | 9.5 c | 2 | 186.2 | 103.8 (L) | 1.0 | 89.2 | 55.8 | 17.2 | ND | > 99.7 | [9] |
D-LpLDHY52V b | E. coli cells | 55.0 c | 5 | 164.6 | 105.2 (D) | 3.0 | 77.6 | 64.7 | 3.49 | 0.41 e | ND | [24] |
LpLDH b | L. plantarum cells | ND | 4 | 120.0 | 85.2 (NT) | 16.0 | 83.3 | 71.0 | 0.9 | 0.03 e | ND | [25] |
LaPPR a | E. coli cells | 19.6 d | 0 | 602.0 | 550.0 (D) | 9.0 | 100 | 91.3 | 10.1 | 0.51 e | > 99.0 | [26] |
D-LrLDH a | E. coli cell-free extract | 0.3 e | 0 | 182.9 | 108.9 (D) | 3.0 | NT | 60.3 | 6.8 | 0.45 e | > 99.0 | [27] |
L-LcLDH1Q88A/I229A a | P. pastoris cultured supernatant | 270.4 f | 0 | 100.0 | 99.8 (L) | 1.8 | 100 | 99.8 | 9.5 | 257.0 | > 99.9 | This study |
a Coupled with GDH; b Coupled with formate dehydrogenase; c Cell-free extract; d Purified enzyme; e Dry cells; f Cultured supernatant; NT means not told. |
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3 结论微生物发酵和酶催化是生物催化法合成PLA的两种重要途径。微生物发酵法由于底物转化率低、反应时间长和无法获得光学纯PLA,而不能满足实际生产。L-乳酸脱氢酶是酶催化生产L-PLA的关键酶,但目前已报道的L-乳酸脱氢酶的酶活力较低,且大多以大肠杆菌表达宿主,存在生成副产物、产率较低和生产效率低等缺点。本研究将突变酶L-LcLDH1Q88A/I229A在毕赤酵母中分泌表达,重组L-LcLDH1Q88A/I229A具有高酶活力;构建reLcLDH1Q88A/I229A与SyGDH的体外辅酶NADH再生的双酶偶联体系不对称还原PPA,并通过优化双酶偶联催化体系显著提高了底物PPA浓度和L-PLA的生产效率。本研究构建的重组L-乳酸脱氢酶突变体的体外辅酶循环体系,在不对称还原PPA中具有高催化活力、不产生副产物、高光学纯度和高生产效率等优点,使其具有较好的工业应用前景。
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