CN116426500A - 一种高酯化能力的脂肪酶突变体及其表达应用 - Google Patents
一种高酯化能力的脂肪酶突变体及其表达应用 Download PDFInfo
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Abstract
本发明提供了一种高酯化能力的脂肪酶突变体及其表达应用,通过半理性理性设计、定点突变技术改造MAS1脂肪酶基因,最后得到具有高酯化能力的脂肪酶突变体,将其应用到了分泌表达系统中。本发明脂肪酶突变体不仅能提升n‑3PUFA甘油三酯的生产效率,同时可以大大节约工厂生产的经济成本,满足n‑3PUFA甘油三酯的工业生产需求。
Description
技术领域
本发明主要涉及理性蛋白质工程进化手段,可对已有的蛋白质结构进行改造,以增强其催化性能。本发明还涉及定点突变后的脂肪酶基因在分泌生产脂肪酶中的应用,属于蛋白质工程以及脂肪酶生产领域。
背景技术
n-3多不饱和脂肪酸(n-3Polyunsaturated fatty acids,n-3PUFA)作为必需氨基酸,因其重要的生理功能受到广泛的关注,如抗心血管疾病、降血脂、降血糖、抗癌、消炎、缓解精神分裂症等。n-3PUFA的存在形式有甘油酯型、游离型和乙酯型。研究表明,游离型n-3PUFA虽易被人体消化吸收,但易氧化生成过氧化物,酸臭味大,难以直接食用。相较于甘油酯型n-3PUFA,乙酯型n-3PUFA更易氧化,且生物利用度低。甘油酯型是n-3PUFA利用的主要存在形式,而天然油脂中n-3PUFA甘油三酯的含量很低。因此,合成富含n-3PUFA甘油酯型产品是当前的发展趋势。
化学法合成甘油酯的过程中需要无机酸、碱为催化剂及分子蒸馏、低温结晶、尿素络合、超临界流体萃取、银离子络合等后处理手段进行除杂。相比较化学法,酶法能够更加高效绿色的催化合成得到n-3PUFA甘油酯。
目前利用酶法制备n-3PUFA甘油酯的方法主要有水解法、酯交换法和酯化法。前两种方法由于受天然鱼油中n-3PUFA含量低的限制,使得n-3PUFA在甘油酯中的含量仍然得不到较大提高,脂肪酶酯化合成方法为此提供了一个重要的技术途径。
MAS1脂肪酶是一个具有很好商业潜力的长链选择性的脂肪酶,其结构与催化机制已经有了清晰的认知,其氨基酸序列如SEQ ID NO.1所示。2016年Wang等(Xiumei Wang,Daoming Li,Weifei Wang,et al.A highly efficient immobilized MAS1 lipase forthe glycerolysis reaction of n-3PUFA-rich ethyl esters.Journal of MolecularCatalysis B:Enzymatic,2016,134:25-31.)研究固定化脂肪酶MAS1催化乙酯型n-3PUFA进行酯交换反应制备甘油型n-3PUFA的能力,结果证明该酶催化能力优于商品化酶Novozym435和Lipozyme RM IM,且甘油酯中n-3PUFA含量更高。2017年Wang等(Xiumei Wang,Daoming Li,Man Qu,et al.Immobilized MAS1 lipase showed high esterificationactivity in the production of triacylglycerols with n-3polyunsaturated fattyacids.Food Chemistry,2017,216:260-267.)将MAS1固定在XAD1180树脂上,在1g脂肪酶,65℃,pH 8.0,甘油:n-3PUFA摩尔比为1:3,反应总体积10.8mL的最优条件下反应24h,固定化MAS1的酯化率高达99.31%。但其仍存在进一步的改造空间,如反应中酶的添加量较大,为9.26%(g/mL),工业生产中成本造价高,反应时间较长,为24h,生产效率有待进一步提升。
因此,进一步对MAS1进行改造,开发一种经济成本低、耗时短且高效制备n-3PUFA甘油三酯的脂肪酶,使该酶更具工业应用和商业价值,是本领域技术人员致力于研发的方向之一。
发明内容
本发明的目的,就是为了解决上述问题而提供了一种能短时高效合成n-3PUFA甘油三酯的脂肪酶突变体,满足n-3PUFA甘油三酯工业化生产需求,节约生产成本,缩短生产时间,提高生产效率。
本发明的目的是这样实现的:
本发明提供了一种高酯化能力的脂肪酶突变体,其氨基酸序列如SEQ ID NO.3所示。
编码权利要求1所述高酯化能力的脂肪酶突变体的基因。
本发明提供了一种重组表达载体,其包含如SEQ ID NO.4所示的核苷酸序列。
还提供了一种宿主细胞,其包含上述的重组表达载体。
本发明还提供了高酯化能力的脂肪酶突变体基因在分泌表达脂肪酶突变体中的应用。
通过半理性设计突变点,得到本发明脂肪酶突变体,即将野生型MAS1脂肪酶第162位的天冬氨酸突变为缬氨酸。
本发明脂肪酶突变体不仅能提升n-3PUFA甘油三酯的生产效率,同时可以大大节约工厂生产的经济成本,满足n-3PUFA甘油三酯的工业生产需求。
附图说明
图1是实施例1中突变体pET22(b)-D162V质粒的核酸电泳图(其中,M:标准分子量Marker;1:D162V突变体);
图2是实施例2中突变体D162V目的蛋白纯化后的蛋白电泳图(其中,1:ProteinMarker;2:蛋白粗酶液;3:蛋白上样穿出液;4:25mM咪唑洗脱液;5:100mM咪唑洗脱液;6:300mM咪唑洗脱液;7:500mM咪唑洗脱液);
图3实施例3中MAS1野生型和突变体D162V甘油三酯生成率的对比图。
具体实施方式
下面结合具体实施例对本发明作进一步的说明,但并不局限于此。下述实施例中所述试剂原料除非注明来源外,均为市售的常见原料,试剂的配制采用常规方法。实施例中未详述的方法均为本领域常规操作。
生物材料:
采用全人工合成的方式合成野生型脂肪酶MAS1基因序列(根据克隆载体性质对其进行密码子优化过的),核苷酸序列如SEQ ID NO.2所示,并构建含有该脂肪酶MAS1基因的pET22b质粒,即pET22b-MAS1质粒。
实施例1含突变体V402F基因的质粒构建
含突变体D162V基因的质粒构建
D162V突变体是用缬氨酸取代脂肪酶MAS1氨基酸序列162位的天冬氨酸,且在C端添加6x His标签。
该突变体以脂肪酶MAS1基因序列为模板,采用PCR的方法进行构建,具体引物设计如下表所示:
以脂肪酶MAS1基因序列为模板,通过上述方法对脂肪酶MAS1基因序列进行定点突变,得到突变体目的基因D162V,具体步骤如下:
1.1pET22b-MAS1原始质粒的扩增
将含有野生型MAS1脂肪酶基因质粒的甘油菌按照1%接种量于10mL LB抗性液体培养基,37℃过夜培养12h左右,第二天抽提质粒。
1.1.1质粒DNA抽提的具体操作
(1)吸取3mL过夜培养好的菌液8000g离心2min后收集菌体,吸去上清液。加入200μL FAPD1对沉淀菌体进行重悬。
(2)加入200μL FAPD2后缓慢翻转5-10次使离心管内液体混匀,室温静置2-4min。
(3)加入350μL FAPD3后缓慢翻转5-10次使离心管内液体混匀。
(4)12000g离心5min后吸取上清液到吸附柱,9000g离心30s。倒掉穿出液,将吸附柱放入同一个收集管中。
(5)加500μL W1 Buffer吸附柱中,9000g离心30s。倒掉穿出液,将吸附柱放入同一个收集管中。
(6)加500μL Wash Buffer到吸附柱中,9000g离心30s。倒掉穿出液,将吸附柱放入同一个收集管中。
(7)将空吸附柱和收集管放入离心机,18000g离心1min,倒掉穿出液。
(8)加50μL Elution Buffer于吸附膜中央,室温静置2min,18000g离心1min后得到的质粒DNA溶液,用Nano drop测定浓度,于-20℃保存或于后续试验中使用。
1.2目的基因D162V的获取及pET22b-D162V质粒的扩增
1.2.1突变体D162V的构建
以MAS1质粒为模板,按照如下体系和条件构建50μL PCR体系并进行PCR。PCR产物进行核酸电泳,电泳电压120V,30-50min,紫外灯下观察,结果如图1所示。脂肪酶MAS1目的基因片段长度939bp,大肠杆菌表达载体PET-22b载体5459bp,所以整个质粒长度在6398bp,从图1中看出构建成功。
体系:
条件:
用上海宝生物公司的Blunting and Kination试剂盒,将以上PCR产物平末端化及磷酸化,反应体系如下所示;其中37℃中孵育15min,70℃孵育5min灭酶。平末端化和磷酸化后的反应体系放于-20℃保存,或者直接用于连接反应。
反应体系:
取平末端化及磷酸化的反应体系,按照如下反应体系添加各组分,混匀,16℃孵育1h后,反应体系于-20℃保存或用于下一步转化。
反应体系:
1.2.2大肠杆菌克隆宿主转化
感受态E.coli DH5α连接体系转化步骤如下:
(1)将保存于-80℃冰箱的感受态放置于冰上解冻并取100μL分装于预冷的1.5mL的无菌离心管中。
(2)取2μL连接体系加入溶解的感受态细胞中,轻弹两下混匀,并置于冰上放置30min。
(3)42℃热激45-90s,快速放在冰上放置2-5min。
(4)加入1mL LB培养基,37℃,200rpm,摇床孵育1h。
(5)取50-100μL转化细胞涂布于含有50μg/mL的Amp抗性平板上,于37℃培养箱倒置培养12-16h。
1.2.3阳性克隆子筛选
在超净台中,挑取平板中3-5个单菌落,轻点至含Amp抗性的固体LB平板上,作好标记,于37℃倒置培养12-16h,其对应的枪头在1mL Amp抗性培养基里涮几下,于37℃,250rpm,摇床孵育8-9h。取孵育好的500μL菌液送样测序,同时剩余500μL菌液加入500μL50%甘油保种,-80℃保存。待公司返还测序结果,用SnapGene软件比对序列。序列比对正确的菌液所对应的菌落挑单,接种于10mL Amp LB液体培养基中,37℃,250rpm摇床过夜,第二天抽提质粒,具体步骤同1.1.1。
实施例2突变体D162V目的蛋白的获取
2.1突变体D162V的表达与纯化
将构建成功的质粒转入大肠杆菌BL21(DE3)中,具体步骤同1.2.2。挑取单菌落于10mL Amp抗性的LB液体培养基中,37℃,250rpm,培养12h。取活化的菌液10mL接种于500mLAmp抗性的LB液体培养基中,37℃,250rpm培养至OD值为0.7,加入IPTG至终浓度0.1mM,诱导发酵12h。发酵完成后,将菌液在4℃,8,000×g条件下离心5min,收集沉淀。
称量菌体沉淀,用10倍(m:V)的破碎缓冲液(20mM PBS,pH 7.4)进行菌体重悬,破碎参数:50%功率,15min。破碎全程在冰上,防止菌体破碎过程中变性失活。破碎后的菌液于4℃,18,000×g条件下高速离心,离心后的上清过0.22μm滤膜除杂。选用Histrap HP 5mL镍柱对带有His标签的蛋白进行特异性吸附,优先用Binding Buffer(20mM PBS,500mMNaCl,pH 7.4)对整个系统进行平衡,将过滤好的上清置于Super loop中,以3mL/min的流速进行上样,收集穿出液,随后再次用Binding Buffer进行平衡,然后用Elution Buffer(20mM PBS,500mM NaCl,500mM IM,pH 7.4)进行梯度洗脱。梯度洗脱程序:5%,20%,60%,100%的Elution Buffer各10个柱体积,收集洗脱缓冲液进行后续的SDS-PAGE蛋白电泳验证、蛋白浓缩和Buffer置换,SDS-PAGE结果如图2。图2中,从1道到8道分别依次是:Marker,粗酶液,上样穿出液,25mM咪唑洗脱液,100mM咪唑洗脱液,300mM咪唑洗脱液,500mM咪唑洗脱液,Marker。发现在咪唑浓度为300mM时蛋白条带单一,蛋白纯度较高。
使用10kDa的超滤管对镍柱纯化后的MAS1蛋白溶液进行超滤脱盐处理,加10mL蛋白溶液至超滤管中,4500rpm离心至超滤管中的溶液至1mL刻度处,加入破碎缓冲液,重复离心3-4次。
用BCA蛋白浓度试剂盒测定纯化后的蛋白浓度,根据蛋白浓度计算0.5mg蛋白对应体积,于冰上分装至2mL EP中,封口膜封好并扎孔,-80℃过夜预冻,用冷冻干燥机对其进行冷冻干燥,得到酶粉。
由此,本实施例最终表达纯化得到突变体D162V目的蛋白。
实施例3n-3PUFA甘油三酯合成中的应用
本实施例以甘油和DHA为底物,分别用MAS1-D162V以及野生型MAS1(WT)催化底物反应生成DHA甘油三酯,考察二者的酯化能力。具体方法如下:
酯化体系:
将冻干的0.5mg酶粉(WT/D162V)加200μL H2O配制成0.0025mg/μL酶液,吸取10μL酶液加入酯化体系,继续按如上体系加样,于40℃,700rpm,反应6h,每次反应上三个平行。反应结束后迅速制样,用正己烷稀释一定倍数,过0.22μm有机相滤膜后进行液相色谱检测。根据如下公式计算得到甘油三酯生成率。
在酶量为0.0025mg,反应时间为6h,反应体积为1mL的反应条件下,结果如图3所示,WT的甘油三酯生成率为24.57%,突变体MAS1-D162V的甘油三酯生成率为53.74%,D162V的甘油三酯生成率是WT的2.18倍,突变体MAS1-D162V较野生型MAS1在短时间内达到了相对高的DHA甘油三酯生成率,且脂肪酶的用量仅为0.0025%(g/mL),因此,该突变体不仅能提升n-3PUFA甘油三酯的生产效率,同时可以大大节约工厂生产的经济成本,满足n-3PUFA甘油三酯的工业生产需求。
以上实施例仅供说明本发明之用,而非对本发明的限制,有关技术领域的技术人员,在不脱离本发明的精神和范围的情况下,还可以作出各种变换或变型,因此所有等同的技术方案也应该属于本发明的范畴,应由各权利要求所限定。
Claims (5)
1.一种高酯化能力的脂肪酶突变体,其特征在于,其氨基酸序列如SEQ ID NO.3所示。
2.编码权利要求1所述高酯化能力的脂肪酶突变体的基因。
3.一种重组表达载体,其包含如SEQ ID NO.4所示的核苷酸序列。
4.一种宿主细胞,其包含如权利要求3中所述的重组表达载体。
5.权利要求2中所述基因在分泌表达脂肪酶突变体中的应用。
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