CN108913671A - 一种ω-转氨酶突变体及其应用 - Google Patents
一种ω-转氨酶突变体及其应用 Download PDFInfo
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- CN108913671A CN108913671A CN201810619322.2A CN201810619322A CN108913671A CN 108913671 A CN108913671 A CN 108913671A CN 201810619322 A CN201810619322 A CN 201810619322A CN 108913671 A CN108913671 A CN 108913671A
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Abstract
本发明公开了一种ω‑转氨酶突变体及其应用,该ω‑转氨酶突变体的氨基酸序列如SEQ ID NO.2所示。本发明ω‑转氨酶突变体由土曲霉(Aspergillus terreus)ω‑转氨酶77位的异亮氨酸、150位的甲硫氨酸、210位的组氨酸和280位的甲硫氨酸分别突变为亮氨酸、半胱氨酸、天冬酰胺和半胱氨酸获得,该ω‑转氨酶突变体的半失活温度(T50 10)为50.3±0.5℃,比野生型提高了11.8℃,在40℃下的半衰期(t1/2)为114.6±2.8min,是野生型的16.6倍,热稳定性显著提高。
Description
技术领域
本发明涉及分子生物学技术领域,尤其涉及一种ω-转氨酶突变体及其应用。
背景技术
手性胺类化合物是具有重要价值的医药及精细化工中间体。目前,超过70%的药物,如 神经类药物、心血管药物、抗高血压药物、抗感染药物及疫苗等都是以手性胺作为中间体来 合成的。
来自于土曲霉(Aspergillus terreus)的ω-转氨酶以酮类化合物为原料,通过立体选择性地 转氨基作用,可以高效生产手性胺,催化氨基供体上的氨基转移到前手性的受体酮,得到手 性胺和副产物酮,反应过程需要磷酸吡哆醛(pyridoxal phosphate,PLP)的参与,催化过程如 下所示:
研究表明,该酶野生型在40℃下的半衰期仅为6.9min,其热稳定性有待进一步提高。
目前,酶稳定性改造的方法有三种,分别是:非理性设计,理性设计和半理性设计三个 方面。其中,基于序列的半理性设计是通过对天然蛋白质序列进行统计学分析,得到的结果 用于指导鉴定可能的靶标位点。较常用的方法是对蛋白质进行一致性突变。一致性突变方法 认为在同源蛋白质中某一个氨基酸位置,一致的氨基酸比不一致的氨基酸对蛋白质稳定性的 平均贡献要高,因此用一致的氨基酸取代不一致的氨基酸,可以提升酶的稳定性。
申请公布号为CN107058256A的发明专利申请文献公开了一种ω-转氨酶突变体,该突变 体获得的方法是利用NCBI数据库和BLAST软件比对筛选获得与ω-转氨酶同源的氨基酸序 列,通过Weblogo程序得到序列一致性结果,并结合ω-转氨酶的序列确定需要突变的氨基酸 残基位点,通过定点突变技术进行实验验证。
根据上述方法,该发明分别提供了第77位氨基酸由异亮氨酸突变为亮氨酸,或第97位 氨基酸由谷氨酰胺突变为谷氨酸,或第210位氨基酸由组氨酸突变为天冬酰胺,或第245位 氨基酸由天冬酰胺突变为天冬氨酸,或第292位氨基酸由甘氨酸突变为天冬氨酸,或者第295 位氨基酸由异亮氨酸突变为缬氨酸的突变体。
此外,二硫键(disulfide bond)是2个巯基被氧化所形成的-S-S-形式的硫原子间的共价 键。在蛋白质中,两个半胱氨酸形成二硫键,能够固定蛋白质的三级结构,降低蛋白质解折 叠熵,提高蛋白质稳定性。
申请公布号为CN105950581A的发明专利申请文献公开了一种引入二硫键的ω-转氨酶突 变体,该突变体是由土曲霉(Aspergillus terreus)ω-转氨酶131位的精氨酸和134位的天冬氨酸 分别突变为半胱氨酸获得。该突变体的半失活温度比野生型提高了1.6℃;在40℃下的半衰 期为10.4min,比野生型延长了3.5min,较野生型提高了50.7%,热稳定性大大提高。
虽然,上述突变体与野生型ω-转氨酶相比能够提高半失活温度以及延长半衰期;但是, 若要在实际生产中更好地加以应用,上述突变体的半失活温度和半衰期仍有待进一步提高。
发明内容
本发明通过组合序列一致性突变和引入二硫键的两种不同理性设计方法,实现了ω-转氨 酶热稳定性的进一步提高,获得了一个具有极佳热稳定性的ω-转氨酶突变体。
首先,本发明利用序列一致性突变筛选热稳定高的ω-转氨酶突变体,具体方法如下:
(1)利用NCBI数据库和BLAST软件比对筛选获得与ω-转氨酶同源的氨基酸序列;
(2)利用Weblogo程序(http://weblogo.berkeley.edu/logo.cgi)得到序列一致性结果, 并结合ω-转氨酶的序列确定需要突变的氨基酸残基位点;
(3)根据氨基酸残基位点设计定点突变引物,以ω-转氨酶基因为模板,进行定点PCR 扩增,纯化后,转化至宿主细胞,获得定点突变文库;
(4)从所述定点突变文库中筛选ω-转氨酶突变体,通过实验确定热稳定性提高的ω-转 氨酶突变体(H210N/I77L)。
与此同时,根据ω-转氨酶的三维结构模型并分析蛋白质结构中B-factor数据,结合键长、 键角、能量等生物信息学特征,使用生物信息学计算软件Disulfide by Design(DbD,http://c ptweb.cpt.wayne.edu/DbD2)以及Disulfide Bonds in Proteins(MODIP,http://caps.ncbs.res.in/ds dbase/modip.html)对该酶进行引入二硫键的理性设计,选出二硫键潜在的引入位点;在ω-转 氨酶突变体H210N/I77L的基础上引入一对额外的二硫键(M150C-M280C),获得ω-转氨酶 突变体(H210N/I77L/M150C/M280C)。
所述ω-转氨酶突变体(H210N/I77L/M150C/M280C)的氨基酸序列如SEQ ID NO.2所示。
本发明又提供了编码所述ω-转氨酶突变体的基因。
所述的基因,核苷酸序列如SEQ ID NO.1所示。
ω-转氨酶突变体(H210N/I77L/M150C/M280C)是指ω-转氨酶(来自土曲霉(Aspergillus terreus),核苷酸序列如SEQ ID NO.3所示,氨基酸序列如SEQ ID NO.4所示)的第77位的 氨基酸由异亮氨酸突变为亮氨酸、第150位的氨基酸由甲硫氨酸突变为半胱氨酸、第210位 的氨基酸由组氨酸突变为天冬酰胺以及第280为的氨基酸由甲硫氨酸突变为半胱氨酸。
本发明还提供了包含所述基因的表达单元。
本发明还提供了包含所述基因的重组载体。重组载体的启动子可以为常用的T7启动子、 Iac启动子或araBAD启动子,在这些启动子的作用下,ω-转氨酶突变体可以直接在大肠杆菌 宿主细胞中实现胞内可溶表达。重组载体所选用的原始载体可以是常用的用于蛋白表达的载 体,比如pET28a。
本发明还提供了包含所述基因的基因工程菌。
本发明还提供了包含所述重组载体的基因工程菌。
可以将目的基因片段整合到基因工程菌的基因组上以获得稳定表达所述ω-转氨酶突变 体蛋白的重组基因工程菌,也可以是通过质粒的形式存在。可以选择常用的用于蛋白表达的 基因工程菌,优选为E.coli BL21(DE3)。
本发明还提供了所述ω-转氨酶突变体在催化(R)-(+)-α-甲基苄胺生成苯乙酮中的应用。 手性选择性为(R)型。
与现有技术相比,本发明具有以下有益效果:
(1)本发明ω-转氨酶突变体由土曲霉(Aspergillus terreus)ω-转氨酶77位的异亮氨酸、 150位的甲硫氨酸、210位的组氨酸和280位的甲硫氨酸分别突变为亮氨酸、半胱氨酸、天冬 酰胺和半胱氨酸获得,该ω-转氨酶突变体的半失活温度(T50 10)为50.3±0.5℃,比野生型 提高了11.8℃,在40℃下的半衰期(t1/2)为114.6±2.8min,是野生型的16.6倍,热稳定性 显著提高。
(2)本发明利用NCBI数据库和BLAST软件比对筛选获得与ω-转氨酶同源的氨基酸序 列,通过Weblogo程序得到序列一致性结果,并结合ω-转氨酶的序列确定需要突变的氨基酸 残基位点,通过定点突变技术进行实验验证,找到序列一致性突变中热稳定提高最多的突变 酶,并在此基础上引入一对额外的二硫键(M150C-M280C),获得的ω-转氨酶突变体(H210N/I77L/M150C/M280C);该方法可有效地提高正突变概率,提高实验效率及可行性,并筛选得到热力学稳定性明显优于野生酶的突变酶。
附图说明
图1为质粒pET28a(+)-ω-AT的基因图谱。
图2为ω-转氨酶序列一致性分析结果图。
图3为突变体在ω-转氨酶三级结构中的位置图(以球表示)及其序列一致性分析结果图。
图4为实施例1中不同突变体酶和野生型酶的半失活温度T50 10结果。
图5为实施例1中不同突变体酶和野生型酶的半衰期t1/2结果。
图6为实施例1中不同突变体酶和野生型酶的最适温度结果。
具体实施方式
下面结合实施例对本发明作进一步说明。实施例所描述的具体物料配比、工艺条件及其 结果仅用于说明本发明,而不应当也不会限制权利要求书的保护范围。
实施例中未注明的实验方法,如感受态细胞制备、转化以及LB培养基配制等参照《分 子克隆实验指南》第三版(J.萨姆布鲁克、D.W.拉塞尔著,黄培堂译,科学出版社,2002)中 的方法进行。
下列实施例中涉及的实验材料如下:
(1)LB培养基:10g/L胰蛋白胨(购于Oxoid),5g/L酵母粉(购于Oxoid),10g/L 氯化钠(购于生工生物工程有限公司(上海)),pH 7.0。LB固体培养基:LB液体培养基加 入2%(质量比)琼脂粉。
(2)Dpn I酶购于Thermo Scientific公司,PrimeStar Max DNA聚合酶、限制性内切酶 Nde I、HindШ、T4DNA连接酶购于TaKaRa公司(宝生物工程大连有限公司,中国)。
引物序列由南京金斯瑞生物科技有限公司合成(中国)。质粒提取试剂盒、DNA琼脂糖 凝胶回收试剂盒、PCR产物纯化试剂盒以及SDS-PAGE凝胶制备试剂盒购于康为世纪生物科 技有限公司公司(中国)。
氯化钠、甘油、氯化钙、咪唑、冰醋酸、磷酸氢二钠、磷酸二氢钠、5-磷酸吡哆醛、考马斯亮蓝蛋白质浓度测定试剂盒、Ni-NTA层析介质、异丙基-β-D-硫代半乳糖苷(IPTG)、硫酸卡那霉素、DNA和蛋白质Marker购于生工生物工程有限公司(上海)。
(3)缓冲液配方:
20mmol/L洗脱缓冲液:50mmol/L磷酸二氢钠,300mmol/L氯化钠,20mmol/L咪唑,pH 8.0;50mmol/L洗脱缓冲液:50mmol/L磷酸二氢钠,300mmol/L氯化钠,50mmol/L咪 唑,pH8.0;100mmol/L洗脱缓冲液:50mmol/L磷酸二氢钠,300mmol/L氯化钠,100mmol/L 咪唑,pH8.0;250mmol/L洗脱缓冲液:50mmol/L磷酸二氢钠,300mmol/L氯化钠,250mmol/L 咪唑,pH8.0。
凝胶染色液:甲醇45.4mL,冰醋酸9.2mL,0.05g考马斯亮蓝R250,溶解于100mL去离子水;凝胶脱色液:甲醇5mL,冰醋酸7.5mL,溶解于100mL去离子水;电泳缓冲液: 3.03gTris,14.4g甘氨酸,1g SDS,用去离子水定容至1L,0.22μm的滤纸抽滤后4℃冷藏。
实施例1
1.序列一致性分析
根据NCBI数据库中Aspergillus terreus的ω-转氨酶基因序列(Genbank:XM_001209325) 以及密码子使用数据(http://www.kazusa.or.jp/codon/)中的Escherichiacoli密码子使用频率分布 表,分析ω-转氨酶的密码子使用情况。密码子优化的ω-转氨酶基因(ω-opt-TA基因),其核苷 酸序列如SEQ ID NO.3所示,其编码的蛋白质共325个氨基酸,氨基酸序列如SEQ ID NO.4 所示。
通过BLAST比对,设置E-value最大值为10-3,序列冗余度不超过0.9,共筛选获得同源 的氨基酸序列91条。依据序列一致性进行突变位点的筛选,筛选的原则是突变的保守性阈值 为0.6,即在多序列比对中该位点氨基酸类型所占的比例达到60%以上,利用Weblogo程序 (http://weblogo.berkeley.edu/logo.cgi)得到序列一致性分析结果(如图2),共筛选出6个突 变位点:H210N、Q97E、G292D、N245D、I295V和I77L(本实施例仅列举H210N、I77L和 两个位点组合后的实验结果)。
2.二硫键位点的选择
将氨基酸序列如SEQ ID NO.4所示的ω-转氨酶的PDB文件(PDB ID:4CE5)分别上传至DbDV 2.12软件(http://cptweb.cpt.wayne.edu/DbD2)和MODIP软件(http://caps.ncbs.res.i n/dsdbase/modip.html)中进行预测易于形成二硫键的位点,MODIP软件预测了153个可以引 入二硫键的位点,并基于形成二硫键的可能性大小,使用A、B、C、D进行排序。DbDV 2. 12软件充分考虑了蛋白质中形成二硫键的各种因素,包括两个半胱氨酸的C-S键旋转角度χ 1,S-S键旋转角度χss,Cα之间的距离rα,Cβ之间的距离rβ的平均范围等,预测出43个易 于形成二硫键的位点。
为了获得小型且有效的突变库,我们选择MODIP软件预测的A等级的二硫键位点与DbDV 2.12软件预测的二硫键位点的重叠点,作为本发明引入二硫键的位点,将其突变为半胱氨酸;选取M150C-M280C位点作为引入二硫键的位点。
3.组合突变
在引入H210N/I77L突变位点的基础上,再引入1对额外的二硫键(M150C-M280C)。
4.克隆
本发明中经密码子优化的ω-转氨酶基因(ω-TA基因)委托通用生物系统(安徽)有限公司进 行全基因合成,基因合成服务中使用pET-28a质粒作为克隆载体,酶切位点分别为NdeI和 Hind III。构建的重组质粒pET-28a-ω-opt-TA转入E.coli BL21(DE3),获得重组菌。
采用定点突变PCR方法依次对ω-转氨酶基因通过定点突变引入相应的突变位点,定点突 变的引物和模板如表1所示。
表1 ω-转氨酶位点定点突变引物的设计
以pET-28a-ω-opt-TA质粒为模板,进行定点PCR扩增。PCR扩增体系为50μL,包含:Prime STAR Max DNA Polymerase 25μL,1μL上游引物(10μM),1μL下游引物(10μM), 1μL质粒模板(100ng/μL),高温灭菌的超纯水补至总体积50μL。
PCR扩增程序为:98℃变性1min后,进入扩增循环,即98℃变性15s,55℃退火15s,72℃延伸3min,共循环30次,最后再72℃延伸7min。PCR产物经过电泳检测,其条带单 一、清晰。
所得定点PCR反应产物用Dpn I在37℃下酶解2h以消除父本模板,酶解产物采用热激法转化入化学感受态细胞E.coli DH 5α,转化液涂布含有卡那霉素(50μg/μL)的LB固体平板得到定点突变文库,于37℃培养12h。
5.突变酶的表达和纯化
从定点突变文库中随机挑取1~3个单菌落,培养并提取质粒,样品送至通用生物系统(安 徽)有限公司测定核苷酸序列,以确定是否引入预期的突变,测序引物为T7通用引物。
引入预期突变的质粒转化入E.coli BL21(DE3)中,挑取单菌落接种至加有5mL LB液 体培养基的试管中,37℃、200r/min条件下培养过夜。将培养好的菌液以1%比例(体积比) 的接种量接种至含50μg/mL卡那霉素的100mL的LB培养基(胰蛋白胨10g,酵母粉5g, 氯化钠10g,调节pH 7.0)中,37℃、180r/min培养至OD600值为0.4~0.6时,加入适量体 积的IPTG(终浓度为0.5mmol/L),然后在25℃、150r/min条件下诱导培养18h后,收集 菌体。
将收集的菌体用磷酸盐缓冲液洗涤两次,后用10%发酵液体积的破胞缓冲液重悬,超声 波破碎细胞,超声破胞工作条件为:功率300W,工作3s,间歇6s,超声8分钟。破胞液于12 000r/min,4℃条件下离心处理30min,收集上清液,即得到含有的ω-转氨酶粗酶液。
采用Ni-NTA亲和层析对所得的粗酶液进行分离纯化。经上样、清洗和洗脱,收集洗脱 液,透析除去小分子即得到纯酶。适当稀释后,以考马斯亮蓝法测定纯酶的浓度。
本实施例获得的ω-转氨酶突变体分别是:
a.突变体I77L(第77位氨基酸由异亮氨酸突变为亮氨酸);
b.突变体H210N(第210位氨基酸由组氨酸突变为天冬酰胺);
c.突变体H210N/I77L(第77位氨基酸由异亮氨酸突变为亮氨酸,且第210位氨基酸由 组氨酸突变为天冬酰胺);
d.突变体M150C-M280C(第150位的氨基酸由甲硫氨酸突变为半胱氨,且第280为的氨基酸由甲硫氨酸突变为半胱氨酸);
e.突变体H210N/I77L/M150C/M280C(第77位的氨基酸由异亮氨酸突变为亮氨酸、第1 50位的氨基酸由甲硫氨酸突变为半胱氨酸、第210位的氨基酸由组氨酸突变为天冬酰胺以及 第280为的氨基酸由甲硫氨酸突变为半胱氨酸)。
6.突变酶活力的测定
以(R)-(+)-α-甲基苄胺和丙酮酸为底物,底物溶液用磷酸盐缓冲液(50mM,pH 8.0)配制,200μL的反应体系中包括180μL底物溶(0.25%DMSO,2.5mM(R)-(+)- α甲基苄胺,2.5mM丙酮酸,0.1mM PLP),20μL纯酶液(约0.3mg/mL)。利用酶标仪检 测波长245nm处OD值随时间的变化曲线。
酶活计算方法如下:
ε为12,000M-1·cm-1,MD190酶标仪测定的数据是mAbs/min。
结果如表2所示。
7.突变酶热稳定性考察
分别将纯化后的野生酶和突变酶在25-55℃水浴中孵育10min,随后迅速放置在冰上 冷却10min。底物溶液用磷酸盐缓冲液(50mmol/L,pH 8.0)配制,200μL的反应体系 中包括180μL的底物溶液(0.25%DMSO、2.5mmol/L(R)-(+)-α-甲基苄胺((R)-α-MBA)、 2.5mmol/L丙酮酸以及0.1mmol/L PLP),20μL纯酶液(约0.3mg/mL),采用MD190酶 标仪检测波长在245nm处相应的酶活力(Purmonen M,Valjakka J,Takkinen K,et al. Moleculardynamics studies on the thermostability of family 11xylanases[J].ProteinEngineering Design and Selection,2007,20(11):551-559.)。
以温度为横坐标,以热处理后与处理前酶活力的比值为纵坐标作图,采用Origin8.0 软件进行Boltzmann S型函数拟合,计算半失活温度(T50 10)。
将野生酶和突变酶在40℃下分别孵育2-50min,孵育结束后迅速放置在冰上冷却10 min,采用上述方法测定酶活力。以时间为横坐标,以热处理后与处理前的比活力的比值为纵坐标作图,通过Origin 8.0软件拟合非线性方程y=exp(-kd·t),一阶速率常数(kd)经非线性回归确定,并计算酶活力降低为50%时所对应的半衰期(t1/2)。
实验结果如表2和图4、5所示。
表2 ω-转氨酶及其突变体的热稳定性结果
8.最适温度
以(R)-(+)-α甲基苄胺和丙酮酸为底物,底物溶液用磷酸盐缓冲液(50mM,pH 8.0)配制, 200μL的反应体系中包括180μL底物溶(0.25%DMSO,2.5mM(R)-(+)-α甲基苄胺,2.5mM 丙酮酸,0.1mM PLP),加入20μL纯酶液(约0.3mg/mL)后(并做三组平行对照),放入不同温度(25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃)的恒温混匀仪 中进行反应(400r/min),3min后放入100℃的水浴中煮沸10min,最后在冰上放置10min 后,利用酶标仪检测波长245nm处的OD值。
实验结果如表2所示。
结果表明,突变体酶H210N、突变体酶I77L和突变体酶M150C-M280C的最适反应温度 均为45℃;突变体酶H210N/I77L的最适反应温度为55℃,与野生酶相比提高了10℃;突变体酶H210N/I77L/M150C-M280C的最适反应温度为55℃,与野生酶相比提高了10℃;但是,突变体酶在较高温条件下具有较好的热力学稳定性。
序列表
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Claims (8)
1.ω-转氨酶突变体,其特征在于,氨基酸序列如SEQ ID NO.2所示。
2.编码如权利要求1所述ω-转氨酶突变体的基因。
3.如权利要求2所述的基因,其特征在于,核苷酸序列如SEQ ID NO.1所示。
4.一种包含如权利要求2或3所述基因的表达单元。
5.一种包含如权利要求2或3所述基因的重组载体。
6.一种包含如权利要求2或3所述基因的基因工程菌。
7.一种包含如权利要求5所述重组载体的基因工程菌。
8.如权利要求1所述ω-转氨酶突变体在催化(R)-(+)-α-甲基苄胺生成苯乙酮中的应用。
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CN110144335A (zh) * | 2019-04-26 | 2019-08-20 | 浙江科技学院 | 一种ω-转氨酶双突变体及其应用 |
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US20220177933A1 (en) * | 2020-12-04 | 2022-06-09 | Zhejiang University Of Science & Technology | Omega-transaminase mutant obtained by dna synthetic shuffling combined mutation and use |
CN114107241A (zh) * | 2020-12-04 | 2022-03-01 | 浙江科技学院 | 一种通过DNA合成改组组合突变获得的ω-转氨酶突变体及应用 |
CN114107241B (zh) * | 2020-12-04 | 2023-12-08 | 浙江科技学院 | 一种通过DNA合成改组组合突变获得的ω-转氨酶突变体及应用 |
CN114181918A (zh) * | 2020-12-04 | 2022-03-15 | 浙江科技学院 | 一种通过DNA合成改组组合突变获得的ω-转氨酶突变体及应用 |
CN114181918B (zh) * | 2020-12-04 | 2023-09-15 | 浙江科技学院 | 一种通过DNA合成改组组合突变获得的ω-转氨酶突变体及应用 |
CN112481230A (zh) * | 2020-12-04 | 2021-03-12 | 浙江科技学院 | 一种通过DNA合成改组组合突变获得的ω-转氨酶突变体及应用 |
CN112481230B (zh) * | 2020-12-04 | 2021-12-07 | 浙江科技学院 | 一种通过DNA合成改组组合突变获得的ω-转氨酶突变体及应用 |
WO2023061237A1 (zh) * | 2021-10-14 | 2023-04-20 | 弈柯莱生物科技(上海)股份有限公司 | 转氨酶及其用于制备西他列汀或其中间体的用途 |
CN115975969A (zh) * | 2021-10-14 | 2023-04-18 | 弈柯莱生物科技(上海)股份有限公司 | 转氨酶及用于制备西他列汀或其中间体的用途 |
CN114134128B (zh) * | 2021-12-07 | 2023-05-23 | 浙江科技学院 | 一种基于祖先序列重建的ω-转氨酶突变体 |
WO2023103947A1 (zh) * | 2021-12-07 | 2023-06-15 | 浙江科技学院 | 一种基于祖先序列重建的ω-转氨酶突变体 |
CN114134128A (zh) * | 2021-12-07 | 2022-03-04 | 浙江科技学院 | 一种基于祖先序列重建的ω-转氨酶突变体 |
CN114525265B (zh) * | 2022-04-21 | 2022-09-30 | 凯莱英医药集团(天津)股份有限公司 | 转氨酶突变体及其应用 |
CN114525265A (zh) * | 2022-04-21 | 2022-05-24 | 凯莱英医药集团(天津)股份有限公司 | 转氨酶突变体及其应用 |
CN116486903A (zh) * | 2023-04-17 | 2023-07-25 | 深圳新锐基因科技有限公司 | 基于同源蛋白序列进化方向结合自由能变提高蛋白稳定性的方法及装置 |
CN116486903B (zh) * | 2023-04-17 | 2023-12-29 | 深圳新锐基因科技有限公司 | 基于同源蛋白序列进化方向结合自由能变提高蛋白稳定性的方法及装置 |
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