CN110791493A - 一种天冬氨酸氨裂合酶突变体及其应用 - Google Patents
一种天冬氨酸氨裂合酶突变体及其应用 Download PDFInfo
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- CN110791493A CN110791493A CN201911182183.2A CN201911182183A CN110791493A CN 110791493 A CN110791493 A CN 110791493A CN 201911182183 A CN201911182183 A CN 201911182183A CN 110791493 A CN110791493 A CN 110791493A
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- ammonia lyase
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Abstract
本发明公开了一种天冬氨酸氨裂合酶突变体,其氨基酸序列为SEQ ID NO:3,与初始型天冬氨酸氨裂合酶AspB相比,显著提高了催化丙烯酸生成β‑丙氨酸的酶活力。采用表达该天冬氨酸氨裂合酶突变体的全细胞催化120g/L丙烯酸底物反应生成β‑丙氨酸时,转化率超过95%,具有工业化应用前景。
Description
技术领域
本发明属于酶催化技术领域,具体地说,涉及一种天冬氨酸氨裂合酶突变体、及其用于酶法催化丙烯酸生产β-丙氨酸的用途。
背景技术
β-丙氨酸有香味、甜味,易溶于水,微溶于乙醇,是自然界中存在的一种天然β型氨基酸。β-丙氨酸是一种天然的非蛋白氨基酸,在生物体内不直接参与蛋白质的合成,但参与多种功能性物质如肌肽、维生素B5的合成,是一种功能性的氨基酸,被广泛应用于医药、食品、化工等行业。β-丙氨酸主要用于合成泛酸、泛酸钙、肌肽、帕米磷酸钠、八柳氮等,也可作为一种膳食补充剂为肌肉提供能量。
β-丙氨酸制备方法有化学法、酶法及发酵法三种方法。其中,化学法和酶法是现在主要的生产方法。化学法包括丙烯腈法、β-氨基丙腈法、琥珀酰亚胺降解法等,但这些方法为高温、高压反应,工艺条件苛刻,且反应过程副产物较多,提取过程复杂,成本高,环境不友好,这些不可避免的缺点导致化学法合成β-丙氨酸在市场上的竞争力越来越弱。酶法催化合成β-丙氨酸主要指的是L-天冬氨酸在L-天冬氨酸α脱羧酶(ADC)催化下脱羧生成β-丙氨酸,该方法催化效率高、条件温和、提取工艺简单环保,长久以来成为β-丙氨酸酶法催化合成研究的热点。但该方法需要使用相对昂贵的底物天冬氨酸,因此导致工艺成本较高,工业化应用方面受到很大的限制。发酵法采用廉洁易得的葡萄糖作为起始原料,一直是人们研究的热点,鲁东大学于2019在大肠杆菌工程菌中实现了以葡萄糖为原料发酵产β-丙氨酸,产量高达50g/L,糖酸转化率达40%(CN201910474753.9),这是已有报道的发酵法产β-丙氨酸的最高产量,尽管如此,由于该方法存在发酵副产物较多、提取工艺复杂、生成成本高等问题,导致该方法还仅限于实验室研究阶段。
2018年中科院微生物研究所吴边实验室采用计算机模拟技术,通过理性改造,成功对Bacillus sp.YM55-1来源的天冬氨酸氨裂合酶(AspB)的底物谱进行了改变,以烯酸为底物实现了多种β型氨基酸的高效合成,其中以巴豆酸为底物合成β-氨基丁酸,底物浓度可以达到300g/L,转化率达到99%,产物β-氨基丁酸的ee值超过99%(Ruifeng Li.et al,Computational redesign of enzymes for regio-and enantioselectivehydroamination.Nat.Chem.Biol.,2018)。
我们对于使用Bacillus sp.YM55-1来源的天冬氨酸氨裂合酶AspB催化合成β-丙氨酸进行了研究,发现该酶能够以丙烯酸或丙烯腈为底物经催化反应得到β-丙氨酸,但转化率较低,判断为AspB酶活力低造成的,因此有必要对该技术进行优化。
发明内容
为了探索使用廉价的丙烯酸或丙烯腈为底物、经酶催化制备β-丙氨酸的工业化可行性,本发明利用基因工程技术来对Bacillus sp.YM55-1来源的天冬氨酸氨裂合酶(AspB)进行改造和筛选,构建针对丙烯酸或丙烯腈高酶活性的天冬氨酸氨裂合酶突变体,从而有利于实现全新酶催化法生产β-丙氨酸的工业化。
为此,本发明通过随机突变、半理性设计等技术对Bacillus sp.YM55-1来源的初始型天冬氨酸氨裂合酶(SEQ ID NO:1)进行改造,获得能够以丙烯酸或丙烯腈作为特异性底物的高酶活的天冬氨酸氨裂合酶突变体,以便高效地将丙烯酸或丙烯腈催化生成β-丙氨酸。
因此,本发明的第一个目的在于提供一种酶活力高的天冬氨酸氨裂合酶突变体。
本发明的第二个目的在于提供编码上述天冬氨酸氨裂合酶突变体的基因。
本发明的第三个目的在于提供包含上述基因的质粒。
本发明的第四个目的在于提供转化了上述质粒的微生物。
本发明的第五个目的在于提供上述天冬氨酸氨裂合酶突变体或微生物在生产β-丙氨酸中的用途。
为了达到上述目的,本发明提供如下天冬氨酸氨裂合酶突变体:
一种天冬氨酸氨裂合酶突变体,其氨基酸序列为:
SEQ ID NO:3,其为SEQ ID NO:1第142位的N替换为V、第188位的H替换为A的突变体,氨基酸序列为:
MNTDVRIEKDFLGEKEIPKDAYYGVQTIRATENFPITGYRIHPELIKSLGIVKKSAALANMEVGLLDKEVGQYIVKAADEVIEGKWNDQFIVDPIQGGAGTSINMNANEVIANRALELMGEEKGNYSKISPNSHVNMSQSTVDAFPTATHIAVLSLLNQLIETTKYMQQEFMKKADEFAGVIKMGRCALQDAVPILLGQEFEAYARVIARDIERIANTRNNLYDINMGATAVGTGLNADPEYISIVTEHLAKFSGHPLRSAQHLVDATQNTDCYTEVSSALKVCMINMSKIANDLRLMASGPRAGLSEIVLPARQPGSSIIPGLVAPVMPEVMNQVAFQVFGNDLTITSASEAGQFELNVMEPVLFFNLIQSISIMTNVFKSFTENCLKGIKANEERMKEYVEKSIGIITAINPHVGYETAAKLAREAYLTGESIRELCIKYGVLTEEQLNEILNPYEMTHPGIAGRK(SEQ ID NO:3)。
一种编码上述天冬氨酸氨裂合酶突变体的基因。
优选地,编码上述天冬氨酸氨裂合酶突变体SEQ ID NO:3的基因可以是下述核苷酸序列:
ATGAACACCGACGTTCGTATCGAAAAAGACTTCCTGGGTGAAAAAGAAATCCCGAAAGACGCTTACTACGGTGTTCAGACCATCCGTGCTACCGAAAACTTCCCGATCACCGGTTACCGTATCCACCCGGAACTGATCAAATCTCTGGGTATCGTTAAAAAATCTGCTGCTCTGGCTAACATGGAAGTTGGTCTGCTGGACAAAGAAGTTGGTCAGTACATCGTTAAAGCTGCTGACGAAGTTATCGAAGGTAAATGGAACGACCAGTTCATCGTTGACCCGATCCAGGGTGGTGCTGGTACCTCTATCAACATGAACGCTAACGAAGTTATCGCTAACCGTGCTCTGGAACTGATGGGTGAAGAAAAAGGTAACTACTCTAAAATCTCTCCGAACTCTCACGTTAACATGTCTCAGTCTACCGTTGACGCTTTCCCGACCGCTACCCACATCGCTGTTCTGTCTCTGCTGAACCAGCTGATCGAAACCACCAAATACATGCAGCAGGAATTCATGAAAAAAGCTGACGAATTCGCTGGTGTTATCAAAATGGGTCGTTGCGCCCTGCAGGACGCTGTTCCGATCCTGCTGGGTCAGGAATTCGAAGCTTACGCTCGTGTTATCGCTCGTGACATCGAACGTATCGCTAACACCCGTAACAACCTGTACGACATCAACATGGGTGCTACCGCTGTTGGTACCGGTCTGAACGCTGACCCGGAATACATCTCTATCGTTACCGAACACCTGGCTAAATTCTCTGGTCACCCGCTGCGTTCTGCTCAGCACCTGGTTGACGCTACCCAGAACACCGACTGCTACACCGAAGTTTCTTCTGCTCTGAAAGTTTGCATGATCAACATGTCTAAAATCGCTAACGACCTGCGTCTGATGGCTTCTGGTCCGCGTGCTGGTCTGTCTGAAATCGTTCTGCCGGCTCGTCAGCCGGGTTCTTCTATCATCCCGGGTCTGGTTGCTCCGGTTATGCCGGAAGTTATGAACCAGGTTGCTTTCCAGGTTTTCGGTAACGACCTGACCATCACCTCTGCTTCTGAAGCTGGTCAGTTCGAACTGAACGTTATGGAACCGGTTCTGTTCTTCAACCTGATCCAGTCTATCTCTATCATGACCAACGTTTTCAAATCTTTCACCGAAAACTGCCTGAAAGGTATCAAAGCTAACGAAGAACGTATGAAAGAATACGTTGAAAAATCTATCGGTATCATCACCGCTATCAACCCGCACGTTGGTTACGAAACCGCTGCTAAACTGGCTCGTGAAGCTTACCTGACCGGTGAATCTATCCGTGAACTGTGCATCAAATACGGTGTTCTGACCGAAGAACAGCTGAACGAAATCCTGAACCCGTACGAAATGACCCACCCGGGTATCGCTGGTCGTAAATAA(SEQ ID NO:4)。
一种包含上述基因的质粒。该质粒包含用于表达上述基因的载体,优选载体是PET系列,比如载体是pET24a,但并不受限于此。
一种转化了上述质粒的微生物,该微生物可作为宿主用于表达上述天冬氨酸氨裂合酶突变体。
优选地,上述微生物选自枯草芽孢杆菌、毕赤酵母、酿酒酵母、大肠杆菌,优选大肠杆菌,更优选大肠杆菌BL21(DE3)。
上述天冬氨酸氨裂合酶突变体或者上述微生物可以用于生产β-丙氨酸。
在生产β-丙氨酸中,以丙烯酸或丙烯腈为底物原料,优先丙烯酸,用上述天冬氨酸氨裂合酶突变体或者微生物作为催化剂来催化反应。
作为一种可选的实施方式,上述微生物可以呈菌体或者其细胞破碎物形式,作为酰化反应的催化剂。
生产β-丙氨酸可采用常规的工艺条件,比如,反应体系中丙烯酸的浓度可选择10~20wt%,优选12wt%;
反应温度选择30~65℃,优选40~60℃,更优选42~50℃,最优选45±0.5℃。
反应体系可以为pH 8.0-10.0。
反应体系中原料丙烯酸或丙烯腈:氨水的摩尔配比可以为1:1.2-1:2。
相较初始型天冬氨酸氨裂合酶SEQ ID NO:1,本发明构建的天冬氨酸氨裂合酶突变体SEQ ID NO:3催化丙烯酸和丙烯腈反应的酶活性有明显提高,当天冬氨酸氨裂合酶突变体应用酶法生产β-丙氨酸时,采用浓度为120g/L的底物,β-丙氨酸生成率可达95%,具有工业化开发和应用前景。
附图说明
图1是本发明构建的重组质粒pET24a-AspB1图谱。
具体实施方式
本发明构建的天冬氨酸氨裂合酶突变体是Bacillus sp.YM55-1来源的初始型天冬氨酸氨裂合酶SEQ ID NO:1的突变体,是SEQ ID NO:1序列中两个氨基酸发生替换(N142V、H188A)后形成的新蛋白质。其中SEQ ID NO:1在文献(Ruifeng Li.et al,Computational redesign of enzymes for regio-and enantioselectivehydroamination.Nat.Chem.Biol.,2018)中有报道,编码基因是序列表中的SEQ ID NO:2。
为表述方便起见,蛋白质的氨基酸缩写既可以使用英文三字母、也可以采用英文单字母,这是本领域技术人员熟知的,这些缩写列于下表中:
表1氨基酸中英文对照及缩写
丙氨酸 | Alanine | A或Ala | 脂肪族类 |
精氨酸 | Arginine | R或Arg | 碱性氨基酸类 |
天冬酰胺 | Asparagine | N或Asn | 酰胺类 |
天冬氨酸 | Aspartic acid | D或Asp | 酸性氨基酸类 |
半胱氨酸 | Cysteine | C或Cys | 含硫类 |
谷氨酰胺 | Glutamine | Q或Gln | 酰胺类 |
谷氨酸 | Glutamic acid | E或Glu | 酸性氨基酸类 |
甘氨酸 | Glycine | G或Gly | 脂肪族类 |
组氨酸 | Histidine | H或His | 碱性氨基酸类 |
异亮氨酸 | Isoleucine | I或Ile | 脂肪族类 |
亮氨酸 | Leucine | L或Leu | 脂肪族类 |
赖氨酸 | Lysine | K或Lys | 碱性氨基酸类 |
蛋氨酸 | Methionine | M或Met | 含硫类 |
苯丙氨酸 | Phenylalanine | F或Phe | 芳香族类 |
脯氨酸 | Proline | P或Pro | 亚氨基酸 |
丝氨酸 | Serine | S或Ser | 羟基类 |
苏氨酸 | Threonine | T或Thr | 羟基类 |
色氨酸 | Tryptophan | W或Trp | 芳香族类 |
酪氨酸 | Tyrosine | Y或Tyr | 芳香族类 |
缬氨酸 | Valine | V或Val | 脂肪族类 |
为了获得酶性能更高的天冬氨酸氨裂合酶,本发明对SEQ ID NO:1的基因序列SEQID NO:2进行点突变。通过易错PCR技术获得一系列突变体,发现有些位点的氨基酸的替换或缺失会导致突变体的酶活力的显著变化。这些位点包括第142位的天冬酰胺和第188位的组氨酸。通过实验发现第142位的天冬酰胺替换为缬氨酸(N142V)、第188位的组氨酸替换为丙氨酸(H188A)所形成的突变体SEQ ID NO:3的酶活性相比初始型酶SEQ ID NO:1有明显提高。
在本发明中,术语“初始(型)”、“初始酶”、“初始型酶”表示相同的意义,都是指天冬氨酸氨裂合酶AspB的初始序列SEQ ID NO:1。为了与突变体相区别和表述方便起见,在本发明中可以将初始型天冬氨酸氨裂合酶称为“初始(型)天冬氨酸氨裂合酶”或者“初始(型)酶”。
本发明的天冬氨酸氨裂合酶突变体的氨基酸数量只有468个,且结构明确,因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。
这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
上述转化体宿主可以使任何适合表达天冬氨酸氨裂合酶突变体的微生物,包括细菌和真菌。优选微生物是枯草芽孢杆菌、毕赤酵母、酿酒酵母、或者大肠杆菌,优选大肠杆菌,更优选大肠杆菌BL21(DE3)。
当作为生物催化剂用于生产β-丙氨酸时,本发明的天冬氨酸氨裂合酶突变体可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶,包括纯化酶、粗酶、发酵液、载体固定的酶、细胞破碎物等;所述菌体的形式包括存活菌体细胞和死亡菌体细胞。
作为另一种可选的实施方式,可以采用表达上述天冬氨酸氨裂合酶突变体SEQ IDNO:3的微生物菌体细胞作为酶催化反应的生物催化剂。菌体的形式包括存活菌体和死亡菌体,当微生物比如枯草芽孢杆菌、毕赤酵母、酿酒酵母或者大肠杆菌不再进行发酵增殖、而是用于酶催化反应时,本身就是一种天然的固定化酶,而且不需要进行破碎处理、甚至提取纯化处理,就可以作为一种酶制剂用于催化反应。由于反应底物和反应产物都是小分子化合物,可以很方便地穿过菌体的生物屏障--细胞膜,因此不需要对菌体进行破碎处理,这在经济方面是有利的。
本发明的天冬氨酸氨裂合酶突变体SEQ ID NO:3的另一个优点是热稳定性高,该机理有待于进一步研究,尽管其本质上是一种蛋白质,而众所周知普通的蛋白酶在较高温度下容易失活变性。例如在该突变体催化丙烯酸或丙烯腈与氨水的反应时,反应温度最高可以达到60℃而不失活,这就为该酶催化反应带来了极大的反应速度优势。因为根据化学和生物学常识,采用的反应温度越高,反应速度就越快。在50-60℃的反应速度会显著地高于常温比如30-40℃下的反应速度。因此,在一种实施方式中,反应温度优选为30-60℃,更优选反应温度为35-58℃、更优选反应温度为40-55℃、更优选反应温度为42-55℃、更优选反应温度为45-52℃、更优选反应温度为45-50℃。
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例
材料和方法
实施例中的全基因合成、引物合成及测序皆由苏州金唯智生物科技有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等,主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,北京,2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
LB培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠,pH7.2。(LB固体培养基另加20g/L琼脂粉。)
TB培养基:24g/L酵母提取物、12g/L胰蛋白胨、16.43g/L K2HPO4.3H2O、2.31g/LKH2PO4、5g/L甘油,pH7.0-7.5。(TB固体培养基另加20g/L琼脂粉。)
底物丙烯酸和产物β-丙氨酸的HPLC测定条件:
Waters symmetry C18,5μm,4.6×250mm;检测波长262nm;流动相A:1.4g Na2HPO4和3.8g Na2B4O7.10H2O用1L水溶解,浓盐酸校正PH8.2;流动相B:甲醇:乙腈:水=45:45:10;操作温度40℃。
梯度程序:
时间 | 流动相A(%) | 流动相B(%) |
0 | 90 | 10 |
17 | 40 | 60 |
25 | 0 | 100 |
实施例1初始天冬氨酸氨裂合酶基因重组大肠杆菌的构建
1.1对于Bacillus sp.YM55-1来源的天冬氨酸氨裂合酶,根据文献(RuifengLi.et al,2018)上已经公布的氨基酸序列即SEQ ID NO:1,以此为基础进行密码子优化,全基因合成基因序列SEQ ID NO:2,并在基因两端设计限制性内切酶位点Nde I和XhoI,亚克隆到载体pET24a(Novagen)的相应位点,获得重组质粒pET24a-AspB1,构建的质粒图谱见图1。
1.2通过电转化将重组质粒pET24a-AspB1转化表达宿主大肠杆菌BL21(DE3)(Invitrogen公司),得到表达初始天冬氨酸氨裂合酶的重组大肠杆菌AspB1。
实施例2通过易错PCR法构建随机突变点库及筛选
2.1易错PCR法构建随机突变点库
以初始型酶的基因SEQ ID NO:2为模板,应用易错PCR技术构建随机突变体库。正向引物AspB1-F为5’-ATGAACACCGACGTTCGTATC-3’,反向引物AspB1-R为5’-TTATTTACGACCAGCGATACCCGG-3’
50μL易错PCR反应体系包括:10ng质粒模板pET24a-AspB1,50pmol一对引物AspB1-F和AspB1-R,1×Taq buffer,0.2mM dGTP,0.2mM dATP,1mM dCTP,1mM dTTP,7mM MgCl2,(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl2,2.5个单位的Taq酶(fermentas公司)。PCR反应条件为:95℃5min;94℃30s,55℃30s,72℃2min/kbp;30个循环;72℃10min。胶回收1.4kbp随机突变片段作为大引物(Axygen DNA凝胶回收试剂盒AP-GX-50),用KOD-plus DNA聚合酶做MegaPrimer PCR:94℃5min;98℃10s,60℃30s,68℃2min/kbp,25个循环;68℃10min。DpnI限制性内切酶(Thermo公司)消化质粒模板,电转化大肠杆菌E.coli BL21(DE3)(Invitrogen公司),得到超过104个克隆的随机突变库。
2.2突变体库的高通量筛选
选取突变体库中的转化子接种到500μL含有50μg/mL卡那霉素LB液体培养基的96孔深孔培养板中,培养过夜,然后取80μl过夜培养物,转接至800μl含有50μg/mL卡那霉素的LB液体培养基中,37℃培养3h后,加入终浓度0.5mM IPTG,降温至25℃,培养过夜。4000rpm离心15min,弃上清,加入100μL含无菌水重悬菌体用于活力测定。
2.3高通量酶活力测定
酶活力定义:在45℃下每分钟催化底物产生1微摩尔(μmol)β-丙氨酸所需要的酶量定义为1个单位(U)。
将上述步骤2.2中100μL菌悬液加入100μL底物反应液(10%丙烯酸,浓氨水校正pH至9.5,在45℃的条件下反应5小时,然后4℃,5000rpm离心10min。取离心上清,检测240nm下的吸光度。
在随机突变库,通过对约3000个突变体克隆筛选,结果显示获得一株克隆AspB-11-D4克隆菌株消耗底物的能力明显增强。
为表述方便起见,实施例中可以将AspB-11-D4克隆菌株产生的突变酶沿用其编号“AspB-11-D4”,本领域技术人员容易理解它们的区别和对应关系。
表2、突变菌株发酵液与初始型酶表达菌株发酵液的酶活力对比结果
菌种编号 | 突变位点 | SEQ ID NO: | 发酵液相对比活 |
AspB1 | - | 1 | 1.0 |
AspB-11-D4 | N142V,H188A | 3 | 53.6 |
实施例3突变菌株AspB-11-D4发酵及转化
3.1摇瓶发酵
从AspB-11-D4的LB培养平板上挑取单菌落,接种至5mL含50μg/mL硫酸卡那霉素的LB液体培养基中,37℃,250rpm培养过夜。取2mL过夜培养物接种至200mL TB培养基中,于37℃,250rpm培养2-3h,至OD600 0.6-0.8时,加入0.1mM IPTG,于28℃,200rpm培养过夜。然后于4℃,10000rpm,离心10min,收集菌体。
3.2初始型酶表达菌株发酵
按照步骤3.1的方法,对初始型酶表达菌株AspB1进行摇瓶发酵,收集菌体。
3.3菌体比活力测定
分别将上述步骤3.1中所得菌体AspB-11-D4、步骤3.2中所得菌体AspB1各自加入到500μL底物反应液(10%丙烯酸,浓氨水校正pH至9.5)中,在45℃的条件下反应30分钟,反应液加入500μL的2M HCl溶液混合均匀后,4℃,12000rpm离心5min,取离心上清,HPLC测定β-丙氨酸的含量,测定结果见表3。
表3、突变体SEQ ID NO:3的菌体酶活力测定结果
菌种编号 | 突变位点 | 氨基酸序列号 | 相对比活 |
AspB1 | —— | 1 | 1.0 |
AspB-11-D4 | N142V,H188A | 3 | 21.3 |
由表3可以看出,相比初始型酶SEQ ID NO:1,本发明的天冬氨酸氨裂合酶突变体SEQ ID NO:3的酶活力提高了20多倍。
实施例4天冬氨酸氨裂合酶突变体生产β-丙氨酸的应用
将12wt%丙烯酸溶液用浓氨水校正pH至9.5,然后加入终浓度5%的AspB-11-D4冻融菌体,于45℃下搅拌反应。反应过程中控制温度45±1℃,反应16个小时。通过HPLC检测反应样品,结果显示,在反应16个小时后,反应体系中β-丙氨酸的生成率超过95%。
综上所述,相比初始天冬氨酸氨裂合酶AspB1,本发明构建的天冬氨酸氨裂合酶突变体SEQ ID NO:3催化丙烯酸反应生成β-丙氨酸的酶活力有了明显提高,提高了20多倍,具有工业化开发和应用前景。
序列表
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Claims (10)
1.一种天冬氨酸氨裂合酶突变体,其氨基酸序列为SEQ ID NO:3。
2.编码如权利要求1所述天冬氨酸氨裂合酶突变体的基因。
3.如权利要求2所述的基因,其特征在于,序列为SEQ ID NO:4。
4.包含如权利要求3所述基因的质粒。
5.转化了如权利要求4所述质粒的微生物。
6.如权利要求5所述的微生物,其特征在于,是所述微生物选自枯草芽孢杆菌、毕赤酵母、酿酒酵母、大肠杆菌。
7.如权利要求6所述的微生物,其特征在于,是所述微生物是大肠杆菌BL21(DE3)。
8.如权利要求1所述天冬氨酸氨裂合酶突变体或者如权利要求6所述微生物在生产β-丙氨酸中的用途。
9.如权利要求8所述的用途,其特征在于,以丙烯酸或丙烯腈为底物、用权利要求1所述天冬氨酸氨裂合酶突变体或者如权利要求6所述微生物催化生产β-丙氨酸。
10.如权利要求9所述的用途,其特征在于,反应体系中底物的浓度为10-20%。
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CN114934037B (zh) * | 2021-08-27 | 2023-06-27 | 上海邦林生物科技有限公司 | 用于生产3-氨基丙腈的天冬氨酸酶突变体 |
CN113979879A (zh) * | 2021-09-26 | 2022-01-28 | 万华化学集团股份有限公司 | 一种高效制备β-氨基丙酸的方法 |
CN116836963A (zh) * | 2023-06-30 | 2023-10-03 | 秦皇岛华恒生物工程有限公司 | 一种天冬氨酸酶突变体及其应用 |
CN116836963B (zh) * | 2023-06-30 | 2024-05-14 | 秦皇岛华恒生物工程有限公司 | 一种天冬氨酸酶突变体及其应用 |
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