CN110951705B - Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell and preparation method and application thereof - Google Patents
Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell and preparation method and application thereof Download PDFInfo
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- CN110951705B CN110951705B CN201911327073.0A CN201911327073A CN110951705B CN 110951705 B CN110951705 B CN 110951705B CN 201911327073 A CN201911327073 A CN 201911327073A CN 110951705 B CN110951705 B CN 110951705B
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- Prior art keywords
- amine dehydrogenase
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- seq
- type amine
- dehydrogenase
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- 239000004474 valine Substances 0.000 claims abstract description 16
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims abstract description 16
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Abstract
本发明涉及一种胺脱氢酶突变体、酶制剂、重组载体、重组细胞及其制备方法和应用。该胺脱氢酶突变体，由野生型胺脱氢酶的第104位的谷氨酸突变为丙氨酸后得到；或者，由野生型胺脱氢酶的第137位的天冬酰胺突变为脯氨酸后得到；或者，由野生型胺脱氢酶的第174位的缬氨酸突变为半胱氨酸后得到；或者，由野生型胺脱氢酶的第197位的缬氨酸突变为天冬氨酸后得到。上述胺脱氢酶的催化效率较高。
The present invention relates to an amine dehydrogenase mutant, an enzyme preparation, a recombinant carrier, a recombinant cell, and a preparation method and application thereof. The amine dehydrogenase mutant is obtained by mutating the glutamic acid at the 104th position of the wild-type amine dehydrogenase to alanine; or, by mutating the asparagine at the 137th position of the wild-type amine dehydrogenase into Obtained after proline; or, obtained by mutating valine at position 174 of wild-type amine dehydrogenase to cysteine; or, by mutating valine at position 197 of wild-type amine dehydrogenase Obtained after aspartic acid. The catalytic efficiency of the above amine dehydrogenases is high.
Description
技术领域technical field
本发明涉及生物技术领域，特别是涉及一种胺脱氢酶突变体、酶制剂、重组载体、重组细胞及其制备方法和应用。The present invention relates to the field of biotechnology, in particular to an amine dehydrogenase mutant, an enzyme preparation, a recombinant vector, a recombinant cell and a preparation method and application thereof.
背景技术Background technique
手性胺在有机合成、香料、医药等方面有广泛的应用，是重要的中间体。手性胺的不同对映异构体的立体结构的差异，使得不同的手性对映体的活性不同，作用于生命体时表现出的生理活性和毒害作用也不同。因此，手性胺逐步成为用于评价药物安全性和药效的重要指标。Chiral amines are widely used in organic synthesis, fragrance, medicine, etc., and are important intermediates. The difference in the three-dimensional structure of different enantiomers of chiral amines makes different chiral enantiomers have different activities, and show different physiological activities and toxic effects when they act on living organisms. Therefore, chiral amines have gradually become an important indicator for evaluating drug safety and efficacy.
目前，制备手性胺的方法主要为化学催化剂和生物酶催化法，相比于化学催化剂，酶催化法具有更佳的催化效果，尤其是在识别手性胺的两种对映体方面，生物酶制剂催化手性药物选择性合成具有显著的技术优势。其中，胺脱氢酶(amine dehydrogenase，AmDH)能够催化前手性酮合成手性胺，是一种有效的、制备手性胺的生物催化剂。然而，现有的胺脱氢酶的催化效率较低，不能满足实际应用的需要。At present, the methods for preparing chiral amines are mainly chemical catalysts and biological enzymatic catalysis. Compared with chemical catalysts, enzymatic catalysis has better catalytic effect, especially in recognizing the two enantiomers of chiral amines. Enzyme preparations catalyze the selective synthesis of chiral drugs with significant technical advantages. Among them, amine dehydrogenase (AmDH) can catalyze the synthesis of chiral amines from prochiral ketones, and is an effective biocatalyst for the preparation of chiral amines. However, the existing amine dehydrogenases have low catalytic efficiency and cannot meet the needs of practical applications.
发明内容SUMMARY OF THE INVENTION
基于此，有必要提供一种催化效率较高的胺脱氢酶。Based on this, it is necessary to provide an amine dehydrogenase with higher catalytic efficiency.
此外，还提供一种胺脱氢酶突变体的应用、酶制剂、重组载体、重组细胞及其制备方法。In addition, an application of an amine dehydrogenase mutant, an enzyme preparation, a recombinant vector, a recombinant cell and a preparation method thereof are also provided.
一种胺脱氢酶突变体，由野生型胺脱氢酶的第104位的谷氨酸突变为丙氨酸后得到；An amine dehydrogenase mutant obtained by mutating the glutamic acid at position 104 of wild-type amine dehydrogenase to alanine;
或者，由野生型胺脱氢酶的第137位的天冬酰胺突变为脯氨酸后得到；Or, it is obtained by mutating the asparagine at position 137 of wild-type amine dehydrogenase to proline;
或者，由野生型胺脱氢酶的第174位的缬氨酸突变为半胱氨酸后得到；Or, it is obtained by mutating the valine at position 174 of wild-type amine dehydrogenase to cysteine;
或者，由野生型胺脱氢酶的第197位的缬氨酸突变为天冬氨酸后得到。Alternatively, it can be obtained by mutating valine at position 197 of wild-type amine dehydrogenase to aspartic acid.
本研究对胺脱氢酶进行了大量的研究，研究结果发现，将野生型胺脱氢酶的第104位的谷氨酸突变为丙氨酸，或者，将野生型胺脱氢酶的第137位的天冬酰胺突变为脯氨酸，或者，将野生型胺脱氢酶的第174位的缬氨酸突变为半胱氨酸，或者，将野生型胺脱氢酶的第197位的缬氨酸突变为天冬氨酸，得到的胺脱氢酶突变体具有较高的催化效率。经试验验证，上述胺脱氢酶突变体的Kcat值为468.34s-1～501.22s-1，说明上述胺脱氢酶突变体具有较高的催化速率，催化效率较高。In this study, a large number of studies on amine dehydrogenase were carried out, and the results found that the glutamate at position 104 of the wild-type amine dehydrogenase was mutated to alanine, or, the 137th position of the wild-type amine dehydrogenase was mutated. Asparagine at position 174 is mutated to proline, or valine at position 174 of wild-type amine dehydrogenase is mutated to cysteine, or, valine at position 197 of wild-type amine dehydrogenase is mutated The amino acid was mutated to aspartic acid, and the resulting amine dehydrogenase mutant had higher catalytic efficiency. Experiments verified that the K cat values of the amine dehydrogenase mutants were 468.34s -1 to 501.22s -1 , indicating that the amine dehydrogenase mutants had higher catalytic rates and higher catalytic efficiency.
其中一个实施例中，所述野生型胺脱氢酶的氨基酸序列如SEQ ID No.1所示。In one embodiment, the amino acid sequence of the wild-type amine dehydrogenase is shown in SEQ ID No.1.
其中一个实施例中，所述野生型胺脱氢酶的核苷酸序列如SEQ ID No.2所示。In one embodiment, the nucleotide sequence of the wild-type amine dehydrogenase is shown in SEQ ID No.2.
一种酶制剂，包括上述胺脱氢酶突变体。An enzyme preparation comprising the above-mentioned amine dehydrogenase mutant.
一种重组载体，包括上述胺脱氢酶突变体的编码序列。A recombinant vector comprising the coding sequence of the above-mentioned amine dehydrogenase mutant.
一种重组细胞，包括上述胺脱氢酶突变体的编码序列。A recombinant cell comprising the coding sequence of the above-mentioned amine dehydrogenase mutant.
上述重组工程菌细胞的制备方法，包括如下步骤：The preparation method of the above-mentioned recombinant engineering bacteria cell, comprises the following steps:
对所述野生型胺脱氢酶的编码序列进行易错PCR扩增，酶切后连接到空载体中，得到胺脱氢酶突变库质粒；Perform error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase, and connect it to an empty vector after enzymatic cleavage to obtain an amine dehydrogenase mutant library plasmid;
将所述胺脱氢酶突变库质粒分别转化到宿主细胞中，得到转化细胞；及The amine dehydrogenase mutant library plasmids are respectively transformed into host cells to obtain transformed cells; and
采用IVC-FACS方法对转化细胞进行高通量筛选，得到所述重组细胞。High-throughput screening of transformed cells by IVC-FACS method is used to obtain the recombinant cells.
其中一个实施例中，所述对所述野生型胺脱氢酶的编码序列进行易错PCR扩增的步骤中，采用序列如SEQ ID No.3～SEQ ID No.4所示的引物对进行易错PCR扩增。In one of the embodiments, in the step of performing error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase, primer pairs whose sequences are shown in SEQ ID No. 3 to SEQ ID No. 4 are used to perform Error-prone PCR amplification.
其中一个实施例中，所述对所述野生型胺脱氢酶的编码序列进行易错PCR扩增的步骤之前，还包括如下步骤：采用序列如SEQ ID No.5～SEQ ID No.6所示的引物对PCR扩增所述野生型胺脱氢酶的编码序列。In one embodiment, before the step of performing error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase, it further includes the following step: using sequences as shown in SEQ ID No.5 to SEQ ID No.6 The indicated primer pair PCR amplified the coding sequence of the wild-type amine dehydrogenase.
上述胺脱氢酶突变体、上述酶制剂、上述重组载体或者上述重组细胞在制备手性胺中的应用。Use of the above-mentioned amine dehydrogenase mutant, the above-mentioned enzyme preparation, the above-mentioned recombinant vector or the above-mentioned recombinant cell in the preparation of chiral amines.
附图说明Description of drawings
图1为野生型胺脱氢酶的蛋白晶体模型的结构示意图。Figure 1 is a schematic structural diagram of a protein crystal model of a wild-type amine dehydrogenase.
具体实施方式Detailed ways
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例及附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。未特别说明，序列表中的碱基序列均为从5’端到3’端的顺序。In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementation disclosed below. Unless otherwise specified, the nucleotide sequences in the sequence listing are in the order from the 5' end to the 3' end.
实施方式的胺脱氢酶突变体具有较高的催化效率，能够在辅酶的作用下催化前手性酮和游离氨不对称合成手性胺，以能够用于制备手性胺。具体地，该胺脱氢酶突变体，由野生型胺脱氢酶的第104位的谷氨酸突变为丙氨酸后得到；或者，由野生型胺脱氢酶的第137位的天冬酰胺突变为脯氨酸后得到；或者，由野生型胺脱氢酶的第174位的缬氨酸突变为半胱氨酸后得到；或者，由野生型胺脱氢酶的第197位的缬氨酸突变为天冬氨酸后得到。The amine dehydrogenase mutant of the embodiment has high catalytic efficiency, and can catalyze the asymmetric synthesis of chiral amines from prochiral ketones and free ammonia under the action of coenzymes, so that they can be used to prepare chiral amines. Specifically, the amine dehydrogenase mutant is obtained by mutating the glutamic acid at the 104th position of the wild-type amine dehydrogenase to alanine; or, it is obtained from the aspartate at the 137th position of the wild-type amine dehydrogenase It is obtained by mutating amide to proline; or, it is obtained by mutating valine at position 174 of wild-type amine dehydrogenase to cysteine; or, it is obtained from valine at position 197 of wild-type amine dehydrogenase obtained by mutating amino acid to aspartic acid.
本研究对胺脱氢酶进行了大量的研究，发现将野生型胺脱氢酶的第104位的谷氨酸突变为丙氨酸，或者，将野生型胺脱氢酶的第137位的天冬酰胺突变为脯氨酸，或者，将野生型胺脱氢酶的第174位的缬氨酸突变为半胱氨酸，或者，将野生型胺脱氢酶的第197位的缬氨酸突变为天冬氨酸，得到的胺脱氢酶突变体具有较高的催化效率。In this study, a large number of amine dehydrogenases were studied, and it was found that the glutamate at position 104 of the wild-type amine dehydrogenase was mutated to alanine, or, the day of the 137th position of the wild-type amine dehydrogenase was mutated. Paragine to proline, or valine at position 174 of wild-type amine dehydrogenase to cysteine, or valine at position 197 of wild-type amine dehydrogenase Aspartic acid, the resulting amine dehydrogenase mutant has higher catalytic efficiency.
其中，野生型胺脱氢酶的氨基酸序列如SEQ ID No.1所示。具体地，如SEQ ID No.1所示的序列为：MEKIRVIIWGLGAMGGGMARMILQKKGMEIVGAIASRPEKSGKDLGEVLDLGLKTGVTISCDPETVLKQPADIVLLATSSFTREVYPQLQRIIASGKNVITIAEEMAYPAYREPELAAKIDKMAKDHGVTVLGTGINPGFVLDTLIIALSGVCMDIKKITARRINDLSPFGTTVMRTQGVGTTVDEFRKGLEEGTIVGHIGFPESISLISEALGLEIDEIREMREPIVSNVYRETPYARVEPGMVAGCKHTGIGYRKGEPVIVLEHPQQIRPELEDVETGDYIEIEGTPNIKLSIKPEIPGGIGTIAIAVNMIPKVISANTGLVTMKDLPVPAALMGDIRKLAKDGVNNA。Wherein, the amino acid sequence of wild-type amine dehydrogenase is shown in SEQ ID No.1.具体地，如SEQ ID No.1所示的序列为：MEKIRVIIWGLGAMGGGMARMILQKKGMEIVGAIASRPEKSGKDLGEVLDLGLKTGVTISCDPETVLKQPADIVLLATSSFTREVYPQLQRIIASGKNVITIAEEMAYPAYREPELAAKIDKMAKDHGVTVLGTGINPGFVLDTLIIALSGVCMDIKKITARRINDLSPFGTTVMRTQGVGTTVDEFRKGLEEGTIVGHIGFPESISLISEALGLEIDEIREMREPIVSNVYRETPYARVEPGMVAGCKHTGIGYRKGEPVIVLEHPQQIRPELEDVETGDYIEIEGTPNIKLSIKPEIPGGIGTIAIAVNMIPKVISANTGLVTMKDLPVPAALMGDIRKLAKDGVNNA。
进一步地，野生型胺脱氢酶的核苷酸序列如SEQ ID No.2所示。具体地，如SEQ IDNo.2所示的序列为：ATGGAAAAAATCCGTGTTATCATCTGGGGTCTGGGTGCTATGGGTGGTGGTATGGCTCGTATGATCCTGCAGAAAAAAGGTATGGAAATCGTTGGTGCTATCGCTTCTCGTCCGGAAAAATCTGGTAAAGACCTGGGTGAAGTTCTGGACCTGGGTCTGAAAACCGGTGTTACCATCTCTTGCGACCCGGAAACCGTTCTGAAACAGCCGGCTGACATCGTTCTGCTGGCTACCTCTTCTTTCACCCGTGAAGTTTACCCGCAGCTGCAGCGTATCATCGCTTCTGGTAAAAACGTTATCACCATCGCTGAAGAAATGGCTTACCCGGCTTACCGTGAACCGGAACTGGCTGCTAAAATCGACAAAATGGCTAAAGACCACGGTGTTACCGTTCTGGGTACCGGTATCAACCCGGGTTTCGTTCTGGACACCCTGATCATCGCTCTGTCTGGTGTTTGCATGGACATCAAAAAAATCACCGCTCGTCGTATCAACGACCTGTCTCCGTTCGGTACCACCGTTATGCGTACCCAGGGTGTTGGTACCACCGTTGACGAATTCCGTAAAGGTCTGGAAGAAGGTACCATCGTTGGTCACATCGGTTTCCCGGAATCTATCTCTCTGATCTCTGAAGCTCTGGGTCTGGAAATCGACGAAATCCGTGAAATGCGTGAACCGATCGTTTCTAACGTTTACCGTGAAACCCCGTACGCTCGTGTTGAACCGGGTATGGTTGCTGGTTGCAAACACACCGGTATCGGTTACCGTAAAGGTGAACCGGTTATCGTTCTGGAACACCCGCAGCAGATCCGTCCGGAACTGGAAGACGTTGAAACCGGTGACTACATCGAAATCGAAGGTACCCCGAACATCAAACTGTCTATCAAACCGGAAATCCCGGGTGGTATCGGTACCATCGCTATCGCTGTTAACATGATCCCGAAAGTTATCTCTGCTAACACCGGTCTGGTTACCATGAAAGACCTGCCGGTTCCGGCTGCTCTGATGGGTGACATCCGTAAACTGGCTAAAGACGGTGTTAACAACGCT。Further, the nucleotide sequence of wild-type amine dehydrogenase is shown in SEQ ID No.2.具体地，如SEQ IDNo.2所示的序列为：ATGGAAAAAATCCGTGTTATCATCTGGGGTCTGGGTGCTATGGGTGGTGGTATGGCTCGTATGATCCTGCAGAAAAAAGGTATGGAAATCGTTGGTGCTATCGCTTCTCGTCCGGAAAAATCTGGTAAAGACCTGGGTGAAGTTCTGGACCTGGGTCTGAAAACCGGTGTTACCATCTCTTGCGACCCGGAAACCGTTCTGAAACAGCCGGCTGACATCGTTCTGCTGGCTACCTCTTCTTTCACCCGTGAAGTTTACCCGCAGCTGCAGCGTATCATCGCTTCTGGTAAAAACGTTATCACCATCGCTGAAGAAATGGCTTACCCGGCTTACCGTGAACCGGAACTGGCTGCTAAAATCGACAAAATGGCTAAAGACCACGGTGTTACCGTTCTGGGTACCGGTATCAACCCGGGTTTCGTTCTGGACACCCTGATCATCGCTCTGTCTGGTGTTTGCATGGACATCAAAAAAATCACCGCTCGTCGTATCAACGACCTGTCTCCGTTCGGTACCACCGTTATGCGTACCCAGGGTGTTGGTACCACCGTTGACGAATTCCGTAAAGGTCTGGAAGAAGGTACCATCGTTGGTCACATCGGTTTCCCGGAATCTATCTCTCTGATCTCTGAAGCTCTGGGTCTGGAAATCGACGAAATCCGTGAAATGCGTGAACCGATCGTTTCTAACGTTTACCGTGAAACCCCGTACGCTCGTGTTGAACCGGGTATGGTTGCTGGTTGCAAACACACCGGTATCGGTTACCGTAAAGGTGAACCGGTTATCGTTCTGGAACACCCGCAGCAGATCCGTCCGGAACTGGAAGACGTTGAAACCGGTGACTACATCGAAATCGAAGGTACCCCGAACATCAAACTGTCTATCAAACCGGAAATCCCGGGTGGTATCGGTACCATCGCTATCGCTGTTAACATGATCCCGAAAGTTATCTCTGCTAACACCGGTCTGGTTACCATG AAAGACCTGCCGGTTCCGGCTGCTCTGATGGGTGACATCCGTAAACTGGCTAAAGACGGTGTTAACAACGCT.
需要说明的是，由于同一氨基酸可由几种不同的密码子来决定，故同一氨基酸可对应不同的编码序列。因此本申请中的如SEQ ID No.1所示的氨基酸序列，除可由如SEQ IDNo.2所示的编码序列所编码，也可由如SEQ ID No.2所示编码序列进行1个或几个核苷酸取代而得到的密码子同义突变的编码序列所编码得到。本领域技术人员可以根据本申请公开的如SEQ ID No.1所示的氨基酸序列，根据现有分子生物学技术，采用cDNA克隆和定点突变的方法或其他适合的方法获得本申请的野生型氨胺脱氢酶的核苷酸序列，因此，编码上述野生型氨胺脱氢酶的核苷酸序列的编码序列并不仅限于SEQ ID NO.2所示的编码序列。如果编码得到的蛋白与本申请的野生型氨胺脱氢酶的核苷酸序列没有明显的功能差异，也包括在本发明的范围内。It should be noted that since the same amino acid can be determined by several different codons, the same amino acid can correspond to different coding sequences. Therefore, the amino acid sequence shown in SEQ ID No. 1 in the present application can not only be encoded by the coding sequence shown in SEQ ID No. 2, but also can be encoded by one or several coding sequences shown in SEQ ID No. 2. The coding sequence of the codon synonymous mutation obtained by nucleotide substitution is obtained. Those skilled in the art can obtain the wild-type amino acid of the present application according to the amino acid sequence disclosed in the present application as shown in SEQ ID No. 1, according to the existing molecular biology technology, using the method of cDNA cloning and site-directed mutagenesis or other suitable methods. The nucleotide sequence of amine dehydrogenase, therefore, the coding sequence of the nucleotide sequence encoding the wild-type aminoamine dehydrogenase is not limited to the coding sequence shown in SEQ ID NO. 2. If there is no obvious functional difference between the encoded protein and the nucleotide sequence of the wild-type aminoamine dehydrogenase of the present application, it is also included in the scope of the present invention.
此外，由于蛋白编码序列的多态性及变异，天然存在的蛋白质会出现基因突变，编码序列中碱基被缺失、替代或增加，或氨基酸的缺失、插入、取代或其它变异，从而导致蛋白质的氨基酸序列出现一个或多个氨基酸被缺失、替代或增加。因此，存在着一些生理和生物活性上基本等同于无变异蛋白质的蛋白。这些结构不同于相应的蛋白质，但与该蛋白质没有明显的功能差异的多肽或蛋白称为功能等同变异体。In addition, due to the polymorphism and variation of the protein coding sequence, the naturally occurring protein will have genetic mutation, the base in the coding sequence is deleted, substituted or added, or the amino acid is deleted, inserted, substituted or other variation, resulting in protein An amino acid sequence occurs where one or more amino acids are deleted, substituted or added. Thus, there are proteins whose physiological and biological activities are substantially equivalent to the unmutated protein. Polypeptides or proteins that are structurally different from the corresponding protein but have no significant functional difference from the protein are called functionally equivalent variants.
功能等同的变异体同样适用于通过缺失、插入和突变等人工手段改变一个或多个密码子，从而向一种蛋白质的氨基酸序列中导入这类变异而制成的多肽。尽管这样能获得更多不同形式的变异体，但所得的变异体作为功能等同变异体的前提是其生理活性基本等同于原始无变异蛋白质的活性。Functionally equivalent variants are also applicable to polypeptides made by artificially altering one or more codons by deletion, insertion, and mutation, thereby introducing such variations into the amino acid sequence of a protein. Although more variants of different forms can be obtained in this way, the premise of the obtained variant being a functionally equivalent variant is that its physiological activity is substantially equivalent to that of the original unmutated protein.
一般的，功能等同变异体的编码序列是同源的，因此，由至少一种改变(如蛋白质的编码序列中一个或多个碱基的缺失、插入或取代或者蛋白质的氨基酸序列中有一个或多个氨基酸缺失、插入或取代)所得的多肽或蛋白一般具有功能上等同于所述蛋白质的活性，因此，由上述编码序列编码的到的多肽或上述氨基酸序列组成的多肽，如果编码得到的蛋白本申请的野生型氨胺脱氢酶的基酸序列没有明显的功能差异，也包括在本申请的范围内。In general, the coding sequences of functionally equivalent variants are homologous and, therefore, are not affected by at least one alteration (such as a deletion, insertion or substitution of one or more bases in the coding sequence of the protein or one or more of the amino acid sequences of the protein). The polypeptide or protein obtained by multiple amino acid deletion, insertion or substitution) generally has the activity equivalent to the protein in function. Therefore, the polypeptide encoded by the above-mentioned coding sequence or the polypeptide composed of the above-mentioned amino acid sequence, if the encoded protein is obtained. The amino acid sequence of the wild-type aminoamine dehydrogenase of the present application has no obvious functional difference, which is also included in the scope of the present application.
在其中一个实施例中，胺脱氢酶突变体的作用底物包括前手性酮和游离氨。In one embodiment, the substrates for the mutant amine dehydrogenase include prochiral ketones and free ammonia.
其中，前手性酮为4酮基戊酸前手性酮。游离氨为氨气游离氨。辅酶为还原型辅酶Ⅰ(NADH)辅酶。Wherein, the prochiral ketone is a 4-ketovaleric acid prochiral ketone. Free ammonia is ammonia gas free ammonia. The coenzyme is reduced coenzyme I (NADH) coenzyme.
上述胺脱氢酶突变体具有较高的催化效率。经试验验证，上述胺脱氢酶突变体的Kcat值为468.34s-1～501.22s-1，说明上述胺脱氢酶突变体具有较高的催化速率，催化效率较高。The above amine dehydrogenase mutants have higher catalytic efficiency. Experiments verified that the K cat values of the amine dehydrogenase mutants were 468.34s -1 to 501.22s -1 , indicating that the amine dehydrogenase mutants had higher catalytic rates and higher catalytic efficiency.
研究发现，野生型胺脱氢酶对底物的亲和性较高，但是由于野生型胺脱氢酶主要在体内比较温和的环境中发挥作用，而工业应用过程要求酶在比较严苛的环境(如高温、极端酸碱度、有机溶剂、非天然底物、产物抑制等)中发挥作用，因此，野生型胺脱氢酶在应用中往往遇到催化效率低等问题。而上述胺脱氢酶突变体的KM值为17.12μM～23.21μM，与野生型胺脱氢酶的KM值相当，说明上述胺脱氢酶突变体对底物仍保持较高的亲和力。The study found that the wild-type amine dehydrogenase has a higher affinity for the substrate, but because the wild-type amine dehydrogenase mainly plays a role in a relatively mild environment in the body, and the industrial application process requires the enzyme to operate in a relatively harsh environment (such as high temperature, extreme pH, organic solvents, non-natural substrates, product inhibition, etc.), therefore, wild-type amine dehydrogenases often encounter problems such as low catalytic efficiency in applications. The K M values of the above amine dehydrogenase mutants were 17.12 μM -23.21 μM, which were comparable to those of the wild-type amine dehydrogenase, indicating that the above amine dehydrogenase mutants still maintained a high affinity for the substrate.
一些研究通过对野生型胺脱氢酶进行改造能够一定程度地提高酶活，但是随着酶活的提高往往伴随着稳定性的降低。上述胺脱氢酶突变体既具有较高的活性，又具有与野生型胺脱氢酶相当的热稳定性。经试验验证，上述胺脱氢酶突变体能够在42℃下保持30min而活性保持不变。Some studies can improve the enzyme activity to a certain extent by modifying the wild-type amine dehydrogenase, but the increase of the enzyme activity is often accompanied by a decrease in stability. The above amine dehydrogenase mutants have both higher activity and thermostability comparable to wild-type amine dehydrogenases. It was verified by experiments that the above-mentioned amine dehydrogenase mutants could be kept at 42°C for 30min and the activity remained unchanged.
综上，上述胺脱氢酶突变体具有较高的催化效率，且对底物具有较高的亲和力，能够在辅酶的作用下催化前手性酮和游离氨不对称合成手性胺，并且具有较优的热稳定性，能够用于制备手性胺，以能够应用于食品、医药等领域。In summary, the above amine dehydrogenase mutants have high catalytic efficiency and high affinity for substrates, and can catalyze the asymmetric synthesis of chiral amines from prochiral ketones and free ammonia under the action of coenzymes, and have With better thermal stability, it can be used to prepare chiral amines, which can be used in food, medicine and other fields.
一实施方式的酶制剂，包括上述实施方式的胺脱氢酶突变体。An enzyme preparation of one embodiment includes the amine dehydrogenase mutant of the above-described embodiments.
其中，酶制剂为可溶性蛋白或者固定化酶。Wherein, the enzyme preparation is a soluble protein or an immobilized enzyme.
在其中一个实施例中，酶制剂还包括辅酶。其中，辅酶为还原型辅酶Ⅰ(NADH)辅酶。In one embodiment, the enzyme preparation further includes a coenzyme. Among them, the coenzyme is reduced coenzyme I (NADH) coenzyme.
上述酶制剂包括上述实施方式的胺脱氢酶突变体，具有较高的催化效率，且对底物具有较高的亲和力，能够在辅酶的作用下催化前手性酮和游离氨不对称合成手性胺，以能够用于制备手性胺，以能够应用于食品、医药等领域。The above-mentioned enzyme preparation includes the amine dehydrogenase mutant of the above-mentioned embodiment, which has high catalytic efficiency and high affinity for the substrate, and can catalyze the asymmetric synthesis of prochiral ketone and free ammonia under the action of coenzyme. Chiral amines can be used to prepare chiral amines, so that they can be used in food, medicine and other fields.
一实施方式的重组载体，包括上述实施方式的脱氢酶突变体的编码序列。The recombinant vector of one embodiment includes the coding sequence of the dehydrogenase mutant of the above-mentioned embodiment.
在其中一个实施例中，重组载体为重组表达载体或重组克隆载体。In one embodiment, the recombinant vector is a recombinant expression vector or a recombinant cloning vector.
在其中一个实施例中，重组载体含有纯化标签。通过设置纯化标签，有利于活性肽的分离纯化。进一步地，纯化标签为His标签、GST标签或SUMO标签。需要说明的是，纯化标签不限于上述指出纯化标签，其他常见的纯化标签也可以作用重组载体的纯化标签。In one embodiment, the recombinant vector contains a purification tag. By setting the purification tag, it is beneficial to the separation and purification of active peptides. Further, the purification tag is His tag, GST tag or SUMO tag. It should be noted that the purification tag is not limited to the purification tag indicated above, and other common purification tags can also be used as the purification tag of the recombinant vector.
在其中一个实施例中，重组载体包括基因工程载体。编码上述脱氢酶突变体的核苷酸插入基因工程载体中。进一步地，基因工程载体为pET-32a载体、pET28a载体、pGEX-6P-1载体、pPIC-9K载体或pPIC-Zα载体。需要说明的是，基因工程载体不限于上述指出基因工程载体，其他常见的基因工程载体也可以作用重组载体的基因工程载体。In one embodiment, the recombinant vector comprises a genetically engineered vector. Nucleotides encoding the above-mentioned dehydrogenase mutants are inserted into genetic engineering vectors. Further, the genetic engineering vector is pET-32a vector, pET28a vector, pGEX-6P-1 vector, pPIC-9K vector or pPIC-Zα vector. It should be noted that the genetic engineering vector is not limited to the genetic engineering vector indicated above, and other common genetic engineering vectors can also be used as the genetic engineering vector of the recombinant vector.
上述重组载体能够较好地保存编码上述脱氢酶突变体的核苷酸，有利于脱氢酶突变体的表达，能够应用于制备手性胺。The above-mentioned recombinant vector can better preserve the nucleotides encoding the above-mentioned dehydrogenase mutants, is beneficial to the expression of the dehydrogenase mutants, and can be applied to the preparation of chiral amines.
一种重组细胞，包括上述实施方式的胺脱氢酶突变体的编码序列。A recombinant cell comprising the coding sequence of the amine dehydrogenase mutant of the above embodiment.
在其中一个实施例中，重组细胞为克隆编码上述胺脱氢酶突变体的编码序列的细胞。In one embodiment, the recombinant cell is a cell that clones the coding sequence encoding the above-mentioned amine dehydrogenase mutant.
在其中一个实施例中，重组细胞为表达编码上述胺脱氢酶突变体的编码序列的细胞。In one embodiment, the recombinant cell is a cell expressing a coding sequence encoding the above-mentioned amine dehydrogenase mutant.
在其中一个实施例中，重组细胞包括受体细胞。编码上述胺脱氢酶突变体的编码序列或上述重组载体位于受体细胞内。In one embodiment, the recombinant cells comprise recipient cells. The coding sequence encoding the above-mentioned amine dehydrogenase mutant or the above-mentioned recombinant vector is located in the recipient cell.
在其中一个实施例中，受体细胞为大肠杆菌、酿酒酵母、毕赤酵母、动物细胞或植物细胞。进一步地，受体细胞为大肠杆菌Escherichia coli 10G、大肠杆菌DH5α、大肠杆菌Top10、大肠杆菌Orgami(DE3)、毕赤酵母GS115或毕赤酵母SMD1168。In one embodiment, the recipient cell is Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, animal cells or plant cells. Further, the recipient cell is Escherichia coli 10G, Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli Orgami (DE3), Pichia pastoris GS115 or Pichia pastoris SMD1168.
需要说明的是，受体细胞不限于上述指出受体细胞，其他常见的受体细胞也可以作用重组细胞的受体细胞。It should be noted that the recipient cells are not limited to the above-mentioned recipient cells, and other common recipient cells can also act on the recipient cells of the recombinant cells.
上述重组细胞能够克隆或表达上述胺脱氢酶突变体，使得能够大规模的制备该胺脱氢酶突变体，并且通过重组细胞定向表达该胺脱氢酶突变体，以能够获得纯度较高的胺脱氢酶突变体，进而有利于胺脱氢酶突变体的应用，因此，上述重组细胞能够用于制备手性胺。The above-mentioned recombinant cells can clone or express the above-mentioned amine dehydrogenase mutants, so that the amine dehydrogenase mutants can be prepared on a large scale, and the amine dehydrogenase mutants can be directionally expressed by the recombinant cells, so as to be able to obtain higher-purity amine dehydrogenase mutants. The amine dehydrogenase mutants are further beneficial to the application of the amine dehydrogenase mutants. Therefore, the above-mentioned recombinant cells can be used to prepare chiral amines.
上述实施方式的重组细胞的制备方法，包括如下步骤S110～S130：The preparation method of the recombinant cell according to the above embodiment includes the following steps S110-S130:
S110：对上述实施方式的野生型胺脱氢酶的编码序列进行易错PCR扩增，酶切后连接到空载体中，得到胺脱氢酶突变库质粒。S110: Perform error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase according to the above embodiment, and then ligate into an empty vector after digestion to obtain an amine dehydrogenase mutant library plasmid.
其中，对上述实施方式的野生型胺脱氢酶的编码序列进行易错PCR扩增的步骤中，采用序列如SEQ ID No.3～SEQ ID No.4所示的引物对进行易错PCR扩增。具体地，如SEQ IDNo.3所示的序列为：5’-ACTGCTCATATGGAAAAAATCCGTGTTATCATC-3’；如SEQ ID No.4所示的序列为：5’-TCAGCTCTCGAGTTAAGCGTTGTTAACACCG-3’。Wherein, in the step of performing error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase according to the above embodiment, the error-prone PCR amplification is performed by using the primer pairs shown in SEQ ID No.3 to SEQ ID No.4. increase. Specifically, the sequence shown in SEQ ID No.3 is: 5'-ACTGCTCATATGGAAAAAATCCGTGTTATCATC-3'; the sequence shown in SEQ ID No.4 is: 5'-TCAGCTCTCGAGTTAAGCGTTGTTAACACCG-3'.
其中，易错PCR的反应体系为：0.05U/μL的DreamTaqTM、250μM的dATP、250μM的dGTP、1050μM的dCTP、1050μM的dTTP、0.4μM的序列如SEQ ID No.3所示的引物、0.4μM的序列如SEQID No.4所示的引物、0.2ng/μL的空载体、0.2mM～0.8mM的氯化锰。Wherein, the reaction system of error-prone PCR is: 0.05U/μL DreamTaq TM , 250 μM dATP, 250 μM dGTP, 1050 μM dCTP, 1050 μM dTTP, 0.4 μM primer whose sequence is shown in SEQ ID No.3, 0.4 The sequences of μM are the primers shown in SEQ ID No. 4, 0.2 ng/μL of empty vector, and 0.2 mM to 0.8 mM of manganese chloride.
其中，易错PCR的反应条件为：95℃，3min，1个循环；95℃、15s，55℃、30s，72℃、1min，30个循环；72℃，5min，1个循环。Among them, the reaction conditions of error-prone PCR were: 95°C, 3min, 1 cycle; 95°C, 15s, 55°C, 30s, 72°C, 1min, 30 cycles; 72°C, 5min, 1 cycle.
其中，空载体为pET-28a质粒。需要说明的是，空载体不限于为pET-28a质粒，也可以为本领域中其他常用的空载体。Wherein, the empty vector is pET-28a plasmid. It should be noted that the empty vector is not limited to the pET-28a plasmid, and can also be other commonly used empty vectors in the art.
在其中一个实施例中，对上述实施方式的野生型胺脱氢酶的编码序列进行易错PCR扩增的步骤之前，还包括如下步骤：采用序列如SEQ ID No.5～SEQ IDNo.6所示的引物对PCR扩增野生型胺脱氢酶的编码序列。此种设置有利于富集野生型胺脱氢酶的编码序列，以有利于通过易错PCR扩增构建胺脱氢酶突变库质粒。In one embodiment, before the step of performing error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase according to the above embodiment, the following step is further included: using sequences as shown in SEQ ID No.5 to SEQ ID No.6 The primer pair shown was used to PCR amplify the coding sequence of wild-type amine dehydrogenase. This setting is beneficial for enriching the coding sequence of wild-type amine dehydrogenase to facilitate the construction of amine dehydrogenase mutant library plasmids by error-prone PCR amplification.
具体地，如SEQ ID No.5所示的序列为：5’-ACTGCTCATATGGAAAAAATCCGTGTTATCATC-3’，其中，带下划线碱基为限制性内切酶NdeI识别位点；如SEQ ID No.6所示的序列为：5’-TCAGCTCTCGAGTTAAGCGTTGTTAACACCG-3’，其中，带下划线碱基为限制性内切酶XhoI识别位点。Specifically, the sequence shown in SEQ ID No.5 is: 5'-ACTGC TCATATG GAAAAAATCCGTGTTATCATC-3', wherein the underlined base is the restriction endonuclease NdeI recognition site; as shown in SEQ ID No.6 The sequence is: 5'-TCAGCT CTCGAG TTAAGCGTTGTTAACACCG-3', wherein the underlined base is the recognition site of restriction endonuclease XhoI.
S120：将胺脱氢酶突变库质粒分别转化到宿主细胞中，得到转化细胞。S120: Transform the amine dehydrogenase mutant library plasmids into host cells respectively to obtain transformed cells.
其中，宿主细胞为大肠杆菌。大肠杆菌表达系统具有遗传背景清楚、目的基因表达水平高、培养周期短、抗污染能力强等优点，是分子生物学研究和生物技术产业化发展进程中的重要工具。进一步地，宿主细胞为大肠杆菌Escherichia coli 10G。需要说明的是，宿主细胞不限于为上述指出的细胞，也可以为本领域中其他常用的宿主细胞。Wherein, the host cell is Escherichia coli. Escherichia coli expression system has the advantages of clear genetic background, high target gene expression level, short culture period, strong anti-pollution ability, etc. It is an important tool in the process of molecular biology research and biotechnology industrialization. Further, the host cell is Escherichia coli 10G. It should be noted that the host cell is not limited to the above-mentioned cells, and can also be other commonly used host cells in the art.
其中，转化的方式为电转化。需要说明的是，转化的方式不限于为电转化，也可以为本领域中其他常用的转化方式。Among them, the conversion method is electrical conversion. It should be noted that the transformation method is not limited to electro-transformation, and can also be other transformation methods commonly used in the field.
S130：采用IVC-FACS方法对转化细胞进行高通量筛选，得到所述重组细胞。S130: Perform high-throughput screening on transformed cells by IVC-FACS method to obtain the recombinant cells.
定向进化是对酶进行分子改造，从而改善其各方面性质的有力工具。其原理是模拟自然界进化原理，在实验室中人工构建目的蛋白的基因突变库，再利用筛选手段挑选出其中符合预期性质的突变体。然而由于突变库中的阳性率较低，因此，常规的基于微孔板或底物平板的筛选方法由于通量低，费时费力，在定向进化筛选中往往不能得到良好效果。本研究通过建立了基于体外区室化-荧光激活细胞分选(IVC-FACS)技术的超高通量筛选方法，能够对酶突变体进行高效快速的筛选，其筛选通量可达每小时一百万个，能够用于对胺脱氢酶随机突变库的高通量筛选，进而获得活性提高的阳性突变体。Directed evolution is a powerful tool for molecular modification of enzymes to improve various aspects of their properties. The principle is to simulate the evolutionary principle of nature, artificially construct a gene mutation library of the target protein in the laboratory, and then use screening methods to select mutants that meet the expected properties. However, due to the low positive rate in the mutation library, conventional screening methods based on microplate or substrate plates often fail to achieve good results in directed evolution screening due to low throughput, time-consuming and labor-intensive. In this study, an ultra-high-throughput screening method based on in vitro compartmentalization-fluorescence-activated cell sorting (IVC-FACS) technology was established, which can efficiently and rapidly screen enzyme mutants, with a screening throughput of up to one hour per hour. Millions, which can be used for high-throughput screening of amine dehydrogenase random mutation library, and then obtain positive mutants with improved activity.
上述重组细胞的制备方法制备的重组细胞能够克隆或表达上述胺脱氢酶突变体，使得能够大规模的制备该胺脱氢酶突变体，并且通过重组细胞定向表达该胺脱氢酶突变体，以能够获得纯度较高的胺脱氢酶突变体，并且具有较优的热稳定性，有利于胺脱氢酶突变体的应用，能够用于制备手性胺。The recombinant cells prepared by the above-mentioned method for preparing recombinant cells can clone or express the above-mentioned amine dehydrogenase mutants, so that the amine dehydrogenase mutants can be prepared on a large scale, and the amine dehydrogenase mutants can be directionally expressed by the recombinant cells, In order to obtain the amine dehydrogenase mutant with higher purity and better thermal stability, it is beneficial to the application of the amine dehydrogenase mutant and can be used to prepare chiral amines.
以下为具体实施例部分。The following is the specific embodiment part.
实施例中采用试剂和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，通常按照常规条件，例如文献、书本中所述的条件或者试剂盒生产厂家推荐的方法实现。实施例中所使用的试剂均为市售。Unless otherwise specified, the reagents and instruments used in the examples are routinely selected in the art. The experimental methods for which specific conditions are not indicated in the examples are usually realized according to conventional conditions, such as conditions described in literatures and books, or methods recommended by the manufacturer of the kit. The reagents used in the examples are all commercially available.
未特别说明，以下实施例中，T4DNA连接酶购于NEB公司；pET28a质粒购于淼灵质粒公司；XhoI酶购于NEB公司；NdeI酶购于NEB公司。Unless otherwise specified, in the following examples, T4 DNA ligase was purchased from NEB Company; pET28a plasmid was purchased from Miaoling Plasmid Company; XhoI enzyme was purchased from NEB Company; NdeI enzyme was purchased from NEB Company.
实施例1Example 1
野生胺脱氢酶的编码序列的克隆Cloning of the coding sequence of wild amine dehydrogenase
野生型胺脱氢酶(GenBank：CP002131.1)的编码序列以大肠杆菌为宿主进行密码子优化，并由苏州金唯智公司合成，采用序列如SEQ ID No.5～SEQ ID No.6所示的引物对PCR扩增野生型胺脱氢酶的编码序列，PCR扩增使用东洋纺(上海)生物科技有限公司的KOD高保真聚合酶，扩增条件为：95℃，2min；然后56℃、20sec，72℃、90sec，共30个循环；最后72℃，10min。其中，野生型胺脱氢酶的氨基酸序列如SEQ ID No.1所示；野生型胺脱氢酶的核苷酸序列如SEQ ID No.2所示。The coding sequence of wild-type amine dehydrogenase (GenBank: CP002131.1) was codon-optimized with Escherichia coli as the host and synthesized by Suzhou Jinweizhi Company. The primer pair was used to PCR amplify the coding sequence of wild-type amine dehydrogenase. The PCR amplification used KOD high-fidelity polymerase from Toyobo (Shanghai) Biotechnology Co., Ltd., and the amplification conditions were: 95°C, 2min; then 56°C, 20sec, 72℃, 90sec, a total of 30 cycles; the last 72℃, 10min. The amino acid sequence of the wild-type amine dehydrogenase is shown in SEQ ID No.1; the nucleotide sequence of the wild-type amine dehydrogenase is shown in SEQ ID No.2.
反应结束后，用质量百分含量为1.5％的琼脂糖凝胶电泳检测PCR产物，得到1.0kb的条带，长度符合预期结果。按照核酸回收试剂盒(购于Takara公司)的使用说明书回收、纯化该目的片段。使用限制性核酸内切酶XhoI和NdeI对回收片段和pET28a质粒进行双酶切，然后用T4DNA连接酶进行连接，将连接产物转化大肠杆菌BL21(DE3)感受态细胞中，涂布于含有卡那霉素(50ug/mL)的LB培养平板上培养12h。挑取阳性菌株，并采用质粒提取试剂盒(购于Takara公司)提取阳性克隆质粒，进行测序。经测序检测结果显示，克隆的胺脱氢酶ANDD-TDO基因序列正确，并且正确接入pET28a质粒，命名重组质粒为pET28a-ANDD-TDO，保存于甘油中。After the reaction, the PCR product was detected by agarose gel electrophoresis with a mass percentage of 1.5%, and a 1.0 kb band was obtained, and the length was in line with the expected result. The target fragment was recovered and purified according to the instruction manual of the nucleic acid recovery kit (purchased from Takara). The recovered fragment and pET28a plasmid were double digested with restriction endonucleases XhoI and NdeI, and then ligated with T4 DNA ligase, and the ligated product was transformed into E. coli BL21 (DE3) competent cells, and coated with kana The cells were cultured for 12 h on LB culture plates with 50 ug/mL. The positive strains were picked, and the positive cloned plasmids were extracted with a plasmid extraction kit (purchased from Takara Company) and sequenced. The results of sequencing showed that the cloned amine dehydrogenase ANDD-TDO gene sequence was correct, and it was correctly inserted into the pET28a plasmid. The recombinant plasmid was named pET28a-ANDD-TDO and stored in glycerol.
实施例2Example 2
胺脱氢酶的表达、纯化及活力测定Expression, Purification and Activity Determination of Amine Dehydrogenase
将甘油管中的含有pET28a-ANDD-TDO重组质粒的工程菌按体积比1％接种到含有100μg/mL卡那霉素的4mL LB培养基试管中，37℃、220rpm培养12h。将培养后的菌液全部转接至含有50μg/mL卡那霉素的1L LB培养基摇瓶中，37℃、220rpm培养约2.5h，使OD600达到0.9左右，加入0.1mM IPTG诱导剂，25℃、200rpm诱导培养16h。将诱导结束后收获的大肠杆菌悬液超声破碎，再经过一步Ni-NTA亲和层析处理后，得到纯度为95％以上的野生型胺脱氢酶。测定野生型胺脱氢酶的活性，结果为：野生型胺脱氢酶的KM值为19.22μM，野生型胺脱氢酶的Kcat值为128.34s-1。其中，参考文献Catal.Sci.Technol.2016:10.1039.C6CY01625A测定野生型胺脱氢酶的活力。The engineered bacteria containing the pET28a-ANDD-TDO recombinant plasmid in the glycerol tube were inoculated into a 4 mL LB medium test tube containing 100 μg/mL kanamycin at a volume ratio of 1%, and cultured at 37° C. and 220 rpm for 12 h. Transfer all the cultured bacterial liquid to a 1L LB medium shake flask containing 50 μg/mL kanamycin, cultivate at 37 °C and 220 rpm for about 2.5 h to make the OD 600 reach about 0.9, add 0.1 mM IPTG inducer, Induction and culture at 25°C and 200rpm for 16h. The Escherichia coli suspension harvested after induction is broken by ultrasonic, and after one-step Ni-NTA affinity chromatography treatment, a wild-type amine dehydrogenase with a purity of more than 95% is obtained. The activity of the wild-type amine dehydrogenase was measured, and the results were as follows: the K M value of the wild-type amine dehydrogenase was 19.22 μM, and the K cat value of the wild-type amine dehydrogenase was 128.34s -1 . Among them, the reference Catal.Sci.Technol.2016:10.1039.C6CY01625A was used to determine the activity of wild-type amine dehydrogenase.
实施例3Example 3
胺脱氢酶的大容量随机突变库的构建Construction of a large-capacity random mutation library of amine dehydrogenases
通过易错PCR(ep-PCR)的方法构建胺脱氢酶的突变库，其中突变率的高低通过调节PCR体系中的锰离子的浓度来实现。具体地，采用对实施例1的野生型胺脱氢酶的编码序列进行易错PCR扩增。易错PCR的反应体系为：0.05U/μL的DreamTaqTM、250μM的dATP、250μM的dGTP、1050μM的dCTP、1050μM的dTTP、0.4μM的序列如SEQ ID No.3所示的引物、0.4μM的序列如SEQ ID No.4所示的引物、0.2ng/μL的空载体、0.2mM～0.8mM的氯化锰；每管总体积为25μL，空载体为pET-28a质粒。易错PCR的反应条件为：95℃，3min，1个循环；95℃、15s，55℃、30s，72℃、1min，30个循环；72℃，5min，1个循环。The mutation library of amine dehydrogenase was constructed by the method of error-prone PCR (ep-PCR), wherein the level of mutation rate was realized by adjusting the concentration of manganese ions in the PCR system. Specifically, the coding sequence of the wild-type amine dehydrogenase of Example 1 was used for error-prone PCR amplification. The reaction system of error-prone PCR is: 0.05U/μL DreamTaq TM , 250 μM dATP, 250 μM dGTP, 1050 μM dCTP, 1050 μM dTTP, 0.4 μM primer with the sequence shown in SEQ ID No.3, 0.4 μM The sequence is shown in the primer of SEQ ID No. 4, the empty vector of 0.2ng/μL, and the manganese chloride of 0.2mM to 0.8mM; the total volume of each tube is 25 μL, and the empty vector is pET-28a plasmid. The reaction conditions of error-prone PCR were: 95℃, 3min, 1 cycle; 95℃, 15s, 55℃, 30s, 72℃, 1min, 30 cycles; 72℃, 5min, 1 cycle.
将易错PCR获得的扩增产物经琼脂糖电泳，切胶纯化回收后，用NdeI和BamHIII进行双酶切，然后用T4连接酶克隆到pET28a质粒上，再将纯化后的连接体系电转化到Escherichia coli 10G感受态细胞中。转化细胞经复苏后接种到50mL的LB培养基(含有100μg/mL的卡那霉素)中，在37℃培养过夜。取培养液质粒回收试剂盒(购于Takara公司)提取质粒，得到突变库质粒。同时，取培养液涂布含有20μg/mL的卡那霉素的LB琼脂糖平板上在37℃培养12h，计算突变库质粒的库容量约为200万个。取若干克隆使用焦磷酸测序的方法测定突变率，发现锰离子浓度为0.6mM时，获得突变率大约为平均每个基因2个变氨基酸残基。突变库的质粒转化宿主BL21(DE3)，接种到含100μg/mL Kan的4mL LB培养基试管中，在37℃、220rpm条件下培养12h；取菌液4mL转接至含50μg/mL Kan的1L LB培养基摇瓶中，在37℃、220rpm条件下培养2.5h，使OD600达到0.9左右，加入0.1mM IPTG诱导剂，在25℃、200rpm条件下诱导培养16h表达后进行筛选，得到表达胺脱氢酶突变库的BL21(DE3)细胞。The amplification product obtained by error-prone PCR was subjected to agarose electrophoresis, purified and recovered by cutting gel, and then double-enzyme digested with NdeI and BamHIII, and then cloned into pET28a plasmid with T4 ligase, and the purified ligation system was electro-transformed into pET28a plasmid. Escherichia coli 10G competent cells. The transformed cells were thawed and then inoculated into 50 mL of LB medium (containing 100 μg/mL of kanamycin), and cultured at 37°C overnight. Take the culture medium plasmid recovery kit (purchased from Takara Company) to extract plasmids to obtain mutant library plasmids. At the same time, the culture solution was coated on LB agarose plates containing 20 μg/mL kanamycin and cultured at 37°C for 12 h, and the library capacity of the mutant library plasmid was calculated to be about 2 million. Several clones were taken to measure the mutation rate by pyrosequencing, and it was found that when the manganese ion concentration was 0.6mM, the mutation rate was about 2 amino acid residues per gene on average. The plasmid of the mutant library was transformed into the host BL21(DE3), inoculated into a 4 mL LB medium test tube containing 100 μg/mL Kan, and cultured at 37 °C and 220 rpm for 12 h; 4 mL of the bacterial solution was taken and transferred to 1 L containing 50 μg/mL Kan In a shake flask of LB medium, culture at 37°C and 220rpm for 2.5h to make the OD600 reach about 0.9, add 0.1mM IPTG inducer, induce and culture for 16h at 25°C and 200rpm, and then screen for expression. BL21(DE3) cells of the hydrogenase mutant library.
实施例4Example 4
胺脱氢酶随机突变库的IVC-FACS高通量筛选IVC-FACS High Throughput Screening of Amine Dehydrogenase Random Mutation Libraries
将表达胺脱氢酶突变库的BL21(DE3)细胞包裹入w/o/w(水包油包水型)二级微液滴进行酶反应。BL21(DE3) cells expressing amine dehydrogenase mutant library were encapsulated into w/o/w (water-in-oil-in-water) secondary microdroplets for enzymatic reaction.
具体地，采用微型膜挤出仪(Avanti Polar Lipids，AL，USA)，配套的两只注射器(Gastight 1001syringe，1mL，Hamilton,NV,USA)以及孔径为8微米的Track-Etch聚碳酸酯膜(Millipore，USA)来制备微液滴。Specifically, a micro-membrane extruder (Avanti Polar Lipids, AL, USA), two matching syringes (Gastight 1001syringe, 1 mL, Hamilton, NV, USA) and a Track-Etch polycarbonate membrane with a pore size of 8 μm ( Millipore, USA) to prepare microdroplets.
首先，将膜固定在膜挤出仪中，然后用注射器吸取0.5mL的油相(油相成分为：含有体积百分含量为2.9％的ABIL EM90乳化剂的轻石蜡油)润洗膜两次。乳化时，将100μL的内水相(即细胞浓度为OD 600:0.5的Escherichia coli BL21(DE3)-CodonPlus细胞悬液)与400μL的油相(油相成分为：含有体积百分含量为2.9％的ABIL EM90乳化剂的轻石蜡油)吸取到同一支注射器中，混合体系经膜挤出仪推注到另一个注射器中，然后再推回到第一个注射器中，这一过程称为一次乳化，生成w/o(油包水型)一级微液。生成的w/o一级微液滴通过显微镜(50i，Nikon，Japan，40×object)实时观察，通过优化乳化次数，使微液滴的直径分布在3～5μm。First, fix the membrane in the membrane extruder, and then use a syringe to draw 0.5 mL of the oil phase (the oil phase composition is: light paraffin oil containing 2.9% ABIL EM90 emulsifier by volume) to rinse the membrane twice . During emulsification, 100 μL of the inner water phase (ie, Escherichia coli BL21(DE3)-CodonPlus cell suspension with a cell concentration of OD 600:0.5) and 400 μL of the oil phase (the oil phase composition is: 2.9% by volume) ABIL EM90 emulsifier (light paraffin oil) is drawn into the same syringe, the mixed system is injected through the membrane extruder into another syringe, and then pushed back into the first syringe, a process called primary emulsification , generate w/o (water-in-oil type) first-level microfluid. The generated w/o first-order microdroplets were observed in real time by a microscope (50i, Nikon, Japan, 40×object). By optimizing the number of emulsifications, the diameters of the microdroplets were distributed between 3 and 5 μm.
制备的w/o一级微液滴通过8μm孔径的膜分散到次水相(即含有体积百分含量为1％TritonX-102的1×PBS，pH7.4)中，生成w/o/w二级微液滴。具体步骤为：将一片新的膜置于膜挤出仪中，用0.5mL次水相润洗两遍。将200μL的w/o一级微液滴和400μL的次水相分别吸取到两支注射器中；具体地，首先，将w/o一级微液滴通过膜挤出仪注入第二支注射器的次水相中，再通过膜挤出仪将混合体系推回原注射器中，完成一次乳化。生成的w/o/w二级微液滴的形态分布通过显微镜进行实时观察，通过优化乳化次数，使得最终w/o/w二级微液滴的直径在10μm左右且大小相对均一。在向0.2mL的含有w/o/w二级微液滴的外水相(外水相为水，w/o/w二级微液滴的含量为80％)中加入0.02mL的荧光底物(即含有10mM荧光素二丁酸酯的二甲基亚砜，购于Sigma公司)，在37℃、1000rpm的金属浴上震荡孵育30min，以进行酶反应，荧光底物荧光素二丁酸酯的终浓度为0.5mM。The prepared w/o primary microdroplets were dispersed into the secondary aqueous phase (i.e., 1×PBS containing 1% TritonX-102 by volume, pH 7.4) through a membrane with 8 μm pore size, resulting in w/o/w Secondary microdroplets. The specific steps are as follows: a new film is placed in a film extruder, and rinsed twice with 0.5 mL of the aqueous phase. 200 μL of w/o primary microdroplets and 400 μL of secondary aqueous phase were respectively drawn into two syringes; specifically, first, the w/o primary microdroplets were injected into the second syringe through a membrane extruder. In the secondary aqueous phase, the mixed system is pushed back into the original syringe by the membrane extruder to complete the primary emulsification. The morphology distribution of the generated w/o/w secondary microdroplets was observed by microscope in real time. By optimizing the number of emulsifications, the final w/o/w secondary microdroplets had a diameter of about 10 μm and were relatively uniform in size. Add 0.02 mL of fluorescent bottom to 0.2 mL of the outer aqueous phase containing w/o/w secondary microdroplets (the outer aqueous phase is water, and the content of w/o/w secondary microdroplets is 80%). (i.e., dimethyl sulfoxide containing 10 mM fluorescein dibutyrate, purchased from Sigma), incubate at 37 °C, 1000 rpm on a metal bath for 30 min with shaking to carry out the enzymatic reaction, the fluorescent substrate fluorescein dibutyric acid The final concentration of ester was 0.5 mM.
用分选型流式细胞仪(BD FACSAriaTM II)检测反应体系的荧光信号，喷嘴的内径为100μm，样品的检测速度10000个细胞/sec具有最高荧光强度的液滴(约占含细胞液滴的0.1％)被分选入空的2mL eppendorf管中，以此为模板将阳性基因进行PCR扩增，PCR反应体系为：0.05U/μL的DreamTaqTM(购于Takara)、250μM的dATP、250μM的dGTP、250μM的dCTP、250μM的dTTP、0.4μM的序列如SEQ ID No.3所示的引物、0.4μM的序列如SEQ ID No.4所示的引物。其中，1000个细胞的分选后体积约为5μL。PCR反应条件为：95℃，3min，1个循环；95℃、15s，55℃、30s，72℃、1min，30个循环；72℃，5min，1个循环。The fluorescence signal of the reaction system was detected by a sorting flow cytometer (BD FACSAria TM II), the inner diameter of the nozzle was 100 μm, and the detection speed of the sample was 10,000 cells/sec. 0.1%) was sorted into an empty 2mL eppendorf tube, and the positive gene was used as a template for PCR amplification. The PCR reaction system was: 0.05U/μL DreamTaq TM (purchased from Takara), 250μM dATP, 250μM dGTP, 250 μM dCTP, 250 μM dTTP, 0.4 μM primer whose sequence is shown in SEQ ID No.3, and 0.4 μM primer whose sequence is shown in SEQ ID No.4. Among them, the post-sort volume of 1000 cells is about 5 μL. PCR reaction conditions were: 95°C, 3 min, 1 cycle; 95°C, 15s, 55°C, 30s, 72°C, 1 min, 30 cycles; 72°C, 5min, 1 cycle.
将PCR产物重新克隆到pET-28a(+)质粒中，并平板培养后，将获得单克隆挑入96孔板中37℃、400rpm进行培养，每个孔含有200μL LB培养基；当细胞OD600达到0.6～0.8时加入1.0mM的IPTG，25℃诱导20h。诱导结束后，3000rpm离心30min收集细胞，弃上清。将细胞通过冻融裂解，向裂解后得到的裂解液中加入200μL的PBS混匀，3000rpm离心30min，取上清，得粗酶液。取10μL粗酶液与10μL的4-硝基苯基丁酸酯的乙腈溶液(4-硝基苯基丁酸酯的浓度为10mM，购于Sigma公司)及180μL的PBS在新的96孔板中37℃反应5min，反应结束后采用分光光度计在波长为405nm下检测不同克隆的酶活性。选择活性高于野生型胺脱氢酶的胺脱氢酶突变体并进行测序，并将阳性突变体经大量培养表达后用镍柱亲和层析进行纯化，得到E104A、N137P、V174C和V197D四种胺脱氢酶突变体。其中，“E104A”表示由野生型胺脱氢酶的第104位的谷氨酸突变为丙氨酸后得到的胺脱氢酶突变体；“N137P”表示由野生型胺脱氢酶的第137位的天冬酰胺突变为脯氨酸后得到的胺脱氢酶突变体；“V174C”表示由野生型胺脱氢酶的第174位的缬氨酸突变为半胱氨酸后得到的胺脱氢酶突变体；“V197D”表示由野生型胺脱氢酶的第197位的缬氨酸突变为天冬氨酸后得到的胺脱氢酶突变体。The PCR product was re-cloned into the pET-28a(+) plasmid, and after plate culture, the obtained single clone was picked into a 96-well plate for cultivation at 37°C and 400 rpm, and each well contained 200 μL of LB medium; when the cell OD600 reached 1.0mM IPTG was added at 0.6-0.8, and induced at 25°C for 20h. After induction, the cells were collected by centrifugation at 3000 rpm for 30 min, and the supernatant was discarded. The cells were lysed by freezing and thawing, 200 μL of PBS was added to the lysed solution obtained after lysis, and the mixture was mixed, centrifuged at 3000 rpm for 30 min, and the supernatant was taken to obtain a crude enzyme solution. Take 10 μL of crude enzyme solution and 10 μL of acetonitrile solution of 4-nitrophenyl butyrate (the concentration of 4-nitrophenyl butyrate is 10 mM, purchased from Sigma) and 180 μL of PBS in a new 96-well plate. The reaction was carried out at 37 °C for 5 min. After the reaction, the enzyme activity of different clones was detected by spectrophotometer at a wavelength of 405 nm. Amine dehydrogenase mutants with higher activity than wild-type amine dehydrogenases were selected and sequenced, and the positive mutants were expressed in large quantities and purified by nickel column affinity chromatography to obtain E104A, N137P, V174C and V197D four Amine dehydrogenase mutants. Among them, "E104A" represents an amine dehydrogenase mutant obtained by mutating the 104th glutamic acid of the wild-type amine dehydrogenase to alanine; "N137P" represents the 137th amino acid dehydrogenase of the wild-type amine dehydrogenase The amine dehydrogenase mutant obtained by mutating the asparagine at position 174 to proline; "V174C" represents the amine dehydrogenase obtained by mutating the valine at position 174 of the wild-type amine dehydrogenase to cysteine Hydrogenase mutant; "V197D" represents an amine dehydrogenase mutant obtained by mutating valine at position 197 of wild-type amine dehydrogenase to aspartic acid.
通过线上软件Swiss-modle构建野生型胺脱氢酶的蛋白晶体模型，详见图1。图1中，方框所框处的结构即为上述四种胺脱氢酶突变体在野生型胺脱氢酶上对应的突变位点。The protein crystal model of wild-type amine dehydrogenase was constructed by the online software Swiss-modle, as shown in Figure 1. In Fig. 1, the structure in the box is the corresponding mutation site of the above four amine dehydrogenase mutants on the wild-type amine dehydrogenase.
测定上述四种胺脱氢酶突变体和野生型胺脱氢酶对前手性酮的动力学参数，参考文献Catal.Sci.Technol.2016:10.1039.C6CY01625A测定酶的动力学参数。测定结果详见表1。其中，前手性酮为XX前手性酮；表1中，“WT”表示野生型胺脱氢酶，“E104A”表示由野生型胺脱氢酶的第104位的谷氨酸突变为丙氨酸后得到的胺脱氢酶突变体；“N137P”表示由野生型胺脱氢酶的第137位的天冬酰胺突变为脯氨酸后得到的胺脱氢酶突变体；“V174C”表示由野生型胺脱氢酶的第174位的缬氨酸突变为半胱氨酸后得到的胺脱氢酶突变体；“V197D”表示由野生型胺脱氢酶的第197位的缬氨酸突变为天冬氨酸后得到的胺脱氢酶突变体。The kinetic parameters of the above four amine dehydrogenase mutants and wild-type amine dehydrogenases for prochiral ketones were determined, and the kinetic parameters of the enzymes were determined by referring to Catal. Sci. Technol. 2016:10.1039.C6CY01625A. The measurement results are shown in Table 1. Among them, the prochiral ketone is the XX prochiral ketone; in Table 1, "WT" means wild-type amine dehydrogenase, and "E104A" means that the glutamic acid at position 104 of the wild-type amine dehydrogenase is mutated to C Amine dehydrogenase mutant obtained after amino acid; "N137P" indicates an amine dehydrogenase mutant obtained by mutating asparagine at position 137 of wild-type amine dehydrogenase to proline; "V174C" indicates Amine dehydrogenase mutant obtained by mutating valine at position 174 of wild-type amine dehydrogenase to cysteine; "V197D" represents a valine at position 197 of wild-type amine dehydrogenase Amine dehydrogenase mutants obtained after mutation to aspartate.
表1野生型胺脱氢酶和胺脱氢酶突变体的酶学性质Table 1 Enzymatic properties of wild-type amine dehydrogenase and amine dehydrogenase mutants
经筛选，得到E104A、N137P、V174C和V197D四种胺脱氢酶突变体，其KM与野生型胺脱氢酶的KM值相当，四种胺脱氢酶突变体的Kcat值明显高于野生型胺脱氢酶的Kcat值，说明上述实施方式的胺脱氢酶突变体具有较高的催化效率，且对底物具有较高的亲和力，能够在辅酶的作用下催化前手性酮和游离氨不对称合成手性胺，以能够用于制备手性胺，以能够应用于食品、医药等领域。After screening, four amine dehydrogenase mutants, E104A, N137P, V174C and V197D , were obtained, and their KM values were comparable to those of wild-type amine dehydrogenases, and the K cat values of the four amine dehydrogenase mutants were significantly higher. Based on the K cat value of the wild-type amine dehydrogenase, it shows that the amine dehydrogenase mutant of the above embodiment has higher catalytic efficiency and higher affinity for the substrate, and can catalyze prochirality under the action of coenzyme. Asymmetric synthesis of chiral amines from ketones and free ammonia can be used to prepare chiral amines, which can be used in food, medicine and other fields.
综上，上述胺脱氢酶突变体具有较高的催化效率，且对底物具有较高的亲和力，能够在辅酶的作用下催化前手性酮和游离氨不对称合成手性胺，并且具有较优的热稳定性，能够用于制备手性胺，以能够应用于食品、医药等领域。In summary, the above amine dehydrogenase mutants have high catalytic efficiency and high affinity for substrates, and can catalyze the asymmetric synthesis of chiral amines from prochiral ketones and free ammonia under the action of coenzymes, and have With better thermal stability, it can be used to prepare chiral amines, which can be used in food, medicine and other fields.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.
序列表sequence listing
<110> 中国科学院苏州生物医学工程技术研究所<110> Suzhou Institute of Biomedical Engineering Technology, Chinese Academy of Sciences
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006015885A1 (en) * | 2004-08-13 | 2006-02-16 | University College Dublin, National University Of Ireland, Dublin | Amino acid dehydrogenase-derived biocatalysts |
| CN107002050A (en) * | 2014-11-25 | 2017-08-01 | 科德克希思公司 | Engineered imine reductases and methods for reductive amination of ketone and amine compounds |
| CN109022381A (en) * | 2018-08-17 | 2018-12-18 | 凯莱英生命科学技术(天津)有限公司 | Amino acid dehydrogenase mutant and its application |
| CN110577941A (en) * | 2019-08-30 | 2019-12-17 | 厦门大学 | A kind of amine dehydrogenase and its application |
Family Cites Families (1)
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006015885A1 (en) * | 2004-08-13 | 2006-02-16 | University College Dublin, National University Of Ireland, Dublin | Amino acid dehydrogenase-derived biocatalysts |
| CN107002050A (en) * | 2014-11-25 | 2017-08-01 | 科德克希思公司 | Engineered imine reductases and methods for reductive amination of ketone and amine compounds |
| CN109022381A (en) * | 2018-08-17 | 2018-12-18 | 凯莱英生命科学技术(天津)有限公司 | Amino acid dehydrogenase mutant and its application |
| CN110577941A (en) * | 2019-08-30 | 2019-12-17 | 厦门大学 | A kind of amine dehydrogenase and its application |
Non-Patent Citations (5)
| Title |
|---|
| "Development of an Amine Dehydrogenase for Synthesis of Chiral Amines";Michael J. Abrahamson et al.;《Angewandte Chemie International Edition》;20120306;第51卷;第3969-3972页 * |
| "Generation of amine dehydrogenases with increased catalytic performance and substrate scope from ε-deaminating L-Lysine dehydrogenase";Vasilis Tseliou et al.;《Nature Communications》;20190816;第10卷;第1-11页 * |
| "Reshaping the Active Pocket of Amine Dehydrogenases for Asymmetric Synthesis of Bulky Aliphatic Amines";Fei-Fei Chen et al.;《ACS Catalysis》;20180215;第8卷;第2622-2628页 * |
| 基于荧光激活细胞分选技术的超高通量酶活性筛选方法及其应用;马富强 等;《生物化学与生物物理进展》;20121231;第39卷(第4期);第299-306页 * |
| 工业蛋白质构效关系的计算生物学解析;陈琦 等;《生物工程学报》;20191025;第35卷(第10期);第1829-1842页 * |
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