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Article(id=1199782978957181130, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1199782966441378761, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2024-0489, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1716220800000, receivedDateStr=2024-05-21, revisedDate=1722268800000, revisedDateStr=2024-07-30, acceptedDate=null, acceptedDateStr=null, onlineDate=1763980153073, onlineDateStr=2025-11-24, pubDate=1733932800000, pubDateStr=2024-12-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763980153073, onlineIssueDateStr=2025-11-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763980153073, creator=13701087609, updateTime=1763980153073, updator=13701087609, issue=Issue{id=1199782966441378761, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='12', pageStart='3179', pageEnd='3412', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763980150088, creator=13701087609, updateTime=1764224975369, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200809838151324146, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1199782966441378761, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200809838151324147, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1199782966441378761, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=3402, endPage=3408, ext={EN=ArticleExt(id=1199782980261609709, articleId=1199782978957181130, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Directed evolution to enhance the catalytic activity of human arginase 1, columnId=null, journalTitle=Acta Pharmaceutica Sinica, columnName=null, runingTitle=null, highlight=null, articleAbstract=

Arginase 1 deficiency (ARG1-D) is a rare genetic metabolic disorder that leads to progressive spastic paralysis, cognitive impairment, and seizures. Recombinant human arginase 1 (rhArg1) is a potential therapeutic agent for this condition, but its clinical application is limited by low activity and short half-life. In this study, we employed directed evolution to address these issues. A random mutation library of rhArg1 was constructed using error-prone PCR, and high-throughput screening was used to identify mutants with enhanced activity. Site-saturation mutagenesis was also performed to investigate the effects of residues R21 and V182 on enzyme activity. Our findings revealed that under reaction conditions devoid of Mn2+, the kcat values of the mutants V182D, V182S, V182H, and R21N increased by 2.0, 1.9, 1.7, and 1.3 times respectively, compared to rhArg1. The kcat/Km values of mutants V182D, V182S, R21D, and R21N were 2.1, 1.7, 1.4, and 1.4 times higher than those of rhArg1, respectively. Additionally, mutants R21D and V182L showed enhanced substrate affinity. Through directed evolution and site-saturation mutagenesis, we successfully obtained rhArg1 mutants with improved activity, thereby enhancing its potential for clinical application.

, correspAuthors=Zhao-yong YANG, Zhi-fei ZHANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Cui-yue FENG, Chen-yu WANG, Meng-jia TANG, Shuai FAN, Zhao-yong YANG, Zhi-fei ZHANG), CN=ArticleExt(id=1199782982107103627, articleId=1199782978957181130, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=定向进化提高人精氨酸酶1的催化活性, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

精氨酸酶1缺乏症(arginase 1 deficiency, ARG1-D) 是一种罕见的遗传性代谢疾病, 导致患者进行性痉挛性瘫痪、认知障碍和癫痫发作。来源于人源的重组人精氨酸酶1 (recombinant human arginase 1, rhArg1) 是其潜在的治疗药物, 但还存在活性低、半衰期短等缺陷限制了其临床应用。本研究采用定向进化的方法, 通过易错PCR构建rhArg1的随机突变文库, 经高通量筛选获得活性提高的突变体, 联合点饱和突变探讨了R21和V182位点对活性的影响。研究发现, 在反应体系中不含Mn2+的条件下, 突变体V182D、V182S、V182H和R21N的kcat值相比于rhArg1提高2.0、1.9、1.7和1.3倍, 突变体V182D、V182S、R21D和R21N的kcat/Km分别是rhArg1的2.1、1.7、1.4和1.4倍。突变体R21D和V182L对底物的亲和力有所增强。本研究通过定向进化和点饱和突变获得了rhArg1活性提高的突变体, 从而提升了其在医疗领域的应用前景。

, correspAuthors=杨兆勇, 张志斐, authorNote=null, correspAuthorsNote=
*杨兆勇, E-mail: ;
张志斐, E-mail:
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Reaction conditions with 0.2 mmol·L<sup>-1</sup> Mn<sup>2+</sup> (A), and without Mn<sup>2+</sup> (B) , figureFileSmall=DroP7YZi7LimfYudsX2VPg==, figureFileBig=wSLWHT7/kb+tCtKrVx5/gA==, tableContent=null), ArticleFig(id=1200378745065435519, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199782978957181130, language=EN, label=null, caption=null, figureFileSmall=coIXJF6EoLCiW0yMOa892g==, figureFileBig=K4zmdrtEnXbG+74JAsqOMg==, tableContent=null), ArticleFig(id=1200378745237401993, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199782978957181130, language=CN, label=Figure 4, caption= Effects of pH (A) and temperature (B) on rhArg1 and its mutants. <i>n</i> = 3, <span class="mag-xml-inline-formula"><tex-math id="M3">$ \stackrel{-}{x} $</tex-math></span> ± <i>s</i> , figureFileSmall=coIXJF6EoLCiW0yMOa892g==, figureFileBig=K4zmdrtEnXbG+74JAsqOMg==, tableContent=null), ArticleFig(id=1200378745417757070, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199782978957181130, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Primer name Primer sequence (5'-3') Primer name Primer sequence (5'-3')
rhArg1-F GAAGGAGATATACATATGAGCGCAAAAAGCCGTACCA V182A-F GGCCTGCGCGATGCGGATCCGGGTGAA
rhArg1-R GTGGTGGTGGTGCTCGAGTTTCGGCGGGTTCAGATAAT V182A-R CGCATCGCGCAGGCCGATATAAACAAT
R21A-F AAAGGCCAGCCGGCTGGTGGTGTTG V182C-F GGCCTGCGCGATTGCGATCCGGGTGAA
R21A-R AGCCGGCTGGCCTTTGCTAAACGGT V182C-R GCAATCGCGCAGGCCGATATAAACAAT
R21C-F AAAGGCCAGCCGTGTGGTGGTGTTG V182D-F GGCCTGCGCGATGACGATCCGGGTGAA
R21C-R ACACGGCTGGCCTTTGCTAAACGGT V182D-R GTCATCGCGCAGGCCGATATAAACAAT
R21D-F AAAGGCCAGCCGGATGGTGGTGTTG V182E-F GGCCTGCGCGATGAGGATCCGGGTGAA
R21D-R ATCCGGCTGGCCTTTGCTAAACGGT V182E-R CTCATCGCGCAGGCCGATATAAACAAT
R21E-F AAAGGCCAGCCGGAAGGTGGTGTTG V182F-F GGCCTGCGCGATTTCGATCCGGGTGAA
R21E-R TTCCGGCTGGCCTTTGCTAAACGGT V182F-R GAAATCGCGCAGGCCGATATAAACAAT
R21F-F AAAGGCCAGCCGTTTGGTGGTGTTG V182G-F GGCCTGCGCGATGGGGATCCGGGTGAA
R21F-R AAACGGCTGGCCTTTGCTAAACGGT V182G-R CCCATCGCGCAGGCCGATATAAACAAT
R21G-F AAAGGCCAGCCGGGTGGTGGTGTTG V182H-F GGCCTGCGCGATCACGATCCGGGTGAA
R21G-R ACCCGGCTGGCCTTTGCTAAACGGT V182H-R GTGATCGCGCAGGCCGATATAAACAAT
R21H-F AAAGGCCAGCCGCATGGTGGTGTTG V182I-F GGCCTGCGCGATATCGATCCGGGTGAA
R21H-R ATGCGGCTGGCCTTTGCTAAACGGT V182I-R GATATCGCGCAGGCCGATATAAACAAT
R21I-F AAAGGCCAGCCGATTGGTGGTGTTG V182K-F GGCCTGCGCGATAAGGATCCGGGTGAA
R21I-R AATCGGCTGGCCTTTGCTAAACGGT V182K-R CTTATCGCGCAGGCCGATATAAACAAT
R21K-F AAAGGCCAGCCGAAAGGTGGTGTTG V182L-F GGCCTGCGCGATCTGGATCCGGGTGAA
R21K-R TTTCGGCTGGCCTTTGCTAAACGGT V182L-R CAGATCGCGCAGGCCGATATAAACAAT
R21L-F AAAGGCCAGCCGCTTGGTGGTGTTG V182M-F GGCCTGCGCGATATGGATCCGGGTGAA
R21L-R AAGCGGCTGGCCTTTGCTAAACGGT V182M-R CATATCGCGCAGGCCGATATAAACAAT
R21M-F AAAGGCCAGCCGATGGGTGGTGTTG V182N-F GGCCTGCGCGATAACGATCCGGGTGAA
R21M-R CATCGGCTGGCCTTTGCTAAACGGT V182N-R GTTATCGCGCAGGCCGATATAAACAAT
R21N-F AAAGGCCAGCCGAATGGTGGTGTTG V182P-F GGCCTGCGCGATCCGGATCCGGGTGAA
R21N-R ATTCGGCTGGCCTTTGCTAAACGGT V182P-R CGGATCGCGCAGGCCGATATAAACAAT
R21P-F AAAGGCCAGCCGCCTGGTGGTGTTG V182Q-F GGCCTGCGCGATCAGGATCCGGGTGAA
R21P-R AGGCGGCTGGCCTTTGCTAAACGGT V182Q-R CTGATCGCGCAGGCCGATATAAACAAT
R21Q-F AAAGGCCAGCCGCAAGGTGGTGTTG V182R-F GGCCTGCGCGATCGGGATCCGGGTGAA
R21Q-R TTGCGGCTGGCCTTTGCTAAACGGT V182R-R CCGATCGCGCAGGCCGATATAAACAAT
R21S-F AAAGGCCAGCCGAGTGGTGGTGTTG V182S-F GGCCTGCGCGATTCGGATCCGGGTGAA
R21S-R ACTCGGCTGGCCTTTGCTAAACGGT V182S-R CGAATCGCGCAGGCCGATATAAACAAT
R21T-F AAAGGCCAGCCGACTGGTGGTGTTG V182T-F GGCCTGCGCGATACGGATCCGGGTGAA
R21T-R AGTCGGCTGGCCTTTGCTAAACGGT V182T-R CGTATCGCGCAGGCCGATATAAACAAT
R21V-F AAAGGCCAGCCGGTTGGTGGTGTTG V182W-F GGCCTGCGCGATTGGGATCCGGGTGAA
R21V-R AACCGGCTGGCCTTTGCTAAACGGT V182W-R CCAATCGCGCAGGCCGATATAAACAAT
R21W-F AAAGGCCAGCCGTGGGGTGGTGTTG V182Y-F GGCCTGCGCGATTACGATCCGGGTGAA
R21W-R CCACGGCTGGCCTTTGCTAAACGGT V182Y-R GTAATCGCGCAGGCCGATATAAACAAT
R21Y-F AAAGGCCAGCCGTATGGTGGTGTTG
R21Y-R ATACGGCTGGCCTTTGCTAAACGGT
), ArticleFig(id=1200378745593917847, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199782978957181130, language=CN, label=Table 1, caption=

Primers used in this study. Underline means mutation sites

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer name Primer sequence (5'-3') Primer name Primer sequence (5'-3')
rhArg1-F GAAGGAGATATACATATGAGCGCAAAAAGCCGTACCA V182A-F GGCCTGCGCGATGCGGATCCGGGTGAA
rhArg1-R GTGGTGGTGGTGCTCGAGTTTCGGCGGGTTCAGATAAT V182A-R CGCATCGCGCAGGCCGATATAAACAAT
R21A-F AAAGGCCAGCCGGCTGGTGGTGTTG V182C-F GGCCTGCGCGATTGCGATCCGGGTGAA
R21A-R AGCCGGCTGGCCTTTGCTAAACGGT V182C-R GCAATCGCGCAGGCCGATATAAACAAT
R21C-F AAAGGCCAGCCGTGTGGTGGTGTTG V182D-F GGCCTGCGCGATGACGATCCGGGTGAA
R21C-R ACACGGCTGGCCTTTGCTAAACGGT V182D-R GTCATCGCGCAGGCCGATATAAACAAT
R21D-F AAAGGCCAGCCGGATGGTGGTGTTG V182E-F GGCCTGCGCGATGAGGATCCGGGTGAA
R21D-R ATCCGGCTGGCCTTTGCTAAACGGT V182E-R CTCATCGCGCAGGCCGATATAAACAAT
R21E-F AAAGGCCAGCCGGAAGGTGGTGTTG V182F-F GGCCTGCGCGATTTCGATCCGGGTGAA
R21E-R TTCCGGCTGGCCTTTGCTAAACGGT V182F-R GAAATCGCGCAGGCCGATATAAACAAT
R21F-F AAAGGCCAGCCGTTTGGTGGTGTTG V182G-F GGCCTGCGCGATGGGGATCCGGGTGAA
R21F-R AAACGGCTGGCCTTTGCTAAACGGT V182G-R CCCATCGCGCAGGCCGATATAAACAAT
R21G-F AAAGGCCAGCCGGGTGGTGGTGTTG V182H-F GGCCTGCGCGATCACGATCCGGGTGAA
R21G-R ACCCGGCTGGCCTTTGCTAAACGGT V182H-R GTGATCGCGCAGGCCGATATAAACAAT
R21H-F AAAGGCCAGCCGCATGGTGGTGTTG V182I-F GGCCTGCGCGATATCGATCCGGGTGAA
R21H-R ATGCGGCTGGCCTTTGCTAAACGGT V182I-R GATATCGCGCAGGCCGATATAAACAAT
R21I-F AAAGGCCAGCCGATTGGTGGTGTTG V182K-F GGCCTGCGCGATAAGGATCCGGGTGAA
R21I-R AATCGGCTGGCCTTTGCTAAACGGT V182K-R CTTATCGCGCAGGCCGATATAAACAAT
R21K-F AAAGGCCAGCCGAAAGGTGGTGTTG V182L-F GGCCTGCGCGATCTGGATCCGGGTGAA
R21K-R TTTCGGCTGGCCTTTGCTAAACGGT V182L-R CAGATCGCGCAGGCCGATATAAACAAT
R21L-F AAAGGCCAGCCGCTTGGTGGTGTTG V182M-F GGCCTGCGCGATATGGATCCGGGTGAA
R21L-R AAGCGGCTGGCCTTTGCTAAACGGT V182M-R CATATCGCGCAGGCCGATATAAACAAT
R21M-F AAAGGCCAGCCGATGGGTGGTGTTG V182N-F GGCCTGCGCGATAACGATCCGGGTGAA
R21M-R CATCGGCTGGCCTTTGCTAAACGGT V182N-R GTTATCGCGCAGGCCGATATAAACAAT
R21N-F AAAGGCCAGCCGAATGGTGGTGTTG V182P-F GGCCTGCGCGATCCGGATCCGGGTGAA
R21N-R ATTCGGCTGGCCTTTGCTAAACGGT V182P-R CGGATCGCGCAGGCCGATATAAACAAT
R21P-F AAAGGCCAGCCGCCTGGTGGTGTTG V182Q-F GGCCTGCGCGATCAGGATCCGGGTGAA
R21P-R AGGCGGCTGGCCTTTGCTAAACGGT V182Q-R CTGATCGCGCAGGCCGATATAAACAAT
R21Q-F AAAGGCCAGCCGCAAGGTGGTGTTG V182R-F GGCCTGCGCGATCGGGATCCGGGTGAA
R21Q-R TTGCGGCTGGCCTTTGCTAAACGGT V182R-R CCGATCGCGCAGGCCGATATAAACAAT
R21S-F AAAGGCCAGCCGAGTGGTGGTGTTG V182S-F GGCCTGCGCGATTCGGATCCGGGTGAA
R21S-R ACTCGGCTGGCCTTTGCTAAACGGT V182S-R CGAATCGCGCAGGCCGATATAAACAAT
R21T-F AAAGGCCAGCCGACTGGTGGTGTTG V182T-F GGCCTGCGCGATACGGATCCGGGTGAA
R21T-R AGTCGGCTGGCCTTTGCTAAACGGT V182T-R CGTATCGCGCAGGCCGATATAAACAAT
R21V-F AAAGGCCAGCCGGTTGGTGGTGTTG V182W-F GGCCTGCGCGATTGGGATCCGGGTGAA
R21V-R AACCGGCTGGCCTTTGCTAAACGGT V182W-R CCAATCGCGCAGGCCGATATAAACAAT
R21W-F AAAGGCCAGCCGTGGGGTGGTGTTG V182Y-F GGCCTGCGCGATTACGATCCGGGTGAA
R21W-R CCACGGCTGGCCTTTGCTAAACGGT V182Y-R GTAATCGCGCAGGCCGATATAAACAAT
R21Y-F AAAGGCCAGCCGTATGGTGGTGTTG
R21Y-R ATACGGCTGGCCTTTGCTAAACGGT
), ArticleFig(id=1200378745732329885, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199782978957181130, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Enzyme Reaction conditions with Mn2+ Enzyme Without Mn2+
kcat/s-1 Km/mmol·L-1 kcat/Km/s-1·mmol-1·L kcat/s-1 Km/mmol·L-1 kcat/Km/s-1·mmol-1·L
rhArg1 198.22 ± 13.33 9.32 ± 2.11 21.27 rhArg1 63.34 ± 1.81 11.94 ± 1.09 5.3
R21N 210.84 ± 6.73 11.17 ± 1.15 18.88 R21N 85.13 ± 1.39 11.14 ± 0.59 7.64
V182A 311.53 ± 43.91 11.72 ± 5.28 26.57 V182A 67.44 ± 1.31 24.05 ± 1.26 2.8
V182I 306.30 ± 18.42 12.77 ± 2.42 23.98 V182I 89.26 ± 2.05 16.45 ± 1.11 5.43
V182P 484.53 ± 26.22 24.95 ± 3.64 19.42 V182P 53.21 ± 1.28 9.69 ± 0.77 5.49
V182Q 271.34 ± 14.75 14.66 ± 2.43 18.51 V182Q 70.08 ± 2.28 15.18 ± 1.49 4.62
V182T 457.33 ± 37.29 13.87 ± 3.49 32.98 V182T 80.68 ± 2.02 14.27 ± 1.09 5.66
R21I 172.75 ± 17.68 8.02 ± 2.86 21.55 R21C 61.94 ± 2.43 10.10 ± 1.31 6.14
R21L 163.40 ± 11.06 10.37 ± 2.31 15.75 R21D 63.44 ± 1.51 8.80 ± 0.71 7.21
R21S 234.86 ± 18.67 10.98 ± 2.83 21.38 V182C 55.72 ± 1.26 10.13 ± 0.76 5.5
V182K 465.90 ± 32.56 44.27 ± 7.16 10.53 V182D 127.12 ± 4.93 11.26 ± 1.36 11.29
V182Y 279.54 ± 62.63 15.04 ± 10.14 18.59 V182H 109.78 ± 3.48 16.64 ± 1.54 6.6
/ / / / V182L 61.50 ± 2.72 9.08 ± 1.37 6.77
/ / / / V182M 82.01 ± 2.25 15.46 ± 1.27 5.31
/ / / / V182N 53.76 ± 2.28 8.33 ± 1.23 6.45
/ / / / V182S 123.16 ± 2.64 13.63 ± 0.90 9.04
/ / / / V182W 60.71 ± 2.34 10.75 ± 1.35 5.65
), ArticleFig(id=1200378745887519141, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199782978957181130, language=CN, label=Table 2, caption=

Kinetic parameters of rhArg1 and its mutants

, figureFileSmall=null, figureFileBig=null, tableContent=
Enzyme Reaction conditions with Mn2+ Enzyme Without Mn2+
kcat/s-1 Km/mmol·L-1 kcat/Km/s-1·mmol-1·L kcat/s-1 Km/mmol·L-1 kcat/Km/s-1·mmol-1·L
rhArg1 198.22 ± 13.33 9.32 ± 2.11 21.27 rhArg1 63.34 ± 1.81 11.94 ± 1.09 5.3
R21N 210.84 ± 6.73 11.17 ± 1.15 18.88 R21N 85.13 ± 1.39 11.14 ± 0.59 7.64
V182A 311.53 ± 43.91 11.72 ± 5.28 26.57 V182A 67.44 ± 1.31 24.05 ± 1.26 2.8
V182I 306.30 ± 18.42 12.77 ± 2.42 23.98 V182I 89.26 ± 2.05 16.45 ± 1.11 5.43
V182P 484.53 ± 26.22 24.95 ± 3.64 19.42 V182P 53.21 ± 1.28 9.69 ± 0.77 5.49
V182Q 271.34 ± 14.75 14.66 ± 2.43 18.51 V182Q 70.08 ± 2.28 15.18 ± 1.49 4.62
V182T 457.33 ± 37.29 13.87 ± 3.49 32.98 V182T 80.68 ± 2.02 14.27 ± 1.09 5.66
R21I 172.75 ± 17.68 8.02 ± 2.86 21.55 R21C 61.94 ± 2.43 10.10 ± 1.31 6.14
R21L 163.40 ± 11.06 10.37 ± 2.31 15.75 R21D 63.44 ± 1.51 8.80 ± 0.71 7.21
R21S 234.86 ± 18.67 10.98 ± 2.83 21.38 V182C 55.72 ± 1.26 10.13 ± 0.76 5.5
V182K 465.90 ± 32.56 44.27 ± 7.16 10.53 V182D 127.12 ± 4.93 11.26 ± 1.36 11.29
V182Y 279.54 ± 62.63 15.04 ± 10.14 18.59 V182H 109.78 ± 3.48 16.64 ± 1.54 6.6
/ / / / V182L 61.50 ± 2.72 9.08 ± 1.37 6.77
/ / / / V182M 82.01 ± 2.25 15.46 ± 1.27 5.31
/ / / / V182N 53.76 ± 2.28 8.33 ± 1.23 6.45
/ / / / V182S 123.16 ± 2.64 13.63 ± 0.90 9.04
/ / / / V182W 60.71 ± 2.34 10.75 ± 1.35 5.65
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冯翠月 1 , 王晨宇 1 , 唐梦佳 1 , 樊帅 2 , 杨兆勇 2, * , 张志斐 1, *
药学学报 | 研究论文 2024,59(12): 3402-3408
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药学学报 | 研究论文 2024, 59(12): 3402-3408
定向进化提高人精氨酸酶1的催化活性
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冯翠月1, 王晨宇1, 唐梦佳1, 樊帅2, 杨兆勇2, * , 张志斐1, *
作者信息
  • 1.华北理工大学药学院, 河北 唐山 063210
  • 2.中国医学科学院、北京协和医学院医药生物技术研究所, 北京 100050

通讯作者:

*杨兆勇, E-mail: ;
张志斐, E-mail:
Directed evolution to enhance the catalytic activity of human arginase 1
Cui-yue FENG1, Chen-yu WANG1, Meng-jia TANG1, Shuai FAN2, Zhao-yong YANG2, * , Zhi-fei ZHANG1, *
Affiliations
  • 1. School of Pharmacy, North China University of Science and Technology, Tangshan 063210, China
  • 2. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
出版时间: 2024-12-12 doi: 10.16438/j.0513-4870.2024-0489
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摘要
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精氨酸酶1缺乏症(arginase 1 deficiency, ARG1-D) 是一种罕见的遗传性代谢疾病, 导致患者进行性痉挛性瘫痪、认知障碍和癫痫发作。来源于人源的重组人精氨酸酶1 (recombinant human arginase 1, rhArg1) 是其潜在的治疗药物, 但还存在活性低、半衰期短等缺陷限制了其临床应用。本研究采用定向进化的方法, 通过易错PCR构建rhArg1的随机突变文库, 经高通量筛选获得活性提高的突变体, 联合点饱和突变探讨了R21和V182位点对活性的影响。研究发现, 在反应体系中不含Mn2+的条件下, 突变体V182D、V182S、V182H和R21N的kcat值相比于rhArg1提高2.0、1.9、1.7和1.3倍, 突变体V182D、V182S、R21D和R21N的kcat/Km分别是rhArg1的2.1、1.7、1.4和1.4倍。突变体R21D和V182L对底物的亲和力有所增强。本研究通过定向进化和点饱和突变获得了rhArg1活性提高的突变体, 从而提升了其在医疗领域的应用前景。

精氨酸酶1缺乏症  /  重组人精氨酸酶1  /  定向进化  /  饱和突变

Arginase 1 deficiency (ARG1-D) is a rare genetic metabolic disorder that leads to progressive spastic paralysis, cognitive impairment, and seizures. Recombinant human arginase 1 (rhArg1) is a potential therapeutic agent for this condition, but its clinical application is limited by low activity and short half-life. In this study, we employed directed evolution to address these issues. A random mutation library of rhArg1 was constructed using error-prone PCR, and high-throughput screening was used to identify mutants with enhanced activity. Site-saturation mutagenesis was also performed to investigate the effects of residues R21 and V182 on enzyme activity. Our findings revealed that under reaction conditions devoid of Mn2+, the kcat values of the mutants V182D, V182S, V182H, and R21N increased by 2.0, 1.9, 1.7, and 1.3 times respectively, compared to rhArg1. The kcat/Km values of mutants V182D, V182S, R21D, and R21N were 2.1, 1.7, 1.4, and 1.4 times higher than those of rhArg1, respectively. Additionally, mutants R21D and V182L showed enhanced substrate affinity. Through directed evolution and site-saturation mutagenesis, we successfully obtained rhArg1 mutants with improved activity, thereby enhancing its potential for clinical application.

arginase 1 deficiency  /  recombinant human arginase 1  /  directed evolution  /  site-saturation mutagenesis
冯翠月, 王晨宇, 唐梦佳, 樊帅, 杨兆勇, 张志斐. 定向进化提高人精氨酸酶1的催化活性. 药学学报, 2024 , 59 (12) : 3402 -3408 . DOI: 10.16438/j.0513-4870.2024-0489
Cui-yue FENG, Chen-yu WANG, Meng-jia TANG, Shuai FAN, Zhao-yong YANG, Zhi-fei ZHANG. Directed evolution to enhance the catalytic activity of human arginase 1[J]. Acta Pharmaceutica Sinica, 2024 , 59 (12) : 3402 -3408 . DOI: 10.16438/j.0513-4870.2024-0489
正文
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精氨酸酶1缺乏症(arginase 1 deficiency, ARG1-D) 是一种因精氨酸酶1 (arginase 1, Arg1) 基因突变导致的常染色体隐性遗传病[1], Arg1的失活会导致精氨酸和其他胍类化合物在血液中过度积累, 影响肝脏尿素循环, 导致尿素生成功能受损[2]。标准治疗包括限制饮食疗法和对症治疗[3, 4], 但均无法解决精氨酸在体内的积累, 且患者依从性差。rhArg1已被证实可以将血液精氨酸水平降低到可接受的范围内, 并显著减少胍类化合物的积累[5-7]。2023年12月, 欧盟正式批准Loargys (pegzilarginase) 用于治疗成人、青少年和2岁及以上儿童的ARG1-D。Loargys是人源的精氨酸酶1, 通过催化精氨酸生成尿素和鸟氨酸来降低患者血浆中精氨酸的浓度。但令人遗憾的是该药物并未引进入国内。同时, 人源精氨酸酶1 (human arginase 1, hArg1) 还是药物靶点, 其涉及30多种严重疾病[8, 9], 从严重的心血管疾病到癌症和罕见的代谢性疾病[10-15]。hArg1已被进一步提出作为治疗肝细胞癌和黑色素瘤的潜在药物[16], 并作为治疗精氨酸营养不良肿瘤的潜在癌症治疗剂进入临床试验[17]。
Arg1是一种含双核锰离子的水解酶, 可催化精氨酸转化为鸟氨酸和尿素, 从而在氮代谢中起关键作用[18, 19]。它存在于细菌、酵母、植物、无脊椎动物和脊椎动物中, 于1904年在哺乳动物肝脏中被发现[20]。在人和哺乳动物的各种组织器官中, Arg有两种亚型, 分别是Arg1和Arg2。其中Arg1 (EC3.5.3.1) 是一种主要表达于肝脏的胞浆异构体, 它调节尿素生成, 而Arg2是一种主要存在于肠道、肾脏和前列腺的线粒体异构体[21-23]。Arg1是一种同源三聚体金属酶, 长度为322个氨基酸残基, 每个单体在其催化核心内配以一个氢氧离子桥接的双核Mn2+簇, 呈α/β-折叠[24]。Arg1的活性会受到精氨酸以及Mn2+的影响, 这使得它区别于尿素循环里的另四种酶(氨甲酰磷酸合成酶、鸟氨酸转氨甲酰基酶、精氨琥珀酸合酶和精氨琥珀酸裂解酶), 进而会对尿素循环产生重要的影响。
虽然hArg1具有免疫原性低的优点, 但其与精氨酸的亲和力较低, 在体内的半衰期较短, 且hArg1能发挥最佳活性的反应条件与生理条件相差很多[15, 25], 限制了其在ARG1-D和癌症治疗中的临床有效性, 因此需要对其进行改造, 从而满足临床应用的需求。本研究采用定向进化的方法提高rhArg1的活性, 通过易错PCR构建rhArg1的随机突变文库, 利用高通量筛选方法筛选活性提高的突变体, 再针对此位点进行点饱和突变进行优化, 对突变体进行活性、酶动力学和热力学参数评价, 期望获得活性和稳定性提高的突变体, 提升rhArg1的成药潜力。
材料与方法
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菌株与试剂  克隆菌株Trans1-T1和表达菌株BL21 (DE3) 均购自北京全式金生物技术有限公司; 表达质粒pET-21a (+) 购自北京安诺伦生物科技有限公司, 质粒提取试剂盒、PCR纯化试剂盒、内切酶购自北京康润诚业生物科技有限公司, 氨苄青霉素(批号: 9314102111)、异丙基-β-D-硫代半乳糖苷(IPTG, 批号: 0487083131) 购自北京兰博利德商贸有限公司; Co2+-NTA填料(批号: 2301151A) 购自日本TaKaRa公司; 30 kDa超滤浓缩管购自美国Millipore公司, 其余试剂均为国产分析纯。
构建随机突变文库和定点突变  人源的Arg1 (NCBI: P05089.2) 经密码子优化后由华大基因合成, 经酶切连接得到表达质粒pET-21a-rhArg1, 以此为模板, 采用QuickMutationTM基因随机突变试剂盒(碧云天, 中国) 构建随机突变文库。点饱和突变由反向PCR完成, 突变引物见表 1, 扩增反应体系为: 模板DNA 1 µL, ddH2O 21 µL, KOD OneTM PCR Master Mix 25 µL, 上下游引物各1.5 µL。PCR条件为: 98 ℃预变性2 min; 98 ℃变性10 s, 60 ℃退火15 s, 68 ℃延伸50 s, 30个循环; 最后68 ℃延伸5 min。
突变文库筛选  将突变体接种在96孔板上, 37 ℃, 200 r·min-1摇至OD600达到0.6~0.8, 然后添加终浓度为1 mmol·L-1 IPTG, 在20 ℃、200 r·min-1条件下发酵12 h, 然后添加细菌裂解酶在37 ℃孵育30 min, 通过3次冻融破碎菌体, 离心吸取上清测定活性。
rhArg1及其突变体的表达与纯化  将测序正确的菌株取1.5 mL菌液提取质粒, 转化至表达菌株大肠杆菌BL21 (DE3) 制备重组表达菌株。按2.5%的接种量接种, 在37 ℃、200 r·min-1培养至OD600为0.6~0.8时加入终浓度为0.2 mmol·L-1 IPTG, 然后在16 ℃、200 r·min-1继续培养14~16 h, 将培养物于4 ℃、6 000 r·min-1下离心6 min, 将收集好的菌体用裂解液(50 mmol·L-1 NaH2PO4, 300 mmol·L-1 NaCl, 10 mmol·L-1咪唑, pH 8.0) 进行重悬, 破碎前加入终浓度为1 mmol·L-1的PMSF溶液。用高压均质器进行菌体破碎, 破碎后的菌体在4 ℃、11 000 r·min-1下离心45 min, 离心后的上清液用0.45 μm滤膜过滤, 取适量Co2+填料装于亲和层析柱中, 用5倍柱体积去离子水清洗两遍, 再加入5倍柱体积裂解液预平衡, 将过滤后的上清液与平衡好的Co2+亲和层析填料混匀, 并在混悬仪上于4 ℃孵育1 h, 将孵育后的填料置于层析柱中, 用10倍柱体积清洗液(50 mmol·L-1磷酸缓冲液, 300 mmol·L-1 NaCl, 20 mmol·L-1咪唑, pH 7.4) 冲洗杂蛋白, 再用5倍柱体积洗脱液(50 mmol·L-1 NaH2PO4, 300 mmol·L-1 NaCl, 250 mmol·L-1咪唑, pH 8.0) 将目的蛋白洗脱下来。使用超滤浓缩管将含有目的蛋白rhArg1的组分汇集并浓缩至适宜浓度。所有突变体的纯度通过12.5%的SDS-PAGE电泳分析, 并采用Bradfoard法测定蛋白质浓度。
rhArg1及其突变体酶活性测定  酶催化总反应体系为200 µL, 将1.4 µmol·L-1蛋白加入到含有10 mmol·L-1 L-精氨酸, 50 mmol·L-1 Tris-HCl (pH 7.4) 和0.2 mmol·L-1 Mn2+中37 ℃反应5 min [不含Mn2+的反应体系是将5.8 µmol·L-1蛋白加入到含有10 mmol·L-1 L-精氨酸和50 mmol·L-1 Tris-HCl (pH 7.4) 中37 ℃反应5 min], 加入30 µL含2-异亚硝基苯丙酮的酸混合物终止反应, 然后100 ℃反应45 min, 置于冰上冷却后12 000 r·min-1离心2 min, 在紫外分光光度计下540 nm处测量吸光度, 并通过标准曲线确定生成尿素的量, 计算rhArg1的酶活。每秒钟催化生成1 mmol尿素所需要的rhArg1量定义为1 U。
rhArg1及其突变体酶动力学测定  为评估酶的动力学参数Km和kcat, 以溶于50 mmol·L-1 Tris-HCl (pH 7.4) 的0.2~100 mmol·L-1 L-精氨酸(或含0.2 mmol·L-1 Mn2+) 作为底物, 分别与适当稀释的酶液反应, 测定酶活性, 利用GraphPad Prism 8.0中Michaelis-Menten非线性拟合作图法计算Km和Vmax值, 并通过测定的蛋白浓度计算出对应的kcat。
rhArg1及其突变体最适反应温度与最适pH值  配制pH分别为5.0、5.5、6.0、6.5的50 mmol·L-1醋酸钠缓冲液, 7.0、7.5、8.0、8.5的50 mmol·L-1 Tris-HCL缓冲液, 9.0、9.5、10.0和10.5的50 mmol·L-1碳酸钠缓冲液, 在37 ℃、不同pH条件下反应5 min, 以最高活性为100%, 计算相对酶活性以确定酶的最适反应pH。将适量酶液与底物在不同温度(30、35、40、45、50、55、60、65、70、75、80、85和90 ℃) 反应5 min, 以最高活性为100%, 测定相对酶活, 以确定酶反应的最适温度。
统计学分析  统计学处理采用GraphPad Prism 8.0软件进行数据分析, 组间对比采用单因素方差分析。数据表示为平均值±标准差, 当P < 0.05时具有统计显著性。
结果与讨论
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1 突变体库的建立和筛选
利用基因随机突变试剂盒构建rhArg1的随机突变文库, 随机挑取20个转化子进行测序, 发现大部分转化子包含2~5个碱基突变, 证明随机突变文库构建成功。利用96孔板在37 ℃、200 r·min-1条件下培养转化子并利用IPTG诱导表达, 然后添加溶菌酶并反复冻融三次裂解细胞, 96孔板离心后吸取上清, 加入包含底物尿素的96孔板中, 反应45 min, 然后与2-异亚硝基苯丙酮显色从而测定活性, 颜色越深表示活性越高。结果如图 1所示, 调取颜色最深的孔对应的转化子进行测序, 确定突变位点的信息, 经过5 000个转化子左右的筛选和测序, 获得了活性提高的突变体R21C和V182Q。
2 rhArg1及其突变体的表达与酶活测定
为了进一步确认筛选突变体的酶学性质, 利用BL21 (DE3) 异源表达rhArg1及其突变体, 经IPTG诱导表达收集菌体、高压破碎离心后, 上清液经Co2+亲和层析纯化(图 2A), 对rhArg1及其突变体进行了酶活测定, Mn2+是rhArg1重要的辅因子[26], 可明显提高蛋白的活性(图 2B), 但rhArg1作为潜在的治疗精氨酸酶1缺乏症的药物, 由于治疗需要长期注射, 而Mn2+对人体有害, 所以在研发过程中需要降低rhArg1对Mn2+的依赖, 所以考察上述突变体时应考量没有额外添加Mn2+条件下的活性。实验结果显示, 在反应体系添加Mn2+的条件下, rhArg1的活性是无添加Mn2+的6倍。突变体R21C和V182Q在无添加Mn2+的条件下, 反应活性分别较rhArg1提高了15%和10%, 在添加Mn2+的条件下, 反应活性分别提高了6%和32%。
3 R21和V182位点的点饱和突变研究
鉴于R21C和V182Q的活性增加, 为了探究21和182位点对活性的影响, 继续采用点饱和突变来研究突变体对活性的影响, 通过反向PCR分别获得R21和V182位点突变为其余18种氨基酸的突变体, 并测量其活性数据, 如图 3A所示, 在添加Mn2+的条件下, 对于21位点, 突变体R21L和R21S的活性相较于rhArg1分别提高了22%和30%。在182位点, 除V182R外, V182位点其余突变体的活性均能保持在60%以上, 其中突变体V182I、V182K、V182P、V182Q和V182T的活性相较于rhArg1分别提高了92%、29%、71%、32%和76%。在无添加Mn2+的条件下, 结果如图 3B所示, 对于第21位精氨酸, 突变体R21N的催化活性最高, 是WT的1.4倍, 突变体R21C和R21N的活性相较于rhArg1分别提高了15%和37%。在V182位点, 突变体的活性提高较为明显, 其中突变体V182A、V182D、V182H、V182I、V182P、V182S和V182T的活性相较于rhArg1分别提高了28%、54%、30%、40%、26%、59%和36%。
4 rhArg1及其突变体酶学性质研究
酶动力学实验结果见表 2, 含Mn2+突变体在酶的催化常数方面, 突变体V182P最高, 达到484.53 s-1; 突变体R21N、R21S、V182A、V182I、V182K、V182P、V182Q、V182T和V182Y的kcat值分别是rhArg1的1.1、1.2、1.6、1.6、2.4、2.4、1.4、2.3和1.4倍。对于米氏常数, R21I较野生型降低14%, 表明突变体对精氨酸的亲和力增大, 而其余突变体的Km值均有不同程度的提高, 表示突变体对底物的亲和力有所下降。在酶的专一性常数方面, 突变体V182T为32.98 s-1·mmol-1·L, 较野生型增大1.6倍, 突变体V182A和V182I的kcat/Km分别是rhArg1的1.2和1.1倍。
不含Mn2+突变体V182D和V182S的催化活性表现最佳, 其kcat分别为127.12和123.16 s-1, 其中突变体V182D在额外添加Mn2+条件下, 活性并没有提高; 突变体R21N、V182A、V182H、V182I、V182M、V182Q和V182T的kcat值分别是rhArg1的1.3、1.1、1.7、1.4、1.3、1.1和1.3倍。在底物亲和力方面, 突变体R21D、V182L、V182N和V182P均有不同程度的降低, 其中突变体V182N的Km值最低, 为8.33 mmol·L-1, 表现出结合精氨酸的能力最强, 而其他突变体的Km值均有不同程度的提高, 表示突变体对底物的亲和力有所下降。最终突变体R21D、R21N、V182D、V182L和V182S的kcat/Km分别是rhArg1的1.4、1.4、2.1、1.3和1.7倍。另外还测定了V182D和V182S的最佳反应pH和温度, 结果如图 4所示, rhArg1和突变体V182D的最适pH为8.5, 而突变体V182S的最适pH为8, 与人体血液pH 7.4~7.6最为接近; rhArg1的最适反应温度为65 ℃, 突变体V182D的最适反应温度为45 ℃, 突变体V182S的最适反应温度为55 ℃。虽最适温度有所降低, 但均高于人体温度。
结论
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本研究成功构建重组表达质粒pET-21a-rhArg1, 并利用大肠杆菌BL21 (DE3) 成功异源表达获得可溶性的rhArg1蛋白。利用易错PCR构建rhArg1随机突变文库, 结合高通量筛选的方法筛选获得了2个对rhArg1活性提高的阳性突变体, 随即对这两个位点进行了点饱和突变, 通过测定酶活性和酶动力学性质来评价突变体, 结果显示在额外添加Mn2+的条件下, 突变体V182K、V182P和V182T的kcat值分别为rhArg1的2.4、2.4和2.3倍, 而不额外添加Mn2+的条件下, 突变体V182D和V182S的kcat值分别为rhArg1的2.0和1.9倍。本研究获得了活性提高的突变体, 并着重研究了有无额外添加Mn2+的酶动力学参数, 为进一步提高rhArg1的成药潜力提供了有力支撑。
作者贡献: 冯翠月撰写了论文; 冯翠月、王晨宇、唐梦佳完成了实验; 冯翠月、樊帅分析了实验数据; 杨兆勇、张志斐负责设计本项实验; 全部作者均阅读并参与修改了本文。
利益冲突: 本文的作者无任何利益冲突。
基金
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  • 国家自然科学基金面上项目(82373767)
  • 中国医学科学院医学与健康科技创新工程(2021-I2M-1-055)
文献
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2024年第59卷第12期
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doi: 10.16438/j.0513-4870.2024-0489
  • 接收时间：2024-05-21
  • 首发时间：2025-11-24
  • 出版时间：2024-12-12
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  • 收稿日期：2024-05-21
  • 修回日期：2024-07-30
基金
国家自然科学基金面上项目(82373767)
中国医学科学院医学与健康科技创新工程(2021-I2M-1-055)
作者信息
    1.华北理工大学药学院, 河北 唐山 063210
    2.中国医学科学院、北京协和医学院医药生物技术研究所, 北京 100050

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*杨兆勇, E-mail: ;
张志斐, E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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