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Article(id=1316714536523125013, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1316714454184739470, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20260069, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1769184000000, receivedDateStr=2026-01-24, revisedDate=null, revisedDateStr=null, acceptedDate=1776528000000, acceptedDateStr=2026-04-19, onlineDate=1791858808879, onlineDateStr=2026-10-13, pubDate=1791043200000, pubDateStr=2026-10-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1791858808879, onlineIssueDateStr=2026-10-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1791858808879, creator=13701087609, updateTime=1791858808879, updator=13701087609, issue=Issue{id=1316714454184739470, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='10', pageStart='4661', pageEnd='5310', issueExtLink='null', onlineDate='null', pubDate='1791043200000', pubDateStr='2026-10-04', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1791858789249, creator='13701087609', updateTime=1791885400527, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1316826070066774161, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1316714454184739470, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1316826070066774162, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1316714454184739470, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=4956, endPage=4973, ext={EN=ArticleExt(id=1316714536871252246, articleId=1316714536523125013, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Effects of site-directed mutagenesis on the enzymatic properties of a thermostable collagenolytic protease, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] To conduct site-directed mutagenesis on the Ca2+-binding sites, Leu253, and protein surface oxidation-sensitive Met residues of TSS, a collagenolytic protease derived from thermophiles, thereby screening out the variants with improved antioxidant activity, thermostability, and catalytic activity and revealing the intrinsic connection among them. [Methods] Site-directed mutagenesis was conducted on the Ca2+-binding sites (Ca1, Ca2, and Ca3) and Leu253 of TSS and the oxidation-sensitive Met residues on the protein surface. The derived variants were further evaluated for H2O2 tolerance, relative activity, and residual activity. Changes in antioxidant activity, catalytic activity, and thermostability after mutations were also studied. [Results] Mutations in amino acid residues at the three Ca2+-binding sites led to decreases in catalytic activity and/or thermostability of TSS. The Leu253 series variants constructed by simulating KP-43 did not enhance the antioxidant activity of TSS. Among the variants of oxidation-sensitive residues, M302L showed increases of 13% and 61% in the catalytic and antioxidant activities, respectively, while remaining unchanged thermostability. Ca2+ increased the catalytic activity and thermostability of TSS at high temperatures in a concentration-dependent manner. A high salinity environment with more than 2 mol/L NaCl prolonged the half-life of TSS at 75 ℃ and inhibited the maturation process of TSS proform. [Conclusion] The antioxidant and catalytic activities are positively correlated in the Leu253 and Met variants of TSS, which conforms to the “Trade-off” mechanism.

, authors=Kui ZHANG*, Min ZHU, Yunyun SU, Xilong WANG, Qian GUO, Zhike WANG, Xiuli LIU, Haiqing LIU, authorsList=Kui ZHANG, Min ZHU, Yunyun SU, Xilong WANG, Qian GUO, Zhike WANG, Xiuli LIU, Haiqing LIU, authorCompany=null, correspAuthors=Kui ZHANG, authorNote=null, correspAuthorsNote=
*E-mail:
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【目的】 针对嗜热微生物来源的热稳定枯草杆菌蛋白酶样丝氨酸蛋白酶(thermostable subtilisin-like serine protease, TSS)的Ca2+结合位点、Leu253位点和蛋白表面氧化敏感Met位点进行定点突变，筛选抗氧化性、热稳定性及催化活性均提高的突变体，并探究三者之间的内在关联。 【方法】 针对TSS的Ca2+结合位点(Ca1、Ca2和Ca3)、Leu253位点和蛋白表面氧化敏感Met残基开展定点突变，通过检测产生的系列突变体对H2O2的耐受性、相对酶活性和在70 ℃保温3 h后的残余活性，系统研究突变体抗氧化活性、催化活性和热稳定性的变化特征。 【结果】 3个Ca2+结合位点突变后，TSS的催化活性和热稳定性均出现不同程度下降；参照KP-43构建的Leu253系列突变体并未增强TSS的抗氧化性；针对氧化敏感残基改造获得的突变体M302L，在保持热稳定性不变、催化活性提升13%的同时抗氧化性提升61%。研究同时发现，Ca2+可提升TSS的高温催化活性和热稳定性，且该作用具有浓度依赖性；2 mol/L及以上的高盐环境可延长TSS在75 ℃的半衰期，并抑制酶原自加工成熟。 【结论】 在TSS的Leu253和Met突变体中，抗氧化性和催化活性呈正相关，符合“Trade-off”机制。

, authors=张魁*, 朱敏, 苏芸芸, 王喜龙, 郭倩, 王志科, 刘秀丽, 刘海卿, authorsList=张魁, 朱敏, 苏芸芸, 王喜龙, 郭倩, 王志科, 刘秀丽, 刘海卿, authorCompany=null, correspAuthors=张魁, authorNote=

作者贡献声明

张魁：总体构思、数据管理与初稿写作；朱敏、苏芸芸、王喜龙：调查研究；郭倩、王志科：提供资源；刘秀丽、刘海卿：项目管理。

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SpringerPlus, 2013, 2: 410., articleTitle=Characterization of a new oxidant-stable serine protease isolated by functional metagenomics, refAbstract=null)], funds=[Fund(id=1316829844290261747, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, awardId=23JRRM742, language=EN, fundingSource=Natural Science Foundation of Gansu Province(23JRRM742), fundOrder=null, country=null), Fund(id=1316829844365759220, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, awardId=23JRRM742, language=CN, fundingSource=甘肃省自然科学基金(23JRRM742), fundOrder=null, country=null), Fund(id=1316829844432868085, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, awardId=XYBYZK2215, language=EN, fundingSource=Doctoral Foundation of Longdong University(XYBYZK2215), fundOrder=null, country=null), Fund(id=1316829844508365558, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, awardId=XYBYZK2215, language=CN, fundingSource=陇东学院博士基金(XYBYZK2215), fundOrder=null, country=null), Fund(id=1316829844583863031, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, awardId=XYBYZK2216, language=CN, fundingSource=陇东学院博士基金(XYBYZK2216), fundOrder=null, country=null), Fund(id=1316829844659360504, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, awardId=XYBYZK2217, language=CN, fundingSource=陇东学院博士基金(XYBYZK2217), fundOrder=null, country=null), Fund(id=1316829844739052281, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, awardId=XYBYZK2229, language=CN, fundingSource=陇东学院博士基金(XYBYZK2229), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1316829838766363314, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, xref=null, ext=[AuthorCompanyExt(id=1316829838774751923, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, companyId=1316829838766363314, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Gansu Key Laboratory of Conservation and Utilization of Biological Resources and Ecological Restoration in Longdong, School of Agriculture and Bioengineering, Longdong University, Qingyang, Gansu, China), AuthorCompanyExt(id=1316829838783140532, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, companyId=1316829838766363314, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=陇东学院 农业与生物工程学院，甘肃省陇东生物资源保护利用与生态修复重点实验室，甘肃 庆阳)])], figs=[ArticleFig(id=1316829843312988903, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=EN, label=Figure 1, caption=Bioinformatics analysis of TSS. A: Schematic representation of the primary structures of the TSS precursor, proform, mature enzyme, N-terminal propeptide deletion protein and N-terminal propeptide; B: Alignment of amino acid sequence around the Ca2+-binding sites of TSS and other S8 family proteases; C: Superimposition of 3D-structures of TSS predicted by two different methods; D: Superimposition of 3D-structures of TSS and KP-43. In A, the signal peptide (S), the N-terminal propeptide (N), the catalytic domain (CD), βJR and PPC domains, and the fused His-tag (H) are indicated. The locations of the active-site residues (D29, H66, and S257) and the three Ca2+-binding sites (Ca1, Ca2 and Ca3) are also shown. pre-TSS: TSS precursor; pro-TSS: TSS proform; mTSS: Mature TSS. In B, the residues serving as Ca2+-binding ligands in KP-43 (AB051423), Myroicolsin (AEC33275), Tk-SP (Q5JIZ5), Pyrolysin (AAB09761), and TSS (OBR56241) are all marked. Vertical arrows indicate the residues chosen for mutational analysis., figureFileSmall=sdKsKK8VwdflSns64347BA==, figureFileBig=jgpCzxBa3862iQYoEWV/cw==, tableContent=null), ArticleFig(id=1316829843388486376, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=CN, label=图1, caption=TSS的生物信息学分析, figureFileSmall=sdKsKK8VwdflSns64347BA==, figureFileBig=jgpCzxBa3862iQYoEWV/cw==, tableContent=null), ArticleFig(id=1316829843505926889, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=EN, label=Figure 2, caption=Enzymatic properties of TSS. A: Effect of Ca2+ concentration on catalytic activity of TSS; B: Thermal inactivation curves of TSS at different salt concentrations; C: Percentage of FSC degradation at different temperatures., figureFileSmall=ujbLEzXktT/roBYFKdMR1Q==, figureFileBig=EJq7zVr6ExM7or7bhUgkVw==, tableContent=null), ArticleFig(id=1316829843564647146, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=CN, label=图2, caption=TSS的酶学性质, figureFileSmall=ujbLEzXktT/roBYFKdMR1Q==, figureFileBig=EJq7zVr6ExM7or7bhUgkVw==, tableContent=null), ArticleFig(id=1316829843648533227, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=EN, label=Figure 3, caption=Effects of Ca2+ and salinity on thermostability and maturation of TSS. A: Effects of Ca2+ on thermostability and maturation of TSS; B: Effects of salinity on thermostability and maturation of TSS; C and D: Investigation of the dual-band phenomena observed for I, M and N. I, M and N indicate the positions of intermediates, mature enzymes and N-terminal propeptides on SDS-PAGE and Western blotting, respectively; Mr represents low molecular weight protein marker. In C, after IPTG, the total proteins from Escherichia coli cells harboring the empty pET-26b plasmid (Control) or expressing each recombinant protein were analyzed by SDS-PAGE and Western blotting (using an anti-His-tag antibody), samples a and b were derived from the conditions in Figure 3B. In D, sample a from Figure 3B was diluted 5-fold with Buffer A and analyzed via SDS-PAGE (S) and gelatin overlay assay (G), both with (lanes 1 and 3) and without (lanes 2 and 4) TCA pre-treatment. Control: The empty pET-26b plasmid; Pro-S257A: The active-site variant of TSS with the catalytic residue Ser257 mutated to Ala; Pro-S257C: The active-site variant of TSS with the catalytic residue Ser257 mutated to Cys; HN-Pro-TSS: recombinant TSS with the His-tag fused to the N-terminus (all other variants harbor a C-terminal His-tag); MTCA and MNon represent the band positions of mTSS in SDS-PAGE (S) and gelatin overlay assay (G) with/without TCA pre-treatment, respectively., figureFileSmall=888jj0P5LyEHMoFDF8EqsQ==, figureFileBig=y4yckAkhtHV1qSXrvvArFA==, tableContent=null), ArticleFig(id=1316829843724030700, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=CN, label=图3, caption=Ca2+ 和盐浓度对TSS热稳定性及加工成熟的影响, figureFileSmall=888jj0P5LyEHMoFDF8EqsQ==, figureFileBig=y4yckAkhtHV1qSXrvvArFA==, tableContent=null), ArticleFig(id=1316829843782750957, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=EN, label=Figure 4, caption=Relative enzymatic activity and thermostability of TSS Ca2+-binding site variants. White bars represent thermostability or residual activity, gray bars represent relative enzymatic activities., figureFileSmall=AC3/ASNyX8l7v1uqMqOPbw==, figureFileBig=uN4pWNv2PYTU8/btoUuzZg==, tableContent=null), ArticleFig(id=1316829843849859822, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=CN, label=图4, caption=TSS的Ca2+ 结合位点定点突变体相对酶活性与热稳定性, figureFileSmall=AC3/ASNyX8l7v1uqMqOPbw==, figureFileBig=uN4pWNv2PYTU8/btoUuzZg==, tableContent=null), ArticleFig(id=1316829843904385775, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=EN, label=Figure 5, caption=Antioxidation properties, enzymatic activity and thermostability of Leu253 and Met variants. A: Antioxidant properties of Leu253 variants; B: Enzymatic activity and thermostability of Leu253 variants; C: Antioxidant properties of Met variants; D: Enzymatic activity and thermostability of Met variants. In A and C, Bars represent the antioxidant activities of different variants. In B and D, White bars represent thermostability or residual activity, while gray bars represent relative enzymatic activity., figureFileSmall=kwAL4AITgj6qU46myWmdUg==, figureFileBig=ucI0RuyxBQoUSEclM7YNlw==, tableContent=null), ArticleFig(id=1316829843975688944, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=CN, label=图5, caption=Leu253 及Met系列突变体的抗氧化性、酶活性及热稳定性, figureFileSmall=kwAL4AITgj6qU46myWmdUg==, figureFileBig=ucI0RuyxBQoUSEclM7YNlw==, tableContent=null), ArticleFig(id=1316829844072157937, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=EN, label=Table 1, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
NamesSequences (5′→3′)NamesSequences (5′→3′)
pro-TSS-FGGAATTCCATATGAAGCGGGAAACAGGGL253A-FCTACGCCTATGCCAGCGGGACAAGC
pro-TSS-RCCGCTCGAGACGGGAGCGGGAL253A-RCTGGCATAGGCGTAGAAGGCGTTG
pre-TSS-FGGCATATGCGGTTGAAATGGCATGCGL253I-FCTACGCCTATATCAGCGGGACAAGC
pro-S257A-FCTATCTCAGCGGGACAGCCATGGCGACGL253I-RCTGATATAGGCGTAGAAGGCGTTG
pro-S257A-RGGCTGTCCCGCTGAGATAGGCGTAGAAGGL253V-FCTACGCCTATGTCAGCGGGACAAGC
pro-S257C-FCTATCTCAGCGGGACATGCATGGCGACGL253V-RCTGACATAGGCGTAGAAGGCGTTG
pro-S257C-RGCATGTCCCGCTGAGATAGGCGTAGAAGGL253C-FCTACGCCTATTGCAGCGGGACAAGC
HN-pro-TSS-RCGCTCGAGGTCGTTGCGCTGTTTTCGL253C-RCTGCAATAGGCGTAGAAGGCGTTG
E187A-FGCCTCCGCAAACGACCGGCCGGAGCL253G-FCTACGCCTATGGCAGCGGGACAAGC
E187A-RGTCGTTTGCGGAGGCGCCCACGGCGL253G-RCTGCCATAGGCGTAGAAGGCGTTG
E187Q-FGCCTCCCAAAACGACCGGCCGGAGCL253S-FCTACGCCTATAGCAGCGGGACAAGC
E187Q-RGTCGTTTTGGGAGGCGCCCACGGCGL253S-RCTGCTATAGGCGTAGAAGGCGTTG
D196S-FGATGAAAGCGAGGCGGACGACCCGL253K-FCTACGCCTATAAGAGCGGGACAAGC
D196S-RGCCTCGCTTTCATCCAGCTCCGGCL253K-RCTCTTATAGGCGTAGAAGGCGTTG
D199A-FGAGGCGGCCGACCCGGACGAGGTAGL253D-FCTACGCCTATGACAGCGGGACAAGC
D199A-RCCGGGTCGGCCGCCTCGTCTTCATCL253D-RCTGTCATAGGCGTAGAAGGCGTTG
D199N-FGAGGCGAACGACCCGGACGAGGTAGM88A-FCAAAGGGGCGGCTCCCGACGCGAAG
D199N-RCCGGGTCGTTCGCCTCGTCTTCATCM88A-RGCCGCCCCTTTGTACTGGCCGTCTG
E203H-FCCGGACCACGTAGCCGCGTTCAGCM88L-FCAAAGGGCTGGCTCCCGACGCGAAG
E203H-RCTACGTGGTCCGGGTCGTCCGCCTCM88L-RGCCAGCCCTTTGTACTGGCCGTCTG
D385A-FCAACGCCTTTTTCCACTACCCCTACGM99A-FCCATTCCGCGCTGGACGAATACGG
D385A-RGTGGAAAAAGGCGTTGCCGTTCAAGGM99A-RGCGCGGAATGGAAGACGAGCTTCG
D385N-FCAACAACTTTTTCCACTACCCCTACGM99L-FCCATTCCCTGCTGGACGAATACGG
D385N-RGTGGAAAAAGTTGTTGCCGTTCAAGGM99L-RGCAGGGAATGGAAGACGAGCTTCG
D393A-FCTACGACGCCGAAGTGGACAACCTCAACM154A-FGGAACATCCGGACGCGACGGCGCTGGTGG
D393A-RCACTTCGGCGTCGTAGGGGTAGTGGM154A-RGCGCCGTCGCGTCCGGATGTTCCCAGAGG
D393N-FCTACGACAACGAAGTGGACAACCTCAACM154L-FCATCCGGACCTGACGGCGCTGGTG
D393N-RCACTTCGTTGTCGTAGGGGTAGTGGM154L-RGTCAGGTCCGGATGTTCCCAGAGAAAC
E394A-FGACGACGCAGTGGACAACCTCAACAACM258A-FGACAAGCGCGGCGACGCCGGTGTTG
E394A-RGTCCACTGCGTCGTCGTAGGGGTAGM258A-RCCGCGCTTGTCCCGCTGAGATAGGC
E394N-FGACGACAATGTGGACAACCTCAACAACM258L-FGACAAGCCTGGCGACGCCGGTGTTG
E394N-RGTCCACATTGTCGTCGTAGGGGTAGM258L-RCCAGGCTTGTCCCGCTGAGATAGGC
E394Q-FGACGACCAAGTGGACAACCTCAACAACM258C-FGACAAGCTGTGCGACGCCGGTGTTG
E394Q-RGTCCACTTGGTCGTCGTAGGGGTAGM258C-RCACAGCTTGTCCCGCTGAGATAGGC
N368D-FCTGGTCAACGACCTGAACCTGCGAGM290A-FGGCGGCGCTCATCAGCGGCGCCGAC
N368D-RCAGGTCGTTGACCAGGGTGCGCTCGM290A-RGAGCGCCGCCTTGATCAATGCC
N370D-FCAACAACCTGGACCTGCGAGTGGTCM290L-FGGCGCTGCTCATCAGCGGCGCCGAC
N370D-RCAGGTCCAGGTTGTTGACCAGGGTGM290L-RGAGCAGCGCCTTGATCAATGCC
D396A-FCGAAGTGGCCAACCTCAACAACGTGM302A-FCGAAGACGCGCGCGTGCAGGGGTTC
D396A-RGAGGTTGGCCACTTCGTCGTCGTAGM302A-RGCGCGTCTTCGTTCAAATCGTCGG
D396N-FCGAAGTGAACAACCTCAACAACGTGM302L-FGAACGAAGACCTACGCGTGCAGG
D396N-RGAGGTTGTTCACTTCGTCGTCGTAGM302L-RCGCGTAGGTCTTCGTTCAAATCG
E402A-FCAACGTGGCGCAGGTCTGGATCAAGM425A-FCGTGCCCGCGGGGCCGCAGCCGTAC
E402A-RGACCTGCGCCACGTTGTTGAGGTTGM425A-RCCGCGGGCACGTTGTAGCCCTCCAC
E402Q-FCAACGTGCAGCAGGTCTGGATCAAGM425L-FCGTGCCCCTGGGGCCGCAGCCGTAC
E402Q-RGACCTGCTGCACGTTGTTGAGGTTGM425L-RCCAGGGGCACGTTGTAGCCCTCCAC
L253M-FCGCCTATATGAGCGGGACAAGCATG
L253M-RGTCCCGCTCATATAGGCGTAGAAGG
), ArticleFig(id=1316829844143461106, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714536523125013, language=CN, label=表1, caption=

本研究所用引物

, figureFileSmall=null, figureFileBig=null, tableContent=
NamesSequences (5′→3′)NamesSequences (5′→3′)
pro-TSS-FGGAATTCCATATGAAGCGGGAAACAGGGL253A-FCTACGCCTATGCCAGCGGGACAAGC
pro-TSS-RCCGCTCGAGACGGGAGCGGGAL253A-RCTGGCATAGGCGTAGAAGGCGTTG
pre-TSS-FGGCATATGCGGTTGAAATGGCATGCGL253I-FCTACGCCTATATCAGCGGGACAAGC
pro-S257A-FCTATCTCAGCGGGACAGCCATGGCGACGL253I-RCTGATATAGGCGTAGAAGGCGTTG
pro-S257A-RGGCTGTCCCGCTGAGATAGGCGTAGAAGGL253V-FCTACGCCTATGTCAGCGGGACAAGC
pro-S257C-FCTATCTCAGCGGGACATGCATGGCGACGL253V-RCTGACATAGGCGTAGAAGGCGTTG
pro-S257C-RGCATGTCCCGCTGAGATAGGCGTAGAAGGL253C-FCTACGCCTATTGCAGCGGGACAAGC
HN-pro-TSS-RCGCTCGAGGTCGTTGCGCTGTTTTCGL253C-RCTGCAATAGGCGTAGAAGGCGTTG
E187A-FGCCTCCGCAAACGACCGGCCGGAGCL253G-FCTACGCCTATGGCAGCGGGACAAGC
E187A-RGTCGTTTGCGGAGGCGCCCACGGCGL253G-RCTGCCATAGGCGTAGAAGGCGTTG
E187Q-FGCCTCCCAAAACGACCGGCCGGAGCL253S-FCTACGCCTATAGCAGCGGGACAAGC
E187Q-RGTCGTTTTGGGAGGCGCCCACGGCGL253S-RCTGCTATAGGCGTAGAAGGCGTTG
D196S-FGATGAAAGCGAGGCGGACGACCCGL253K-FCTACGCCTATAAGAGCGGGACAAGC
D196S-RGCCTCGCTTTCATCCAGCTCCGGCL253K-RCTCTTATAGGCGTAGAAGGCGTTG
D199A-FGAGGCGGCCGACCCGGACGAGGTAGL253D-FCTACGCCTATGACAGCGGGACAAGC
D199A-RCCGGGTCGGCCGCCTCGTCTTCATCL253D-RCTGTCATAGGCGTAGAAGGCGTTG
D199N-FGAGGCGAACGACCCGGACGAGGTAGM88A-FCAAAGGGGCGGCTCCCGACGCGAAG
D199N-RCCGGGTCGTTCGCCTCGTCTTCATCM88A-RGCCGCCCCTTTGTACTGGCCGTCTG
E203H-FCCGGACCACGTAGCCGCGTTCAGCM88L-FCAAAGGGCTGGCTCCCGACGCGAAG
E203H-RCTACGTGGTCCGGGTCGTCCGCCTCM88L-RGCCAGCCCTTTGTACTGGCCGTCTG
D385A-FCAACGCCTTTTTCCACTACCCCTACGM99A-FCCATTCCGCGCTGGACGAATACGG
D385A-RGTGGAAAAAGGCGTTGCCGTTCAAGGM99A-RGCGCGGAATGGAAGACGAGCTTCG
D385N-FCAACAACTTTTTCCACTACCCCTACGM99L-FCCATTCCCTGCTGGACGAATACGG
D385N-RGTGGAAAAAGTTGTTGCCGTTCAAGGM99L-RGCAGGGAATGGAAGACGAGCTTCG
D393A-FCTACGACGCCGAAGTGGACAACCTCAACM154A-FGGAACATCCGGACGCGACGGCGCTGGTGG
D393A-RCACTTCGGCGTCGTAGGGGTAGTGGM154A-RGCGCCGTCGCGTCCGGATGTTCCCAGAGG
D393N-FCTACGACAACGAAGTGGACAACCTCAACM154L-FCATCCGGACCTGACGGCGCTGGTG
D393N-RCACTTCGTTGTCGTAGGGGTAGTGGM154L-RGTCAGGTCCGGATGTTCCCAGAGAAAC
E394A-FGACGACGCAGTGGACAACCTCAACAACM258A-FGACAAGCGCGGCGACGCCGGTGTTG
E394A-RGTCCACTGCGTCGTCGTAGGGGTAGM258A-RCCGCGCTTGTCCCGCTGAGATAGGC
E394N-FGACGACAATGTGGACAACCTCAACAACM258L-FGACAAGCCTGGCGACGCCGGTGTTG
E394N-RGTCCACATTGTCGTCGTAGGGGTAGM258L-RCCAGGCTTGTCCCGCTGAGATAGGC
E394Q-FGACGACCAAGTGGACAACCTCAACAACM258C-FGACAAGCTGTGCGACGCCGGTGTTG
E394Q-RGTCCACTTGGTCGTCGTAGGGGTAGM258C-RCACAGCTTGTCCCGCTGAGATAGGC
N368D-FCTGGTCAACGACCTGAACCTGCGAGM290A-FGGCGGCGCTCATCAGCGGCGCCGAC
N368D-RCAGGTCGTTGACCAGGGTGCGCTCGM290A-RGAGCGCCGCCTTGATCAATGCC
N370D-FCAACAACCTGGACCTGCGAGTGGTCM290L-FGGCGCTGCTCATCAGCGGCGCCGAC
N370D-RCAGGTCCAGGTTGTTGACCAGGGTGM290L-RGAGCAGCGCCTTGATCAATGCC
D396A-FCGAAGTGGCCAACCTCAACAACGTGM302A-FCGAAGACGCGCGCGTGCAGGGGTTC
D396A-RGAGGTTGGCCACTTCGTCGTCGTAGM302A-RGCGCGTCTTCGTTCAAATCGTCGG
D396N-FCGAAGTGAACAACCTCAACAACGTGM302L-FGAACGAAGACCTACGCGTGCAGG
D396N-RGAGGTTGTTCACTTCGTCGTCGTAGM302L-RCGCGTAGGTCTTCGTTCAAATCG
E402A-FCAACGTGGCGCAGGTCTGGATCAAGM425A-FCGTGCCCGCGGGGCCGCAGCCGTAC
E402A-RGACCTGCGCCACGTTGTTGAGGTTGM425A-RCCGCGGGCACGTTGTAGCCCTCCAC
E402Q-FCAACGTGCAGCAGGTCTGGATCAAGM425L-FCGTGCCCCTGGGGCCGCAGCCGTAC
E402Q-RGACCTGCTGCACGTTGTTGAGGTTGM425L-RCCAGGGGCACGTTGTAGCCCTCCAC
L253M-FCGCCTATATGAGCGGGACAAGCATG
L253M-RGTCCCGCTCATATAGGCGTAGAAGG
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定点突变对一种耐热胶原蛋白水解酶酶学特性的影响
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张魁 * , 朱敏 , 苏芸芸 , 王喜龙 , 郭倩 , 王志科 , 刘秀丽 , 刘海卿
微生物学报 | 研究报告 2026,66(10): 4956-4973
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微生物学报 |研究报告 2026 , 66 (10) : 4956 -4973
定点突变对一种耐热胶原蛋白水解酶酶学特性的影响
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张魁* , 朱敏, 苏芸芸, 王喜龙, 郭倩, 王志科, 刘秀丽, 刘海卿
作者信息
  • 陇东学院 农业与生物工程学院，甘肃省陇东生物资源保护利用与生态修复重点实验室，甘肃 庆阳
通讯作者:
张魁
作者简介:

作者贡献声明

张魁：总体构思、数据管理与初稿写作；朱敏、苏芸芸、王喜龙：调查研究；郭倩、王志科：提供资源；刘秀丽、刘海卿：项目管理。

Effects of site-directed mutagenesis on the enzymatic properties of a thermostable collagenolytic protease
Kui ZHANG* , Min ZHU, Yunyun SU, Xilong WANG, Qian GUO, Zhike WANG, Xiuli LIU, Haiqing LIU
Affiliations
  • Gansu Key Laboratory of Conservation and Utilization of Biological Resources and Ecological Restoration in Longdong, School of Agriculture and Bioengineering, Longdong University, Qingyang, Gansu, China
    • Corresponding Author:
    *E-mail:
出版时间: 2026-10-04 doi: 10.13343/j.cnki.wsxb.20260069
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【目的】 针对嗜热微生物来源的热稳定枯草杆菌蛋白酶样丝氨酸蛋白酶(thermostable subtilisin-like serine protease, TSS)的Ca2+结合位点、Leu253位点和蛋白表面氧化敏感Met位点进行定点突变，筛选抗氧化性、热稳定性及催化活性均提高的突变体，并探究三者之间的内在关联。 【方法】 针对TSS的Ca2+结合位点(Ca1、Ca2和Ca3)、Leu253位点和蛋白表面氧化敏感Met残基开展定点突变，通过检测产生的系列突变体对H2O2的耐受性、相对酶活性和在70 ℃保温3 h后的残余活性，系统研究突变体抗氧化活性、催化活性和热稳定性的变化特征。 【结果】 3个Ca2+结合位点突变后，TSS的催化活性和热稳定性均出现不同程度下降；参照KP-43构建的Leu253系列突变体并未增强TSS的抗氧化性；针对氧化敏感残基改造获得的突变体M302L，在保持热稳定性不变、催化活性提升13%的同时抗氧化性提升61%。研究同时发现，Ca2+可提升TSS的高温催化活性和热稳定性，且该作用具有浓度依赖性；2 mol/L及以上的高盐环境可延长TSS在75 ℃的半衰期，并抑制酶原自加工成熟。 【结论】 在TSS的Leu253和Met突变体中，抗氧化性和催化活性呈正相关，符合“Trade-off”机制。

胶原蛋白水解酶  /  定点突变  /  催化活性  /  热稳定性  /  抗氧化性

[Objective] To conduct site-directed mutagenesis on the Ca2+-binding sites, Leu253, and protein surface oxidation-sensitive Met residues of TSS, a collagenolytic protease derived from thermophiles, thereby screening out the variants with improved antioxidant activity, thermostability, and catalytic activity and revealing the intrinsic connection among them. [Methods] Site-directed mutagenesis was conducted on the Ca2+-binding sites (Ca1, Ca2, and Ca3) and Leu253 of TSS and the oxidation-sensitive Met residues on the protein surface. The derived variants were further evaluated for H2O2 tolerance, relative activity, and residual activity. Changes in antioxidant activity, catalytic activity, and thermostability after mutations were also studied. [Results] Mutations in amino acid residues at the three Ca2+-binding sites led to decreases in catalytic activity and/or thermostability of TSS. The Leu253 series variants constructed by simulating KP-43 did not enhance the antioxidant activity of TSS. Among the variants of oxidation-sensitive residues, M302L showed increases of 13% and 61% in the catalytic and antioxidant activities, respectively, while remaining unchanged thermostability. Ca2+ increased the catalytic activity and thermostability of TSS at high temperatures in a concentration-dependent manner. A high salinity environment with more than 2 mol/L NaCl prolonged the half-life of TSS at 75 ℃ and inhibited the maturation process of TSS proform. [Conclusion] The antioxidant and catalytic activities are positively correlated in the Leu253 and Met variants of TSS, which conforms to the “Trade-off” mechanism.

collagenolytic protease  /  site-directed mutagenesis  /  catalytic activity  /  thermostability  /  antioxidant activity
张魁, 朱敏, 苏芸芸, 王喜龙, 郭倩, 王志科, 刘秀丽, 刘海卿. 定点突变对一种耐热胶原蛋白水解酶酶学特性的影响. 微生物学报, 2026 , 66 (10) : 4956 -4973 . DOI: 10.13343/j.cnki.wsxb.20260069
Kui ZHANG, Min ZHU, Yunyun SU, Xilong WANG, Qian GUO, Zhike WANG, Xiuli LIU, Haiqing LIU. Effects of site-directed mutagenesis on the enzymatic properties of a thermostable collagenolytic protease[J]. Acta Microbiologica Sinica, 2026 , 66 (10) : 4956 -4973 . DOI: 10.13343/j.cnki.wsxb.20260069
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胶原蛋白和胶原蛋白水解短肽作为兼具组织修复、抗纤维化和良好生物相容性等多重生物化学与医学功能的生物材料，具备极高的应用价值与市场发展潜力[1-4]。胶原蛋白水解酶已被广泛应用于食品、医疗、制药、化妆品和纺织工业等领域[5-6]；部分细菌胶原蛋白水解酶作为毒力因子参与人类疾病进程，近几十年来也受到越来越多的关注[7-10]。溶组织梭菌胶原酶(Clostridium histolyticum collagenase, ChC)是首个被鉴定和系统表征的细菌胶原酶，目前已成为新发现胶原蛋白水解酶研究的标准参照酶[11-12]，也是可实现商品化获取的微生物胶原蛋白水解酶[13]。以ChC为代表的中温胶原蛋白水解酶热稳定性较差，仅在生理条件下(37 ℃和中性pH)表现出胶原蛋白水解活性，在高温环境下降解胶原蛋白存在明显局限性，实际应用场景受到诸多限制[14]。与之相反，分离自嗜热微生物的胶原蛋白水解酶可在高温条件下降解胶原蛋白，一方面可提升胶原蛋白降解效率，另一方面也能有效降低反应过程中的微生物污染风险[15-17]，因此热稳定胶原蛋白水解酶在皮革生产、食品加工、化妆品加工、纺织品处理和医药等领域具备更为重要的科学研究价值与应用前景。
热稳定枯草杆菌蛋白酶样丝氨酸蛋白酶(thermostable subtilisin-like serine protease, TSS)是一种新发现的嗜热耐盐枯草杆菌蛋白酶(subtilisin)样胶原蛋白水解酶，兼具优良的耐盐碱特性，在60-80 ℃均表现出较高的胶原蛋白水解活性[18]。TSS的低明胶水解活性、“马鞍形”活性曲线以及对不同溶解状态胶原蛋白α1和α2链的降解位点特异性等特征，与其他已报道的S8家族细菌胶原蛋白水解酶存在明显差异[18-19]，同时可耐受多种极端环境条件，因此TSS这类热稳定胶原蛋白水解酶在胶原蛋白废弃物处理和皮革工业中的应用前景十分广阔[15]。胶原蛋白水解酶TSS来源于短芽孢杆菌属(Brevibacillus sp.) WF146[20]，该菌株最适生长温度约为58 ℃，可在以鱼鳞胶原蛋白为唯一碳氮源的培养基中正常生长。TSS与其他S8家族来源的胶原蛋白水解酶具有较高的结构域组成相似性，前期实验结果也证实其在高温条件下具备胶原蛋白降解能力。生物信息学分析显示，TSS的成熟酶(mature enzyme, mTSS)含有3个预测的Ca2+结合位点，且Ca2+和NaCl均对其高温催化活性和热稳定性具有调控作用。本研究拟针对其Ca2+结合位点、Leu253位点和蛋白表面氧化敏感Met位点开展定点突变，以期筛选获得抗氧化性、热稳定性及胶原蛋白降解活性均提升的突变体，并探究TSS中三者之间的内在关联，为开发新型胶原蛋白类固体废弃物降解用蛋白酶提供理论依据。
1 材料与方法
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1.1 材料
1.1.1 菌株
短芽孢杆菌属(Brevibacillus sp.) WF146 (CCTCC AB209297)于55 ℃在LB培养基中培养，用于基因组DNA提取。大肠杆菌(Escherichia coli) DH5α和大肠杆菌BL21(DE3) 于37 ℃在LB培养基中培养，分别用于基因克隆与表达，培养过程中根据实验需求添加终浓度30 μg/mL的卡那霉素。
1.1.2 主要试剂和仪器
限制性内切酶(Nde I和Xho I)，ThermoFisher Scientific公司；2×T8 High-Fidelity Master Mix，北京擎科生物科技股份有限公司；T4 DNA连接酶，北京全式金生物技术有限公司；Azocasein和ChC，Sigma-Aldrich (Shanghai) Trading有限公司；低分子量蛋白质Marker，生工生物工程(上海)股份有限公司；质粒pET-26b、抗His标签单克隆抗体，Novagen公司；山羊抗小鼠IgG二抗，亚科因(武汉)生物技术有限公司；细菌基因组DNA提取试剂盒，天根生化科技(北京)有限公司；DEAE-SepharoseTM Fast Flow、Sepharose Fast Flow层析柱，GE Healthcare Bio-Sciences公司。
PCR仪、台式高速离心机，Eppendorf公司；双层空气恒温振荡器，哈尔滨市东联电子技术开发有限公司；水平电泳槽，北京六一生物科技有限公司；紫外可见分光光度计，上海菁华科技仪器有限公司；超声波细胞破碎仪，宁波新芝生物科技股份有限公司。
1.2 质粒构建与定点突变
参照细菌基因组DNA提取试剂盒说明书提取Brevibacillus sp. WF146的基因组DNA，采用表1所列引物分别扩增携带自身信号肽的TSS前体(precursor-TSS和pre-TSS)基因、不含自身信号肽的TSS酶原(proform-TSS和pro-TSS)基因，将扩增片段插入pET-26b载体的Nde I-Xho I酶切位点，构建重组质粒pET26b-pre-TSS和pET26b-pro-TSS。PCR反应体系(25.0 μL)：2×T8 High-Fidelity Master Mix 12.5 µL，上、下游引物(10 µmol/L)各1.0 µL，DNA模板0.5 µL，ddH2O 10.0 µL。PCR反应条件：95 ℃预变性5 min；95 ℃变性30 s，58 ℃退火30 s，72 ℃延伸2 min，共30个循环；72 ℃终延伸5 min。以pET26b-pro-TSS为模板，扩增编码TSS的N端前肽缺失蛋白(delta N-terminal propeptide, ΔN)及N端前肽(N-terminal propeptide, N)结构域的DNA片段，插入pET-26b载体构建对应重组蛋白的表达质粒。以pET26b-pro-TSS为模板，使用表1所列诱变引物，采用QuikChange site-directed mutagenesis (SDM)方法[21]构建pro-TSS活性位点突变体(pro-TSS-S257A/C和ΔN/S257A)、Ca2+结合位点突变体、Leu253和氧化敏感Met残基突变体的表达质粒。所有重组质粒均以pET-26b为载体，在大肠杆菌BL21(DE3)中实现重组蛋白异源表达。
1.3 蛋白表达与纯化
将携带不同重组质粒的大肠杆菌BL21(DE3)细胞于37 ℃、185 r/min培养90 min，至菌液OD600达到约0.7时加入终浓度0.4 mmol/L的IPTG诱导重组蛋白表达，随后在30 ℃下继续培养4 h。培养液于4 ℃、8 000 r/min离心5 min后收集菌体，将菌体重悬于缓冲液(50.0 mmol/L Tris-HCl, 10.0 mmol/L CaCl2, pH 7.5)中，冰浴条件下采用直径3 mm的变幅杆进行超声波细胞破碎，探头浸入液面下5-10 mm，超声功率设置为100 W，采用脉冲模式工作：超声1 s、间歇4 s，总超声时长5 min。破碎完成后的细胞裂解液于4 ℃、12 000 r/min离心10 min，分别收集可溶性上清组分和不可溶性细胞沉淀组分。收集的上清液采用(NH4)2SO4进行50%-80%饱和度的分段盐析处理，随后采用经相同缓冲液平衡的DEAE-SepharoseTM Fast Flow层析柱进行离子交换层析，纯化成熟TSS (mTSS)。其余融合His标签的重组蛋白，采用经相同缓冲液平衡的Ni2+螯合Sepharose Fast Flow层析柱(可选择添加3.0 mol/L NaCl)，通过亲和层析法从可溶性上清组分中纯化获得。
1.4 蛋白酶生物信息学分析
1.4.1 两种方法预测TSS三级结构后的结构叠加图
通过在线工具RoseTTAFold (https://robetta.bakerlab.org/submit.php)预测得到的TSS三级结构以红色显示[22]；以KP-43晶体结构(PDB登录号1WMF)为模板进行同源模建得到的TSS三级结构以绿色显示[18,23]，使用Swiss-PdbViewer软件对2种方法预测的TSS三级结构进行叠加比对，TSS催化三联体(D-H-S)的侧链以黑色标识。
1.4.2 TSS与KP-43三级结构叠加图
使用SpdbViewer软件对TSS和KP-43的三级结构进行叠加比对，TSS与KP-43的催化结构域(catalytic domain, CD)与β-果冻卷结构域(β-jelly roll domain, βJR)分别以浅蓝色与紫色显示，KP-43的对应结构域分别以绿色与灰色显示，TSS催化三联体(D-H-S)的侧链以黑色标识。
1.5 蛋白酶活性测定
1.5.1 蛋白酶比活力测定
600 µL反应体系包含300 µL浓度为5 μg/mL的酶液和终质量分数0.50%的Azocasein底物，于60 ℃反应30 min后，将反应体系转移至冰浴中冷却5 min终止反应，随后加入等体积的40%三氯乙酸(trichloroacetic acid, TCA)，室温静置15 min沉淀未反应底物，12 000 r/min离心10 min后取上清液测定OD335值，计算蛋白酶样品的比活力。
酶活力单位(U/mg)定义为：在Buffer A (50.0 mmol/L Gly-NaOH, pH 9.0, 10.0 mmol/L CaCl2)中，1 min内使反应体系经12 000 r/min离心10 min后的上清液在OD335处的吸光值升高0.01所需要的酶量，即为1个酶活力单位。
1.5.2 蛋白酶相对活性测定
各突变体酶液在Buffer A中测定各自蛋白酶比活力，以野生型mTSS的比活力为100%，计算各突变体的相对酶活性。
1.6 酶学性质测定
1.6.1 蛋白酶Ca2+ 浓度依赖性测定
在50.0 mmol/L Tris-HCl (pH 7.5)缓冲体系中，以终质量分数0.25%的Azocasein为底物，测定浓度为5.0 μg/mL的mTSS在2.0 mmol/L EDTA或0.0-20.0 mmol/L梯度浓度Ca2+条件下的酶活性，以10.0 mmol/L Ca2+条件下测得的酶活性为100%，计算不同条件下的相对酶活性，所有数据均为3次平行实验结果的平均值。
1.6.2 不同盐浓度下蛋白酶热失活曲线测定
将浓度为5.0 μg/mL的mTSS溶于添加不同浓度NaCl的Buffer A中，于75 ℃水浴条件下进行保温处理，在预设时间间隔点取样，取出后立即置于冰水混合物中冷却终止热失活进程。待最后一组样品保温完成后，统一取出所有冷却样品，以终质量分数0.25%的Azocasein为底物测定残余酶活性，以未进行75 ℃保温处理的初始酶活性为100%，计算不同盐浓度条件下的残余酶活性，采用Origin2021软件绘制热失活曲线，所有数据均为3次平行实验结果的平均值。
1.6.3 不同温度下TSS对鱼鳞胶原蛋白(fish scale collagen, FSC)的降解效率测定
称取60 mg FSC，采用Buffer A润洗3次去除杂质后，加入终浓度10 mg/L的mTSS，置于3 mL Buffer A反应体系中，在30-70 ℃梯度温度条件下振荡反应30 min。反应结束后12 000 r/min离心10 min，收集上清液测定OD280值。另称取20 mg FSC，加入过量ChC胶原酶完全降解，测定降解产物的OD280值，折算得到60 mg FSC完全降解后的理论OD280值，将该值设为100%，计算不同温度下mTSS对FSC的降解效率。
1.6.4 Ca2+ 对TSS热稳定性及加工成熟的影响
将含pro-TSS表达产物的细胞可溶性组分，在添加3.0 mol/L NaCl的条件下进行镍柱亲和层析，洗脱所得样品经透析处理后，分别置于不含(-)或添加(+) 10.0 mmol/L CaCl2的50.0 mmol/L Tris-HCl (pH 7.5)缓冲体系中，于60 ℃条件下保温1-4 h，取样后进行SDS-PAGE检测。剩余保温后的样品同时以终质量分数0.25%的Azocasein为底物测定酶活性，以保温1 h条件下测得的酶活性为100%，计算不同条件下的相对酶活性。
1.6.5 盐浓度对TSS热稳定性及加工成熟的影响
将含pro-TSS表达产物的细胞可溶性组分，在添加3.0 mol/L NaCl的条件下进行镍柱亲和层析，洗脱所得样品用含不同浓度NaCl (0.0-5.0 mol/L NaCl)的50.0 mmol/L Tris-HCl (pH 7.5, 10.0 mmol/L CaCl2)缓冲液稀释10倍，于60 ℃条件下保温1 h后取样进行SDS-PAGE检测。剩余保温后的样品同时以终质量分数0.25%的Azocasein为底物测定酶活性，以2.0 mol/L NaCl条件下测得的酶活性为100%，计算不同盐浓度条件下的相对酶活性。
1.7 蛋白酶热稳定性测定方法
将浓度为5.0 μg/mL的重组蛋白溶于Buffer A中，于70 ℃条件下保温3 h，随后以终质量分数0.25%的Azocasein为底物测定残余酶活性，以未进行70 ℃保温处理的初始样品活性为100%，计算相对酶活性，所有数据均为3次平行实验结果的平均值。
1.8 蛋白酶抗氧化性测定方法
将浓度为5.0 μg/mL的重组蛋白溶于Buffer A中，添加终浓度50.0 mmol/L的H2O2，于30 ℃水浴条件下保温30 min，随后立即转移至冰水混合物中冷却终止氧化反应，以终质量分数0.25%的Azocasein为底物测定残余酶活性，以未进行H2O2处理的初始样品活性为100%，计算相对酶活性，所有数据均为3次平行实验结果的平均值。
1.9 SDS-PAGE和免疫印迹分析
采用含12%聚丙烯酰胺的Tris-Gly缓冲体系进行SDS-PAGE分析。为避免样品制备与电泳过程中蛋白酶发生自降解，蛋白样品预先用终质量分数20%的TCA沉淀，经丙酮充分洗涤后再进行SDS-PAGE上样分析。免疫印迹检测依次使用稀释比例1:10 000的抗His标签单克隆抗体、稀释比例1:50 000的山羊抗小鼠IgG二抗孵育显色。
1.10 明胶印迹活性分析
配制含终质量分数0.50%明胶的聚丙烯酰胺凝胶，方法如下：2%明胶储存液1.25 mL、30%丙烯酰胺储存液2 mL，同时补加3.30 μL的TEMED和50 μL的10% APS储存液，其余用ddH2O补足至5 mL后混匀制胶。将待检测蛋白样品制样后进行SDS-PAGE电泳。电泳结束后切下分离胶，用ddH2O漂洗5 min，随后用添加0.25% Triton X-100的Buffer A漂洗3次，每次漂洗时长20 min，最后用Buffer A反复润洗数次，彻底去除残留的Triton X-100。在保鲜膜上平铺预先用Buffer A充分浸泡的3层滤纸，依次放置凝胶、另一层分离胶与3层滤纸，组装完成“三明治”结构后用保鲜膜完全包裹，置于60 ℃条件下保温反应1 h。反应结束后取出凝胶，按常规考马斯亮蓝染色-脱色流程处理后扫描保存图像。
2 结果与分析
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2.1 胶原蛋白水解酶TSS的生物信息学分析
TSS前体(pre-TSS)由信号肽(signal peptide, S)、N端前肽(N)、催化结构域(CD)、β-果冻卷结构域(βJR)和肽酶前体C末端结构域(prepeptidase C-terminal domain, PPC)组成(图1A)。TSS成熟酶(mTSS)与一株抗氧化碱性蛋白酶KP-43具有较高的氨基酸序列一致性，其中催化结构域相似度为49%，βJR结构域相似度为44%。结合TSS与其他S8家族蛋白酶Ca2+结合位点的氨基酸多序列比对结果(图1B)及KP-43的晶体结构，可预测TSS前体含有3个Ca2+结合位点(Ca1、Ca2和Ca3)，其中Ca1位于催化功能区，Ca2和Ca3位于βJR结构域。
分别采用基于人工智能的蛋白质结构预测方法，包括基于Rosetta框架的转换、模板与比对折叠模型(rosetta transform, template, and align fold, RoseTTAFold)与同源模建方法(SWISS-MODEL)预测TSS的三级结构，结果显示2种方法得到的模型在分子整体结构与结构域分布特征上均呈现良好的匹配度(图1C)。鉴于TSS与KP-43具有较高的氨基酸序列一致性，本研究进一步使用SpdbViewer软件对预测得到的TSS三级结构与KP-43晶体结构进行叠加比对，结果显示二者在三级结构层面同样具有较高的匹配度(图1D)。值得注意的是，TSS与KP-43的催化结构域相似度较高，βJR结构域则存在一定的结构差异，这与二者氨基酸序列一致性的差异特征完全吻合。
2.2 TSS的酶学性质
在添加2.0 mmol/L金属螯合剂EDTA的反应体系中，mTSS的催化活性极低，表明TSS的蛋白酶催化活性依赖Ca2+参与，这与2.1节中TSS含有3个预测Ca2+结合位点的结果相互印证。随着体系中Ca2+浓度逐步升高，mTSS的活性同步提升，在10.0-20.0 mmol/L Ca2+条件下达到活性峰值(图2A)。上述结果证实该酶是一种Ca2+浓度依赖性蛋白酶。值得注意的是，当缓冲液中不额外添加Ca2+时，测得的相对酶活性较含2.0 mmol/L EDTA的对照组高出约35%，推测是由于TSS在大肠杆菌中异源表达过程中，其分子上的多个强Ca2+结合位点结合了部分来源于培养基的Ca2+，这些结合态Ca2+在后续的培养与纯化过程中仍稳定保留在酶分子内部，只有加入EDTA才能将其完全螯合去除。
本研究进一步在75 ℃、pH 9.0条件下分析不同盐浓度对mTSS热失活进程的影响(图2B)。在0.0 mol/L NaCl条件下，mTSS保温4 h后残余酶活约为20%；随着NaCl浓度升高至1.0-4.0 mol/L区间，mTSS保温4 h后的残余活性明显提升，在4.0 mol/L NaCl条件下达到最高值约80%，说明盐浓度升高可显著增强TSS的热稳定性。综上可知，TSS是一种Ca2+浓度依赖性蛋白酶，其催化活性与热稳定性的维持均需要Ca2+的参与。
前期研究发现TSS对大分子底物胶原蛋白(collagen)、偶氮胶原蛋白(azo dye-impregnated collagen, Azocoll)与小分子底物N-琥珀酰-丙氨酸-丙氨酸-脯氨酸-苯丙氨酸-对硝基苯胺(N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide, suc-AAPF-pNA)呈现出差异化的降解行为[18]，本研究进一步使用鱼鳞胶原蛋白FSC模拟其对胶原蛋白类固体废弃物的降解效果(图2C)。在30-70 ℃区间内，TSS对FSC的降解效率呈非线性变化特征，整体随温度升高逐步提升，这也进一步印证了TSS属于嗜热型胶原蛋白水解酶的判定。因此，TSS具备作为胶原蛋白类固体废弃物降解用蛋白酶的潜力，拥有极高的研究与应用价值。
2.3 Ca2+ 和盐浓度对TSS高温活性及加工成熟的影响
将pro-TSS表达产物的细胞可溶性组分在无额外Ca2+的环境中于60 ℃保温1 h后，样品中的蛋白发生大量降解；而补加10.0 mmol/L Ca2+的pro-TSS对应组分在相同条件下保温1 h后未发生明显降解，且全部完成酶原自加工成熟过程(图3A)；延长保温时间至4 h，蛋白条带仍未发生明显变化。这表明Ca2+可显著提升TSS的热稳定性，同时可促进其酶原(pro-TSS)或中间体(intermediate, I)的加工成熟。
本研究进一步探究了在10.0 mmol/L Ca2+存在的条件下，盐浓度对TSS加工成熟过程的影响(图3B)。pro-TSS表达产物的细胞可溶性组分在0.0-2.0 mol/L NaCl环境中于60 ℃保温1 h后，全部或大部分完成自加工成熟，催化活性在2.0 mol/L NaCl条件下达到峰值；未进行60 ℃保温处理的样品，以及在NaCl浓度高于3.0 mol/L的环境中保温的样品，均未检测到明显的蛋白酶活性；同时在2.0 mol/L NaCl处理组的样品中可检测到中间体(I)与N端前肽(N)的特征蛋白条带。上述结果表明：(1) pro-TSS的自加工成熟过程需要在含Ca2+的高温(如60 ℃)环境中进行，且Ca2+可显著加速该自加工成熟进程；(2)高浓度NaCl (3.0-5.0 mol/L)可抑制pro-TSS的自加工成熟；(3)在2.0 mol/L NaCl/10.0 mmol/L Ca2+的条件下，Ca2+的促进作用与高盐的抑制作用达到动态平衡，酶活性水平达到最高点，同时自加工产生的中间体与被切除的N端前肽(经历部分降解)均可被检测到。
值得注意的是，I、M和N在SDS-PAGE检测胶中均呈现为双条带特征(图3A、3B)，为明确该现象的产生机制，本研究开展了针对性验证实验。已有研究表明，将枯草杆菌蛋白酶类(subtilases)的催化Ser残基突变为Ala后，可使蛋白酶完全丧失催化活性；而将催化Ser残基突变为Cys后，可大幅降低蛋白酶活力但无法完全抑制其自加工活性，这类突变体在自加工切除N端前肽后难以进一步降解游离的N端前肽，因此无法完成完全成熟过程[24-25]，据此可通过构建SA/SC突变体解析subtilases的自加工成熟机制。本研究分别构建了TSS的SA/SC突变体(图3C)，其中pro-S257A在SDS-PAGE与免疫印迹分析中仅显示1条相对分子质量为110 kDa的条带，对应TSS的酶原形式；而pro-S257C样品中额外出现了78 kDa与22 kDa的特征条带，推测是由于TSS酶原发生自加工，切除22 kDa的N端前肽后生成78 kDa的中间体所致；由于pro-S257C的His-tag融合在C端，因此被切除的N端前肽无法在免疫印迹分析中被检测到(图3C)。
图3B中的样品a对应TSS的成熟酶(M)，由于TSS自加工成熟过程需要先后切除N端前肽与C端PPC结构域[18]，因此M无法在免疫印迹分析中被检测到(图3C)。本研究利用TSS具备明胶水解活性，且经/不经TCA预处理后在SDS-PAGE中的迁移率存在差异的特性，设计开展明胶印迹活性分析实验(图3D)，结果显示MTCA与MNon在SDS-PAGE和明胶印迹活性分析中均呈现双条带特征，说明MNon的2条条带均为具有催化活性的成熟酶(M)形式，MTCA的2条条带也符合该特征，同理可解释图3A中M的双条带现象。本研究推测，mTSS在较低浓度条件下(图3D中样品a经5倍稀释)会呈现双条带，而在较高浓度条件下(图3B中样品a) 2条条带发生重叠而显示为单一条带，图3A中M因蛋白浓度较低呈现双条带的结果，进一步印证了上述推测的合理性。
图3B中样品b的I组分在SDS-PAGE中呈现双条带，在免疫印迹分析中呈现多条带(图3C)，这表明pro-TSS表达产物的细胞可溶性组分即使在高盐的抑制作用下，仍发生了部分加工成熟，生成了多个不同的中间体，本研究将样品b中与pro-S257C产生的78 kDa产物相对分子质量一致的条带定义为中间体(I)。由于pro-TSS的His-tag融合在C端，因此SDS-PAGE中可检测到的2条N端前肽条带无法在免疫印迹分析中被识别(图3C)。本研究进一步构建了N端融合His-tag的HN-pro-TSS，由于其发生了部分加工成熟，在免疫印迹分析中同样呈现多条带特征，本研究将样品b中与pro-S257C产生的22 kDa产物相对分子质量一致的条带定义为N端前肽(N)。
综上所述，78 kDa产物为TSS的中间体(I)，58 kDa产物为其成熟酶(M)，22 kDa产物为酶原(pro-TSS)自加工切除下的N端前肽(N) (图3A-3C)。
2.4 Ca2+ 结合位点对TSS高温活性及热稳定性的影响
2.4.1 TSS相关Ca2+ 结合位点定点突变体的构建
结合TSS与KP-43、Myroicolsin、Tk-SP和Pyrolysin的Ca2+结合位点氨基酸多序列比对结果(图1B)及KP-43的晶体结构，可预测TSS共含有3个Ca2+结合位点(Ca1、Ca2和Ca3)，其中Ca1位于催化结构域，Ca2和Ca3位于βJR结构域。本研究对每个Ca2+结合位点内的配位氨基酸残基进行定点突变[18]，构建了系列重组突变体(图1A、1B)。其中针对Ca1位点构建了6个突变体(E187A、E187Q、D196S、D199A、D199N和E203H)；针对Ca2位点构建了7个突变体(D385A、D385N、D393A、D393N、E394A、E394N和E394Q)；针对Ca3位点构建了6个突变体(N368D、N370D、D396A、D396N、E402A和E402Q)。将上述突变体分别在大肠杆菌中异源表达，纯化获得各重组蛋白样品后开展系统的酶学性质检测。
2.4.2 Ca1位点的作用
为明确Ca1位点对TSS热稳定性的调控作用，将该位点的系列突变体在70 ℃条件下保温3 h后测定残余酶活性，并与野生型mTSS进行平行对比。结果显示，野生型mTSS经该热处理后仍可保留80%以上的残余活性；Ca1位点各突变体(D196S、D199N和E203H)的残余活性均低于野生型mTSS，其中E187A、E187Q和D199A在70 ℃保温3 h后已完全丧失催化活性(图4)。
本研究进一步测定了上述突变体的相对酶活性，结果显示Ca1位点不同突变体的催化活性相较于mTSS呈现出差异化的变化趋势(图4)。例如，E187A的催化活性仅为mTSS的45%左右，且在70 ℃保温3 h后完全失活，推测该突变体的分子结构稳定性急剧下降，直接干扰了其正常催化功能的发挥。D196S的热稳定性明显下降，但其相对酶活性较野生型提升约30%，该现象可通过酶活性与稳定性的“代偿(Trade-off)”机制进行解释：酶分子的结构刚性与柔性呈负相关，结构刚性下降会伴随分子柔性提升，反之亦然，在酶学性质层面即表现为热稳定性与催化活性此消彼长的关联特征[26]。
对于其余突变体(E187Q、D199A、D199N和E203H)，其热稳定性下降的同时催化活性较mTSS出现小幅降低，该结果同样可通过“Trade-off”机制解释：这类突变体分子柔性提升带来的活性增益，不足以抵消结构稳定性下降对酶催化功能造成的负面影响，因此最终酶活性未超过野生型水平。上述结果表明，Ca1位点对TSS的高温催化活性与热稳定性均具有关键调控作用。
2.4.3 Ca2和Ca3位点的作用
与Ca1位点不同，Ca2和Ca3位点均位于TSS的βJR结构域内(图1A)。Ca2与Ca3位点所有突变体的热稳定性和催化活性均低于野生型mTSS (图4)。该结果表明，Ca2和Ca3位点对TSS的热稳定性与高温催化活性均具有重要影响，其作用机制可能是通过调控βJR结构域的自身稳定性，或调控βJR结构域与催化功能区的相互作用，最终影响酶分子的整体结构稳定性与催化功能。
TSS的Ca3位点在多种同源酶的βJR结构域中高度保守，例如KP-43[23]、Myroicolsin[27-28]、Tk-SP[29]以及Pyrolysin[30]的对应PPC结构域中均存在该保守Ca2+结合位点(图1B)。值得注意的是，其他同源酶中该位点保守存在的2个Asp残基，在TSS的Ca3位点中被替换为Asn残基(Asn368和Asn370) (图1B)。将TSS中的Asn368与Asn370分别突变为Asp残基后，获得的突变体N368D和N370D的热稳定性较突变前进一步下降(图4)，这说明在Ca3位点额外引入带负电荷的羧基基团会破坏TSS的分子结构稳定性，进而干扰酶分子的整体构象与催化功能。曾静等[31-32]的研究发现，来源于Pyrococcus furiosus的Pyrolysin，其PPC结构域中对应Ca2+结合位点的Asp818和Asp820残基替换为Asn残基后，酶的热稳定性显著提升，该位点恰好对应TSS Ca3位点的Asn368和Asn370残基，其机制可能是Asp突变为Asn后消除了该Ca2+结合位点内不合理的静电斥力。由此推测，TSS通过引入不带电荷的Asn替代Asp作为Ca3位点的配位残基，消除了位点内不适宜的静电斥力，最终实现酶热稳定性的提升，这可能是TSS适应高温环境的一种特殊进化策略。
2.5 TSS系列突变体抗氧化性与高温催化活性及热稳定性的关系
TSS与抗氧化蛋白酶KP-43具有较高的氨基酸序列一致性，参考KP-43已报道的抗氧化分子机制[23,33]，本研究针对TSS的特定位点Leu253及蛋白分子表面易被氧化的Met残基构建系列突变体，通过测定各突变体的酶活力相关参数，系统探究TSS抗氧化性与催化活性、热稳定性之间的内在关联。
已有研究表明，subtilases家族蛋白酶的催化残基Ser通常紧邻一个Met残基，该Met残基被氧化后会阻碍水解反应四面体中间体的形成，进而抑制酶的催化活性；KP-43的催化残基Ser255紧邻Met256，在其上游不远处还存在另一个Met251。Nonaka等[23]认为在氧化胁迫条件下，KP-43中的Met251会优先被氧化，从而延缓Met256的氧化进程，最终提升KP-43的整体抗氧化能力；同时氧化型KP-43中Met256上的亚砜氧原子与氧离子洞周围水分子的距离较远，进一步强化了KP-43的抗氧化特性。在TSS中，催化残基Ser257紧邻Met258，而与KP-43的Met251相对应的位点在TSS中为Leu253[18]。基于该序列特征，本研究构建了TSS针对Leu253位点的系列突变体，同时对TSS分子表面全部7个Met残基进行逐一突变构建系列突变体，以期筛选出抗氧化性能提升的TSS突变体。
结果显示(图5A、5B)，在抗氧化性发生改变的Leu253系列突变体中，L253M和L253C的催化活性和热稳定性与野生型mTSS相比无明显变化；L253A、L253I、L253G和L253S的两项酶学性质均出现不同程度的下降；L253V的热稳定性下降幅度远大于催化活性的下降幅度；L253D的催化活性显著降低，同时热稳定性仅出现小幅下降；L253K的抗氧化性与热稳定性均有所提升，但催化活性仅为野生型的15%。上述结果表明，参考KP-43的抗氧化机制将TSS催化Ser257上游的Leu253突变为其他氨基酸残基时，受突变残基本身的电荷特性与空间位阻效应影响，突变体酶分子刚性提升带来的稳定性增益不足以抵消分子柔性下降对酶催化功能产生的负面效应。综上，在Leu253系列突变体中，TSS的抗氧化性与酶分子稳定性存在关联。
通过替换酶分子表面氧化敏感残基Met构建的系列突变体中(图5C、5D)，M99A和M258A的抗氧化能力较mTSS提升约3倍，热稳定性仅小幅下降，但二者的催化活性仅为野生型的10%左右。类似地，M88L、M99L、M154A、M258L/C和M302A也呈现出同样的性质特征。这表明在TSS蛋白表面的Met位点引入突变会对酶分子的整体构象产生较大影响，推测其抗氧化能力的提升是由于该位点被氧化后在一定程度上维持了酶分子结构的完整性与稳定性。因此在Met系列突变体中，TSS的抗氧化能力同样与酶分子稳定性密切相关。其中突变体M302L在热稳定性保持不变、催化活性提升13%的同时，抗氧化能力较mTSS提高61%，说明该突变体的酶分子结构未发生破坏性改变，同时实现了抗氧化性能的提升。由此可见，对TSS特定位点的氧化敏感残基Met302进行替换，可在不降低热稳定性的前提下有效提升该酶的抗氧化能力。
综上所述，蛋白结构柔性是酶发挥催化活性的基础，蛋白结构刚性是酶维持热稳定性的核心，热稳定性与催化活性之间完全符合“Trade-off”代偿机制，因此在Leu253和Met两类抗氧化突变体中，TSS的抗氧化性与催化活性也基本符合该“Trade-off”机制。
3 讨论与结论
收起
TSS的一级结构分析结果显示，其酸性氨基酸残基(Asp和Glu)占比高达16.4%，直接导致其等电点(pI)低至4.38。低pI的mTSS中，大量酸性氨基酸残基分布于酶分子表面，使得分子大部分区域带负电荷，活性位点周边区域的负电荷分布尤为密集；同时碱性氨基酸残基也集中分布在分子表面，在局部形成正电荷富集区域。经计算，mTSS分子表面共形成5个键长≤4 Å的盐桥和19个键长为4-8 Å的长距离离子对，其中21个离子对分布于酶分子表面，剩余3个位于催化功能区与βJR结构域的夹缝区域[18]。已有大量研究证实，分子表面的离子对与离子网络是调控蛋白酶热稳定性的关键因素[34-36]，极端嗜热蛋白酶的晶体结构解析结果也进一步表明，离子对在维持极端嗜热酶的结构稳定性中发挥重要作用[37-38]。TSS分子表面密集的正负电荷区域与丰富的离子对网络，共同赋予了该蛋白酶较高的结构刚性，使其能够有效适应高温环境。
TSS同时展现出优异的耐盐特性，随着NaCl浓度逐步升高至4.0 mol/L，其热稳定性持续提升，这种高耐盐表型在其他嗜热微生物来源的胶原蛋白水解酶中尚未见报道[39-44]。Ding等[45]研究发现，高浓度NaCl可提升来源于Thermoactinomyces vulgaris CDF枯草杆菌蛋白酶样蛋白酶Als (QBK13760)的热稳定性。已有研究证实，分子表面的酸性氨基酸残基可在蛋白外围形成致密的水化膜，这是嗜盐蛋白酶在高盐环境下维持结构稳定性的核心机制；但嗜盐蛋白酶在低盐条件下会因分子表面过多负电荷基团间的静电斥力引发构象破坏，最终导致结构失活[46-49]。由此可见，高比例的酸性氨基酸残基赋予TSS更多形成Ca2+结合位点的潜在可能，同时大幅增加了分子内离子对与离子网络的数量。在该层面上，TSS与Als均采用了类似嗜盐蛋白酶的进化策略，通过提升酸性氨基酸残基占比来适应高盐环境[18,45-46,50]，同时同步实现了酶热稳定性的提升。
TSS的3个Ca2+结合位点(Ca1、Ca2和Ca3)对该酶的成熟加工、催化活性维持与结构稳定性均具有关键调控作用[18]。参与形成Ca2+结合位点的氨基酸残基发生改变后，会影响酶分子对Ca2+的结合能力，同时破坏位点周边的局部静电平衡，进而干扰酶分子的结构刚性，以及催化功能区与βJR结构域之间的相互作用，最终表现为酶活性或热稳定性的改变。绝大多数Ca2+结合位点定点突变体的活性与稳定性之间的关联特征均可通过“Trade-off”代偿机制得到合理解释[26,51]，即催化活性的提升通常伴随热稳定性的下降，反之热稳定性的提升也往往以催化活性的降低为代价。
与TSS具有较高氨基酸序列一致性、进化距离较近的蛋白酶KP-43具备优异的抗氧化性能[18,23,52]，其抗氧化机制已被明确：在氧化胁迫条件下，Met251优先发生氧化，从而延缓了Met256的氧化进程，最终实现KP-43整体抗氧化能力的提升[23]。本研究参考KP-43的抗氧化机制构建的TSS Leu253位点系列突变体，并未获得抗氧化能力提升的突变体，其原因是该位点的突变直接干扰了酶分子的结构刚性与催化柔性的平衡。本研究进一步将mTSS分子表面7个易被氧化的Met残基分别突变为Ala、Leu和Cys，构建了系列突变体，酶学性质检测结果显示，与其他突变体相比，突变体M302L的抗氧化性和催化活性较mTSS分别提高61%和13%，且热稳定性与野生型完全相当。这说明对TSS特定位点的氧化敏感残基Met302进行替换，可在完全不降低酶热稳定性的前提下，有效提升其抗氧化性能。因此在TSS的两类抗氧化突变体(Leu253系列与Met系列突变体)中，其抗氧化性与催化活性之间的关联特征也基本符合“Trade-off”代偿机制。
综上所述，本研究针对TSS的多个关键功能位点开展系统的定点突变研究，得到以下核心结论：(1)验证了TSS中预测的3个Ca2+结合位点(Ca1、Ca2和Ca3)的功能有效性，对其中任意配位氨基酸残基进行突变，均会导致TSS的热稳定性与催化活性降低；(2)在保持热稳定性不变的前提下，筛选获得了催化活性提升13%、抗氧化能力提升61%的优良突变体M302L；(3)酶学性质表征结果证实，TSS是一种Ca2+依赖性蛋白酶，NaCl和Ca2+均可提升该酶的热抵抗能力并促进其催化活性，但二者在TSS酶原的自加工成熟过程中发挥完全相反的调控作用：Ca2+起促进作用，高浓度NaCl起抑制作用。系统解析TSS抗氧化性、高温催化活性与热稳定性之间的内在关联，对推动该酶在高温高盐、强氧化等极端环境下实现胶原蛋白类固体废弃物的降解与高值化循环利用具有重要意义，同时可为胶原蛋白活性短肽的绿色生产、海洋来源鱼鳞胶原蛋白固体废弃物的无害化处理等应用领域提供直接的实验参考与理论支撑。
基金
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  • 甘肃省自然科学基金(23JRRM742)
  • 陇东学院博士基金(XYBYZK2215)
  • 陇东学院博士基金(XYBYZK2216)
  • 陇东学院博士基金(XYBYZK2217)
  • 陇东学院博士基金(XYBYZK2229)
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doi: 10.13343/j.cnki.wsxb.20260069
  • 接收时间：2026-01-24
  • 首发时间：2026-10-13
  • 出版时间：2026-10-04
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  • 收稿日期：2026-01-24
  • 录用日期：2026-04-19
基金
Natural Science Foundation of Gansu Province(23JRRM742)
甘肃省自然科学基金(23JRRM742)
Doctoral Foundation of Longdong University(XYBYZK2215)
陇东学院博士基金(XYBYZK2215)
陇东学院博士基金(XYBYZK2216)
陇东学院博士基金(XYBYZK2217)
陇东学院博士基金(XYBYZK2229)
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    陇东学院 农业与生物工程学院，甘肃省陇东生物资源保护利用与生态修复重点实验室，甘肃 庆阳

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species		 占总种数比例
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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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