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Article(id=1226956554696569675, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250178, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1741104000000, receivedDateStr=2025-03-05, revisedDate=null, revisedDateStr=null, acceptedDate=1746460800000, acceptedDateStr=2025-05-06, onlineDate=1770458838389, onlineDateStr=2026-02-07, pubDate=1756915200000, pubDateStr=2025-09-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770458838389, onlineIssueDateStr=2026-02-07, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770458838389, creator=13701087609, updateTime=1770458838389, updator=13701087609, issue=Issue{id=1226956547847275311, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='9', pageStart='3821', pageEnd='4232', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1770458836757, creator=13701087609, updateTime=1770459153781, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1226957877613605816, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1226957877613605817, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=4151, endPage=4161, ext={EN=ArticleExt(id=1226956557758411691, articleId=1226956554696569675, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a carbon source, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] To achieve efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a substitute for glucose as the carbon source. [Methods] We enhanced the laccase production in C. cinerea by either exogenous addition of the invertase GspInv or endogenous co-expression of GspInv to hydrolyze sucrose in molasses. The fermentation conditions were optimized based on the laccase activity. [Results] With 40 g/L molasses as the carbon source, strain CcPIE5-14 achieved the laccase activity of (11.9±1.2) U/mL, while sucrose remained unutilized in the fermentation liquid. Upon addition of exogenous GspInv, sucrose in the fermentation liquid was hydrolyzed into fructose and glucose, and strain CcPIE5-14 exhibited the peak laccase activity of (14.8±0.7) U/mL in the medium with 30 g/L molasses. Co-expression of GspInv in CcPIE5-14 generated the engineered strain CcPIE5-14-GspInv-12, which demonstrated the maximum laccase activity of (28.1±2.4) U/mL in the mKjalke medium. When using molasses as the carbon source for laccase production, strain CcPIE5-14-GspInv-12 achieved the peak laccase activity of (20.1±2.7) U/mL, which represented a 2.24-fold increase over that of the parental strain CcPIE5-14. Following fermentation condition optimization, strain CcPIE5-14-GspInv-12 attained the maximum laccase activity of (44.6±2.6) U/mL, which marked a 2.22-fold enhancement over the pre-optimization level. [Conclusion] The engineered strain CcPIE5-14-GspInv-12, co-expressing laccase and invertase, demonstrates efficient production of the alkaline laccase PIE5 in C. cinerea with cost-effective molasses as the carbon source.

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*E-mail:
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【目的】 利用廉价糖蜜替代葡萄糖作为碳源，在真菌灰盖鬼伞(Coprinopsis cinerea)中高效表达碱性真菌漆酶PIE5。 【方法】 通过外源添加蔗糖酶GspInv或在C. cinerea中共表达GspInv分解糖蜜中的蔗糖；以漆酶活力为参考，优化菌株发酵条件。 【结果】 以40 g/L糖蜜为碳源时，菌株CcPIE5-14表达漆酶活力为(11.9±1.2) U/mL，发酵液中蔗糖未被利用。添加外源蔗糖酶GspInv后，发酵液中的蔗糖被水解为果糖与葡萄糖，菌株CcPIE5-14在30 g/L糖蜜条件下漆酶活力最高，达到(14.8±0.7) U/mL。在CcPIE5-14中共表达GspInv获得菌株CcPIE5-14-GspInv-12，在mKjalke培养基中最高漆酶活性为(28.1±2.4) U/mL。利用糖蜜为碳源发酵制备漆酶时，共表达菌株CcPIE5-14-GspInv-12的最高漆酶活性为(20.1±2.7) U/mL，较出发菌株CcPIE5-14提高了2.24倍。发酵条件优化后，菌株CcPIE5-14-GspInv-12的最高漆酶活性为(44.6±2.6) U/mL，是优化前的2.22倍。 【结论】 本研究成功构建了漆酶和蔗糖酶共表达菌株，菌株CcPIE5-14-GspInv-12能以廉价糖蜜为碳源高效表达碱性真菌漆酶PIE5。

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作者贡献声明

孟新悦：文稿撰写；王先华：实验操作；赵慧芳：数据收集与分析；刘娟娟：数据分析及文稿审阅；方泽民：实验设计及文稿审阅。

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The result represents the mean±SD of three replicates., figureFileSmall=FhIrQfBB7oJePqoeVcX5ng==, figureFileBig=GyDDpvMk/hUfSRYtBhFt9g==, tableContent=null), ArticleFig(id=1226964059950593010, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956554696569675, language=CN, label=图2, caption=共表达菌株在mKjalke培养基中的蔗糖酶活力分析, figureFileSmall=FhIrQfBB7oJePqoeVcX5ng==, figureFileBig=GyDDpvMk/hUfSRYtBhFt9g==, tableContent=null), ArticleFig(id=1226964060072227832, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956554696569675, language=EN, label=Figure 3, caption=Shake flask fermentation of recombinant strains for laccase production. A: Laccase activity of co-expressesed strains in mKjalke medium; B: Analysis of residual sugar content in fermentation supernatant; C: Laccase activity and biomass of co-expressed strains in molasses. 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The data were the mean±SD of the three replicates., figureFileSmall=URwC+fP5JRszZpJZ5ucCsw==, figureFileBig=cVZKAj630q9UktVjnLs2rw==, tableContent=null), ArticleFig(id=1226964060441325581, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956554696569675, language=CN, label=图4, caption=发酵条件优化。A：糖蜜浓度；B：氮源种类；C：工业蛋白胨浓度；D：工业酵母浸粉浓度；E：氮源复配比例；F：初始pH；G：发酵温度。, figureFileSmall=URwC+fP5JRszZpJZ5ucCsw==, figureFileBig=cVZKAj630q9UktVjnLs2rw==, tableContent=null), ArticleFig(id=1226964060550377496, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956554696569675, language=EN, label=Figure 5, caption=Preparation of laccase PIE5 in a 1 L bioreactor. 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以糖蜜为碳源在真菌灰盖鬼伞中高效表达碱性漆酶PIE5
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孟新悦 1, 2 , 王先华 1, 2 , 赵慧芳 1, 2 , 刘娟娟 1, 2 , 方泽民 1, 2
微生物学报 | 研究报告 2025,65(9): 4151-4161
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微生物学报 | 研究报告 2025, 65(9): 4151-4161
以糖蜜为碳源在真菌灰盖鬼伞中高效表达碱性漆酶PIE5
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孟新悦1, 2, 王先华1, 2, 赵慧芳1, 2, 刘娟娟1, 2, 方泽民1, 2
作者信息
  • 1 安徽大学 生命科学学院，安徽 合肥
  • 2 生物催化与现代生物制造安徽省重点实验室，安徽 合肥
Efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a carbon source
Xinyue MENG1, 2, Xianhua WANG1, 2, Huifang ZHAO1, 2, Juanjuan LIU1, 2, Zemin FANG1, 2
Affiliations
  • 1 School of Life Sciences, Anhui University, Hefei, Anhui, China
  • 2 Anhui Key Laboratory of Biocatalysis and Modern Biomanufacturing, Hefei, Anhui, China
出版时间: 2025-09-04 doi: 10.13343/j.cnki.wsxb.20250178
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摘要
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【目的】 利用廉价糖蜜替代葡萄糖作为碳源，在真菌灰盖鬼伞(Coprinopsis cinerea)中高效表达碱性真菌漆酶PIE5。 【方法】 通过外源添加蔗糖酶GspInv或在C. cinerea中共表达GspInv分解糖蜜中的蔗糖；以漆酶活力为参考，优化菌株发酵条件。 【结果】 以40 g/L糖蜜为碳源时，菌株CcPIE5-14表达漆酶活力为(11.9±1.2) U/mL，发酵液中蔗糖未被利用。添加外源蔗糖酶GspInv后，发酵液中的蔗糖被水解为果糖与葡萄糖，菌株CcPIE5-14在30 g/L糖蜜条件下漆酶活力最高，达到(14.8±0.7) U/mL。在CcPIE5-14中共表达GspInv获得菌株CcPIE5-14-GspInv-12，在mKjalke培养基中最高漆酶活性为(28.1±2.4) U/mL。利用糖蜜为碳源发酵制备漆酶时，共表达菌株CcPIE5-14-GspInv-12的最高漆酶活性为(20.1±2.7) U/mL，较出发菌株CcPIE5-14提高了2.24倍。发酵条件优化后，菌株CcPIE5-14-GspInv-12的最高漆酶活性为(44.6±2.6) U/mL，是优化前的2.22倍。 【结论】 本研究成功构建了漆酶和蔗糖酶共表达菌株，菌株CcPIE5-14-GspInv-12能以廉价糖蜜为碳源高效表达碱性真菌漆酶PIE5。

灰盖鬼伞  /  糖蜜  /  蔗糖酶  /  碱性真菌漆酶  /  高效表达

[Objective] To achieve efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a substitute for glucose as the carbon source. [Methods] We enhanced the laccase production in C. cinerea by either exogenous addition of the invertase GspInv or endogenous co-expression of GspInv to hydrolyze sucrose in molasses. The fermentation conditions were optimized based on the laccase activity. [Results] With 40 g/L molasses as the carbon source, strain CcPIE5-14 achieved the laccase activity of (11.9±1.2) U/mL, while sucrose remained unutilized in the fermentation liquid. Upon addition of exogenous GspInv, sucrose in the fermentation liquid was hydrolyzed into fructose and glucose, and strain CcPIE5-14 exhibited the peak laccase activity of (14.8±0.7) U/mL in the medium with 30 g/L molasses. Co-expression of GspInv in CcPIE5-14 generated the engineered strain CcPIE5-14-GspInv-12, which demonstrated the maximum laccase activity of (28.1±2.4) U/mL in the mKjalke medium. When using molasses as the carbon source for laccase production, strain CcPIE5-14-GspInv-12 achieved the peak laccase activity of (20.1±2.7) U/mL, which represented a 2.24-fold increase over that of the parental strain CcPIE5-14. Following fermentation condition optimization, strain CcPIE5-14-GspInv-12 attained the maximum laccase activity of (44.6±2.6) U/mL, which marked a 2.22-fold enhancement over the pre-optimization level. [Conclusion] The engineered strain CcPIE5-14-GspInv-12, co-expressing laccase and invertase, demonstrates efficient production of the alkaline laccase PIE5 in C. cinerea with cost-effective molasses as the carbon source.

Coprinopsis cinerea  /  molasses  /  invertase  /  fungal alkaline laccase  /  efficient expression
孟新悦, 王先华, 赵慧芳, 刘娟娟, 方泽民. 以糖蜜为碳源在真菌灰盖鬼伞中高效表达碱性漆酶PIE5. 微生物学报, 2025 , 65 (9) : 4151 -4161 . DOI: 10.13343/j.cnki.wsxb.20250178
Xinyue MENG, Xianhua WANG, Huifang ZHAO, Juanjuan LIU, Zemin FANG. Efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a carbon source[J]. Acta Microbiologica Sinica, 2025 , 65 (9) : 4151 -4161 . DOI: 10.13343/j.cnki.wsxb.20250178
正文
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漆酶(EC 1.10.3.2)，是一类能够催化酚类与非酚类化合物氧化的含铜多酚氧化酶，属于多铜氧化酶家族(multicopper oxidases, MCOs)中最大的群体[1]。真菌、植物、细菌和昆虫等的基因组中均包含漆酶基因，但已发现的漆酶超过80%来自真菌和细菌[2]。目前已从平菇(Pleurotus ostreatus)[3-4]、变色栓菌(Trametes versicolor)[3-4]、枯草芽孢杆菌(Bacillus subtilis)[5]等微生物中克隆、纯化并表征了超过100个漆酶蛋白。其中，真菌漆酶主要在酸性条件下发挥活性(pH 3.5-5.5)，具有氧化还原电势高、比活力高、底物范围广等优势[6-7]。细菌漆酶主要在中性及碱性条件下具有良好催化活性(pH>6.0)，具有热稳定性好的特性，但催化比活力低、底物范围窄[3-4]。基于不同来源漆酶的优缺点，获得在碱性条件下具有良好催化性能的真菌漆酶一直是漆酶研究的主要方向之一。
漆酶在众多领域具有应用价值[8-9]。例如，Selina等[10]发现漆酶可用于污染土壤中毒性化合物及传统油田和化石燃料中多环芳烃的分解；Pramanik等[11]利用漆酶降解了一些有毒偶氮染料(如酚红、亚甲蓝、结晶紫等)；Gutiérrez-Escobar等[12]发现在葡萄酒酿造中添加固定化的漆酶能够提取酚类副产物，改善葡萄酒的颜色与口感。然而，漆酶产量不足一直是限制其大规模应用的主要瓶颈。野生菌株分泌漆酶活性低、产酶真菌培养时间长、产酶效率低、生产成本高等均是限制因素[13]。通过基因异源高效表达、菌株选择与改良、培养基成分优化、发酵条件优化等策略是提高漆酶产量和降低生产成本的关键步骤[14]。
糖蜜是从甘蔗中提取蔗糖后产生的下游副产物[15]，主要由水(20%-28%)、总糖(57%-71%)、粗蛋白(5%-9%)、有机酸(3%-9%)和无机盐(2%-8%)组成[16]，可作为微生物发酵的良好廉价碳源。Yan等[17]在制备生物柴油时使用糖蜜替代葡萄糖，使制备成本降低了60%。据统计，我国每年生产约400万t糖蜜，其中大部分被直接丢弃，导致了大量资源的浪费和环境污染[17]。
真菌漆酶PIE5是Yin等[18]在灰盖鬼伞(Coprinopsis cinerea)来源漆酶Lcc9基础上，通过定向进化获得的第一个在碱性条件下(pH 8.5)具有良好催化活性的真菌漆酶。在以20 g/L葡萄糖为碳源、C. cinerea FA2222为宿主细胞同源过表达PIE5时，菌株CcPIE5-14的漆酶活力达到24.2 U/mL，获得的漆酶在染料废水处理中具有潜在应用价值[15]。本研究使用廉价糖蜜代替葡萄糖，以期通过对过表达菌株CcPIE5-14的改造和发酵优化实现真菌漆酶PIE5的高效表达。
1 材料与方法
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1.1 菌株
PIE5表达菌株CcPIE5-14保存于本实验室。菌株在37 ℃培养。
1.2 培养基
Yeast malt glucose培养基(g/L)：酵母提取物4.0，葡萄糖4.0，麦芽浸膏10.0，用于菌株培养。mKjalke培养基(g/L)：酵母提取物10.0，葡萄糖20.0，磷酸二氢钾2.0，二水氯化钙0.5，七水硫酸镁0.05，用于菌株液体发酵种瓶制备和漆酶发酵。
1.3 主要试剂和仪器
2,2′-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐[2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, ABTS]购自阿拉丁试剂(上海)有限公司；糖蜜购自广西博宣食品有限公司；质粒提取试剂盒购自北京全式金生物技术有限公司；真菌基因组快速抽提试剂盒购自生工生物工程(上海)股份有限公司；其他化学试剂均为国产或进口分析纯。
发酵罐购自上海保兴生物设备工程有限公司。
1.4 蔗糖酶(Gongronellasp. invertase, GspInv)的表达、纯化及糖蜜的预处理
GspInv在毕赤酵母GS115中的过表达制备及纯化参照Zhou等[19]的方法进行。纯化的GspInv保存于4 ℃备用。糖蜜预处理时向不同浓度糖蜜发酵培养基中加入60 U GspInv，在37 ℃、120 r/min条件下培养CcPIE5-14一周。
1.5 蔗糖酶GspInv过表达载体的构建
载体pYSK7主要由双孢蘑菇(Agaricus bisporus) gpd Ⅱ启动子、C. cinerea的lcc1编码序列和lcc1启动子组成[20]。将载体中的lcc1序列替换成GspInv序列，构建GspInv过表达载体。GspInv的克隆及其与pYSK7载体酶切片段的同源重组连接参照Zhou等[19]的方法进行，最终获得pYSK7-GspInv过表达载体。为研究GspInv胞内和胞外过表达对漆酶PIE5表达的影响，在GspInv序列上游添加了信号肽序列ATGTCCAGGAAACTTTTCTCTCTCGCCTACTTGGCTGTTGTACTGGTCAGT，获得过表达载体pYSK7-GspInv-s。酿酒酵母Y1H的转化及阳性克隆验证参考YeastmarkerTM Yeast Transformation System 2 User Manual[21]。获得的阳性克隆在YPDA液体培养基(参考YeastmarkerTM Yeast Transformation System 2 User Manual配制)[21]中30 ℃恒温培养2 d后，参考TIANprep Yeast Plasmid DNA Kit提取重组质粒并转入大肠杆菌JM109中扩增，用于菌株CcPIE5-14的转化。
1.6 GspInv过表达CcPIE5-14菌株的构建
菌株CcPIE5-14原生质体的制备以及GspInv过表达质粒与潮霉素筛选标记质粒的共转化参考王先华等[20]的方法。转化后的原生质体涂布于固体再生培养基中，在37 ℃培养24 h后，向培养皿中添加5 mL含有100 μg/mL潮霉素的再生培养基，继续在37 ℃培养3 d。随后，挑取活性生长的单菌落，接种至YMG固体培养基中培养至菌落直径>1 cm。将菌株的菌丝刮取至无菌EP管中，按照真菌基因组快速抽提试剂盒的方法提取真菌基因组。以提取的真菌基因组为模板，使用引物GspInvF (5′-ATGGTGCTT GCTGATCCT-3′)和GspInvR (5′-TCAAGGGC GATTGAACG-3′)，通过PCR反应进行阳性转化子的鉴定，并计算阳性转化率。PCR反应体系(20 μL)：2×ApexHF FS PCR Master Mix 10 μL，上、下游引物(10 μmol/L)各1 μL，DNA模板5 μL，ddH2O 3 μL。PCR反应条件：95 ℃预变性5 min；95 ℃变性30 s；68 ℃退火30 s；72 ℃延伸1 min，共30个循环；72 ℃终延伸5 min。引物合成、测序均由生工生物工程(上海)股份有限公司完成。
1.7 C.cinerea 菌株的培养
挖取4块大小约为0.5 cm2的C. cinerea菌块接种至YMG液体培养基中，37 ℃、120 r/min培养4 d。在无菌环境下，通过高速均浆制备发酵种子液。将种子液按照5%的体积分数接种至mKjalke液体培养基或不同浓度糖蜜培养基(将mKjalke培养基中的葡萄糖替换为糖蜜)中，37 ℃、120 r/min连续培养7 d，每隔24 h取样并监测目标酶蛋白活力的变化。
1.8 漆酶、蔗糖酶活力测定
漆酶活力测定采用ABTS法，在1 mL反应体系中添加950 μL酒石酸钠缓冲液(pH 4.0)、33 μL ABTS (终浓度500 μmol/L)和17 μL适当稀释的酶液，涡旋振荡3 s，30 ℃水浴中反应3 min后立即置于冰上30 s以终止反应，测定OD420。漆酶活力计算如公式(1)所示。酶活(enzyme activity, E)单位定义为1 min内转化1 μmol/L底物所需的酶量。
E (U/L)=OD420×稀释倍数×555.56
蔗糖酶活力测定步骤如下：在0.6 mL反应体系中添加430 μL乙酸-乙酸钠缓冲液(50 mmol/L，pH 5.0)、120 μL 1 mol/L蔗糖溶液和50 μL适当稀释的酶液，混匀后在45 ℃水浴中孵育5 min，利用葡萄糖试剂盒(葡萄糖氧化酶法)测定反应体系中生成的葡萄糖量，进而计算蔗糖酶的酶活力。酶活单位(U)定义为1 min内转化1 μmol/L底物所需的酶量。
1.9 发酵液残糖的HPLC测定
不同时刻发酵液中残糖的测定使用HPLC法，使用示差检测器进行检测，色谱柱型号为Aminex HPX-Ca (9 μm，7.8 mm×300 mm)，柱温80 ℃，检测器温度55 ℃，流动相为超纯水，流速0.5 mL/min，进样量20 μL。配制不同浓度的葡萄糖、果糖和蔗糖标准品，制备相应的标准曲线。通过标准曲线计算不同时刻发酵液中残糖的含量。
1.10 发酵条件优化
以mKjalke培养基为基础，采用单因素法对发酵培养基进行优化。具体优化条件如下：(1) 氮源种类(A玉米浆、B Aobox酵母浸粉、C工业酵母浸粉、D大豆蛋白胨、E工业蛋白胨、F多聚蛋白胨、G牛肉膏)；(2) 氮源浓度(5、8、10、12、15 g/L)；(3) 发酵温度(25、29、34、37 ℃)；(4) 培养基初始pH (5.0、5.5、6.0、6.5、7.0、7.7)。
1.11 PIE5的1 L发酵罐发酵制备
使用1.10节优化获得的发酵培养基开展PIE5的发酵制备。1 L的发酵罐初始装液量为650 mL。种子液按照1.7节方法制备，并以5%的接种量接种至发酵罐内。发酵初始阶段，设定发酵温度为37 ℃，通气量为5 m3/h，搅拌叶轮转速为150 r/min，待发酵设备各参数稳定后，设定发酵罐内溶氧(dissolved oxygen, DO)为100%。当DO降至40%以下时，启动溶氧关联，自动控制压缩空气和氧气的通气量，使罐内的DO稳定在30%-40%之间。每12 h取发酵上清液测定漆酶活力。
2 结果与分析
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2.1 CcPIE5-14以蔗糖酶处理糖蜜为碳源时表达漆酶PIE5活力
糖蜜是微生物发酵的良好碳源和氮源。本研究考察了糖蜜浓度对PIE5表达的影响。当糖蜜浓度为40 g/L时CcPIE5-14表达的漆酶活性最高，达到(11.9±1.2) U/mL。当糖蜜浓度超过40 g/L时漆酶表达活力迅速下降(图1A)。对发酵体系中的主要糖组分及浓度进行分析，发现培养基中蔗糖浓度维持在(22.3±0.6) g/L。随着培养时间的延长，葡萄糖和果糖浓度逐渐下降，在发酵的第4天降低至0 g/L (图1B)。这些结果表明CcPIE5-14不能利用蔗糖，因此对糖蜜中的蔗糖进行水解预处理可能有效提高漆酶表达活力。
来自卵形孢球托霉菌(Gongronella butleri)W5的高效蔗糖酶GspInv可以高效水解蔗糖为葡萄糖和果糖[19]。向培养CcPIE5-14体系中加入60 U GspInv以促进蔗糖分解。结果显示，当糖蜜浓度为30 g/L时漆酶活力最高，达到(14.8±0.7) U/mL，而此时未添加蔗糖酶GspInv的培养体系中漆酶活力仅为(6.2±0.4) U/mL (图1A)。与直接使用糖蜜相比，经GspInv预处理后发酵体系中的最高漆酶活性提高了1.39倍。发酵体系中的蔗糖在GspInv作用下浓度逐渐下降，而葡萄糖和果糖浓度逐渐升高(图1C)。上述结果说明添加的蔗糖酶有效水解了糖蜜中的蔗糖为葡萄糖和果糖，后者被CcPIE5-14利用，用于菌体生长和漆酶的合成与分泌。
2.2 蔗糖酶GspInv表达载体的转化及阳性转化子的验证
为了在菌株CcPIE5-14中实现GspInv的共表达，将表达载体pYSK7-GspInv-s和pYSK7-GspInv分别与潮霉素抗性筛选载体PCRⅡ-hygB共转化至CcPIE5-14原生质体中。经潮霉素筛选最终获得pYSK7-GspInv-s和pYSK7-GspInv转化子10株和54株。经PCR验证，分别获得7株和38株阳性转化子，阳性率分别为70.0%和70.4%。
随机挑选阳性转化子菌株，在mKjalke培养基中培养以验证蔗糖酶蛋白的表达能力。结果显示(图2)，CcPIE5-14-GspInv-s阳性转化子菌株的最高蔗糖酶活性为(25.3±2.3) U/mL，最低为(11.0±0.5) U/mL；CcPIE5-14-GspInv阳性转化子菌株的最高蔗糖酶活性为(46.5±2.5) U/mL，最低为(12.8±1.9) U/mL。结果表明，2种类型的gspinv表达载体均成功转入菌株CcPIE5-14并正确表达，然而信号肽的加入并未有效增加胞外蔗糖酶活力。
2.3 共表达菌株的漆酶活力测定
重组菌株在mKjalke培养基中的漆酶合成能力研究结果显示，重组菌株与CcPIE5-14的发酵周期一致，均在第7天达到最高漆酶活性(图3A)。其中，共表达菌株CcPIE5-14-GspInv-12的漆酶活力最高，达到(28.1±2.4) U/mL；CcPIE5-14-GspInv-3的活力为(25.8±0.4) U/mL。与出发菌株的最高酶活力(21.3±1.0) U/mL相比，分别提高了0.32倍和0.21倍。
使用30 g/L糖蜜替代mKjalke培养基中的葡萄糖(图3C)，共表达菌株CcPIE5-14-GspInv-12的最高漆酶活性为(20.1±2.7) U/mL，CcPIE5-14-GspInv-s-1的最高漆酶活性为(14.8±0.4) U/mL。与CcPIE5-14的酶活力(6.2±0.4) U/mL相比，分别提高了2.24倍和1.39倍。
对共表达菌株CcPIE5-14-GspInv-12发酵液上清中残糖含量的测定结果显示，发酵液中的蔗糖已逐步被转化为葡萄糖和果糖，且葡萄糖和果糖进一步被重组菌株利用(图3B)。对发酵终点的菌丝体干重分析结果显示(图3C)，蔗糖酶GspInv转入CcPIE5-14菌株后，PIE5表达菌株的生物量均有不同程度的提高。然而生物量与漆酶活力并不呈正比例关系，表明合适的蔗糖酶表达量对于平衡菌体生物量和漆酶表达是必需的。
2.4 碳源、氮源、pH以及温度优化
以CcPIE5-14-GspInv-12为出发菌株，以mKjalke培养基为基础，将碳源替换为糖蜜进行发酵培养基成分的优化。结果显示(图4A)，当糖蜜浓度为40 g/L时PIE5发酵酶活性最高，达到(25.1±1.9) U/mL。随着糖蜜浓度的增加菌体的干重逐渐增加，而酶活性逐渐降低。因此选用40 g/L的糖蜜作为最终碳源浓度进行后续的优化实验。
氮源种类优化结果显示，以工业酵母浸粉和工业蛋白胨作为单一氮源时效果最佳，漆酶活力分别为(30.3±1.5) U/mL和(31.0±1.0) U/mL (图4B)。使用玉米浆、Aobox酵母浸粉和多聚蛋白胨作为单一氮源时也获得了较高的漆酶活力，分别为(24.7±2.3)、(26.3±1.9)、(23.3±0.1) U/mL。因此选择工业蛋白胨和工业酵母浸粉2种氮源进行最适浓度优化。当2种氮源的浓度为10 g/L时漆酶活性最高。其中，以工业蛋白胨为氮源时漆酶活性为(28.3±1.1) U/mL (图4C)，而以工业酵母浸粉为氮源时漆酶活性为(30.2±1.2) U/mL (图4D)。
本研究表明，使用复合氮源有利于菌体生长和漆酶表达。因此继续保持总浓度在10 g/L，使用工业酵母浸粉和工业蛋白胨作为复合氮源，对二者的添加比例进行优化。结果显示，当工业酵母浸粉与工业蛋白胨的比例为2:1和3:1时漆酶活性最高，分别为(34.9±0.2) U/mL和(34.4±0.5) U/mL，高于单一氮源时的酶活性(图4E)。由于工业酵母浸粉与工业蛋白胨的比例为2:1和3:1时，PIE5的产量相差不大，为了降低培养基的成本，在后续的研究中使用工业酵母浸粉:工业蛋白胨=2:1的复合比例，即6.7 g/L工业酵母浸粉和3.3 g/L工业蛋白胨进行后续实验。
不同初始pH对PIE5表达的影响显著(图4F)。随着pH的升高，PIE5酶活力呈先上升后下降的趋势。当初始pH为6.0时漆酶活性达到最高点，为(44.6±2.6) U/mL。当初始pH值超过6.5后，漆酶活性和生物量均有所降低，说明在偏酸性的环境更有利于菌株CcPIE5-14-GspInv-12的培养，并且也更有利于PIE5的表达。
培养温度对漆酶PIE5表达的影响研究结果显示(图4G)，当发酵温度为37 ℃时漆酶活性最高，为(40.8±3.3) U/mL；其次是25 ℃，酶活为(32.0±0.6) U/mL。在29 ℃和34 ℃发酵时酶活较低，分别为(22.2±3.4) U/mL和(24.6±0.6) U/mL。当菌体在37 ℃下培养时，由于温度适宜菌体生物量快速积累并且表达大量漆酶，在第6天即达到最高酶活。在25 ℃下培养时，虽然单个菌丝球表达漆酶的效率较高，但总体生物量增长速度缓慢，在第8天才达到最高酶活。因此37 ℃培养时发酵周期更短，为了减少发酵成本，在后续的优化实验中选用37 ℃作为PIE5的发酵温度。
2.5 1 L发酵罐中发酵验证重组菌株的PIE5合成能力
为了进一步验证重组菌株CcPIE5-14-GspInv-12的漆酶PIE5合成能力，在基于摇瓶优化获得的最佳培养基配方和培养条件下，利用1 L发酵罐进行发酵验证。漆酶活性在37 ℃培养发酵至84 h时达到最高点，为(38.2±2.9) U/mL (图5A)。在发酵过程中，由于菌株代谢以及部分菌丝球的裂解(图5B)，导致发酵液的pH逐渐上升。菌丝球的形态变化与发酵液pH增长趋势一致。
3 讨论与结论
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糖蜜中的糖主要是蔗糖(48.8%)，只有少量的葡萄糖(5.29%)和果糖(8.07%)[22]。当以蔗糖作为碳源时，C. cinerea生长状况不佳[23]。与这一现象一致，本研究中残糖浓度测定结果显示，CcPIE5-14不能利用蔗糖。因此在这种生长不良的条件下仅检测到极少量的漆酶活性是合理的，同时也提示对糖蜜中的蔗糖进行水解预处理可能有效提高漆酶表达活力。通过GspInv水解糖蜜中的蔗糖为果糖和葡萄糖，漆酶发酵活力提高至(14.8±0.7) U/mL。进一步通过在菌株中共表达GspInv降低外源添加蔗糖酶的成本，最终筛选获得的菌株漆酶活力达到(20.1±2.7) U/mL，比对照提高了2.24倍。Ma等[24]在利用糖蜜生产二十二碳六烯酸的过程中，在裂殖壶菌(Schizochytrium sp.)中表达了高效蔗糖酶后产物产量提高了2.57倍。
目前，灰盖鬼伞产漆酶的最高酶活因培养条件和优化策略的不同而有所差异。通过基因编辑或异源表达技术[25]，灰盖鬼伞的漆酶产量可能进一步提升。Bao等[26]在毛头鬼伞(Coprinus comatus)中异源表达漆酶Lac1时，漆酶活力在第6天达到0.55 U/mL。蔗糖酶在C. cinerea中异源表达，可能从碳源利用、代谢调控、基因表达调控、协同作用等方面促进漆酶酶活[27]。蔗糖酶基因gspinv在灰盖鬼伞胞内和胞外表达，对漆酶活力的影响可能因基因整合位点的不同而有所差异[28-29]。蔗糖酶在胞内表达可以改变代谢途径，蔗糖的水解产物果糖可以调节苯丙烷代谢途径，更快地激活漆酶合成的上游通路[29]。胞内表达蔗糖酶影响次级代谢产物的合成(如对羟基苯甲酸)，进而诱导漆酶基因表达，提高漆酶产量[30]。胞内表达蔗糖酶得到的水解产物葡萄糖和果糖，为菌株提供更多可利用的碳源，促进菌体生长和代谢活动，从而间接提高漆酶的合成与分泌[31]。此外，蔗糖酶会受到最终产物葡萄糖和果糖的抑制。本研究表明，蔗糖酶的胞内表达可显著提高漆酶活性，该结果与已有文献报道相吻合。
本研究构建了高效蔗糖酶GspInv表达载体，并将其转入CcPIE5-14中，成功获得了共表达GspInv的重组菌CcPIE5-14-GspInv-12。重组菌以糖蜜发酵漆酶时，最高漆酶活性达到(20.1±2.7) U/mL，相较于CcPIE5-14提高了2.24倍。通过发酵条件优化，PIE5的表达量进一步提高，达到(44.6±2.6) U/mL，是优化前的2.22倍。通过对重组菌株的改造，实现了CcPIE5-14以廉价糖蜜为碳源条件下PIE5的高水平表达。
基金
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  • 国家自然科学基金(32370133)
  • 安徽省杰出青年基金(2008085J12)
文献
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2025年第65卷第9期
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doi: 10.13343/j.cnki.wsxb.20250178
  • 接收时间：2025-03-05
  • 首发时间：2026-02-07
  • 出版时间：2025-09-04
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  • 收稿日期：2025-03-05
  • 录用日期：2025-05-06
基金
National Natural Science Foundation of China(32370133)
国家自然科学基金(32370133)
Science Fund for Distinguished Young Scholars of Anhui Province(2008085J12)
安徽省杰出青年基金(2008085J12)
作者信息
    1 安徽大学 生命科学学院，安徽 合肥
    2 生物催化与现代生物制造安徽省重点实验室，安徽 合肥
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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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