CN115141841A - 一种毕赤酵母突变菌株及其在海藻酸裂解酶生产中的应用 - Google Patents
一种毕赤酵母突变菌株及其在海藻酸裂解酶生产中的应用 Download PDFInfo
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- CN115141841A CN115141841A CN202210888125.7A CN202210888125A CN115141841A CN 115141841 A CN115141841 A CN 115141841A CN 202210888125 A CN202210888125 A CN 202210888125A CN 115141841 A CN115141841 A CN 115141841A
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- pichia pastoris
- alginate lyase
- mutant strain
- nju
- alginate
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Abstract
本发明涉及基因工程技术领域，具体涉及一种毕赤酵母突变菌株及其在海藻酸裂解酶生产中的应用。所述突变菌株为毕赤酵母NJU‑12（Pichia pastoris NJU‑12），已于2022年5月30日保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2022758。所述突变菌能大幅度提高海藻酸裂解酶的表达量，其摇瓶发酵上清液中海藻酸裂解酶酶活高达3006 U/ml，比出发菌提高了57%，有利于降低该酶的生产成本，促进其在工业领域中的广泛应用。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种毕赤酵母突变菌株及其在海藻酸裂解酶生产中的应用。
背景技术
来自海洋的植物-褐藻相对于陆生植物有许多优点而受到广泛关注。褐藻是主要的养殖藻类，2012年世界范围内（33个国家）捕捞和养殖的藻类的产量分别是1.1和23.8万湿吨（1000公斤）。由于褐藻含高水平的碳水化合物，是一种有前景的原料生物质源，其中含量最多的碳水化合物为海藻酸，其次是昆布多糖，甘露醇和岩藻多糖。
海藻酸已被广泛用于多种日用品的加工与生产中。在食品领域作为食品凝胶剂、增稠剂和品质改良剂。目前利用海藻酸制成的食品多达百种。在保健领域，海藻酸制成含有抗药性的薄膜来提高食品的保质期和安全性。海藻酸还可减缓脂肪、糖和胆盐的吸收，能够降低血清胆固醇、血中甘油三酯和血糖，预防高血压、糖尿病和肥胖等。在医药领域，海藻酸盐在药物或蛋白质递送，细胞封装，组织再生，手术和伤口处理等方面引起了很多关注。作为释放或包埋药物及细胞的微胶囊，也可作为支架材料。在农业领域中，海藻酸可作为农药的缓释剂、杀菌剂和稳定剂。
海藻酸是一种天然高分子化合物，可通过物理化学或生物手段进行处理，断裂糖苷键使其变为寡糖和/或单糖单元，实现化学结构、物理性质或外观变化。其中糖苷键的水解是海藻酸降解的首要因素。
相比传统的物理化学方法，海藻酸裂解酶反应条件温和、特异性高，可作为工具酶制备一系列具有生物活性的海藻酸寡糖。这些海藻酸寡糖有特殊的生物活性，在医药、动植物、微生物、能源等方面蕴含着巨大的前景。
在医药上，海藻酸寡糖可导致人单核细胞产生细胞毒性细胞因子。8种PSS(丙二醇海藻酸钠)和PGMS(丙二醇甘露糖醛酸硫酸酯)是两类低分子量海藻酸衍生物，具有抗氧化和预防心血管和脑血管疾病的作用。在动植物上，海藻酸寡糖能够调节植物和微生物的生理过程，增强植物的发芽和枝条的生长；加速植物根系生长和提高产黄青霉培养物中青霉素的产量；海藻酸寡糖可诱导小麦根系产生一氧化氮(NO)，增加小麦根部横截面的中柱面积，从而促进小麦根系的生长与延伸；当小鼠注射寡糖时，它们诱导了粒细胞集落刺激因子的增加。在微生物上，酶解产生的AOS能促进双歧杆菌和乳酸菌这两种肠道有益菌的生长，有益于人体健康。 在能源领域，2014年成功构建了能利用褐藻糖类，并高效转化为乙醇的酿酒酵母代谢工程菌株，该工程菌株能够利用甘露醇和DEH进行乙醇发酵，最终乙醇产量为4.6%(v/v)(36.2g/L)，糖类的乙醇转化率达最大理论值的 83%。
海藻酸裂解酶通过β-消除反应断裂海藻酸分子间的1,4-糖苷键。海藻酸裂解酶大多数来自海洋，例如海藻类，软体动物，微生物和海洋细菌。根据剪切方式分为内切型海藻酸裂解酶和外切型海藻酸裂解酶，根据底物特异性可以分为 polyM特异性、polyG特异性以及对PolyM和PolyG都具有活性的PolyMG裂解酶。内切型海藻酸裂解酶切割海藻酸聚合物的糖苷键生成的主要产物为不饱和寡糖（二糖、三糖和四糖），并在其非还原端的C4和C5之间形成双键，而外切型海藻酸裂解酶则是将寡糖再进一步降解生成不饱和单糖。海藻酸裂解酶可以被分为7个多糖裂解酶家族PL5，6，7，14，15，17和18。目前发现的海藻酸内切酶有一百多种，上面的七个多糖裂解酶家族均含有，根据NCBI Pubmed上的文献搜索，从在Sphingomonas sp. A1中首次发现外切型海藻酸裂解酶A1-IV后，陆续在来自15种细菌中发现18 种外切型海藻酸裂解酶，这些酶分别属于PL17、PL15家族、PL6家族和PL7家族。
随着海藻酸及其寡糖越来越多地应用于化工、医药、农业、分子生物学、海洋生物学等领域，海藻酸裂解酶日益成为海洋生物资源开发的竞争热点。高特异性、高活性的海藻酸裂解酶的研究与开发具有重要的理论意义和应用价值，有着广泛的应用前景。
发明内容
本发明的目的是提供一种毕赤酵母突变菌株及其在海藻酸裂解酶生产中的应用。所述突变菌是通过紫外诱变方法筛选获得的海藻酸裂解酶高产菌株，可广泛应用于海藻酸裂解酶的生产。
本发明一方面涉及一种重组质粒，其携带有海藻酸裂解酶基因。
所述海藻酸裂解酶基因的核苷酸序列为SEQ ID NO：1，编码的氨基酸序列为SEQID NO：2。
本发明一方面涉及一种毕赤酵母工程菌，其携带有上述重组质粒。
本发明还涉及一种毕赤酵母突变菌株，是以上述毕赤酵母工程菌为出发菌，通过紫外诱变的方法获得的。
所述突变菌株为毕赤酵母NJU-12（Pichia pastoris NJU-12），已于2022年5月30日保藏于中国武汉 武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M2022758。
本发明还涉及一种发酵生产海藻酸裂解酶的方法，是以上述毕赤酵母突变菌为发酵菌株。
有益效果
本发明首先构建得到重组表达海藻酸裂解酶基因的工程菌毕赤酵母NJU，其摇瓶发酵上清液中海藻酸裂解酶酶活最高达到1915U/ml；然后以毕赤酵母NJU为出发菌，通过紫外诱变筛选获得突变菌株毕赤酵母NJU-12，能大幅度提高海藻酸裂解酶的表达量，其摇瓶发酵上清液中海藻酸裂解酶酶活高达3006 U/ml，比出发菌提高了57%，取得了意料不到的技术效果。
所述突变菌株可作为海藻酸裂解酶的发酵生产菌株，有利于降低该酶的生产成本，促进其在工业领域中的广泛应用。
具体实施方式
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001)和CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本发明所记载的技术方案的基础上，采用本领域其它常规的方法、实验方案和试剂，而不限于本发明具体实施例的限定。
菌株与载体：毕赤酵母GS115、载体pPIC9k、G418购自Invitrogen公司。
试剂：DNA聚合酶购买自Takara公司，T4连接酶、限制性内切酶购自Fermentas公司，质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司。其余试剂均为国产分析纯。
下面结合具体实施方式，对本发明做进一步阐述。
实施例1、海藻酸裂解酶基因NJU的克隆
以海藻酸裂解酶基因（GenBank号为ASA33933.1）的氨基酸序列为基础，分析该海藻酸裂解酶的氨基酸序列，去除其自身的信号肽，再根据毕赤酵母的密码子偏好性，对其进行密码子优化，由华大基因公司进行全基因合成。申请人将该海藻酸裂解酶基因命名为NJU，其核苷酸序列为SEQ ID NO:1，编码的氨基酸序列为SEQ ID NO:2。
采用PCR反应克隆海藻酸裂解酶基因NJU片段，引物和反应条件如下：
引物1(F)：GCGCGAATTCGCGGAAGTGAATCTGGTGAATCCGA（下划线为EcoR I酶切位点）；
引物1(R)：TAAAGCGGCCGCTTACGGAAATTCATAGCCATCATGG（下划线为Not I酶切位点）。
PCR条件为：94℃变性5min；然后94℃变性30s，56℃复性30s，72℃延伸1min30s，35个循环后，72℃保温10min。NJU基因全长1278bp。
实施例2、表达重组海藻酸裂解酶基因NJU的毕赤酵母工程菌的构建
1、重组质粒的构建
将克隆得到的海藻酸裂解酶基因NJU，用限制性内切酶EcoR I和Not I进行双酶切，100 μl酶切体系如下：
| 反应组分 | 体积 |
| NJU的 PCR产物 | 40 μl |
| 10×H buffer | 10 μl |
| 10×BSA | 10 μl |
| <i>Eco</i>R I | 5 μl |
| <i>Not</i> I | 5 μl |
| ddH<sub>2</sub>O | 30 μl |
37℃酶切4 h后，琼脂糖凝胶电泳回收。
将表达载体pPIC9K用限制性内切酶EcoR I和Not I进行双酶切，100 μl酶切体系如下：
| 反应组分 | 体积 |
| pPIC9K | 40 μl |
| 10×H buffer | 10 μl |
| 10×BSA | 10 μl |
| <i>Eco</i>R I | 5 μl |
| <i>Not</i> I | 5 μl |
| ddH<sub>2</sub>O | 30 μl |
37℃酶切4 h后，琼脂糖凝胶电泳回收。
将经EcoR I和Not I双酶切的NJU片段与表达载体pPIC9K连接，构建表达载体pPIC9K-NJU。连接体系如下：
| 反应组分 | 体积 |
| 表达载体pPIC9K双酶切产物 | 5 μl |
| NJU基因双酶切产物 | 3 μl |
| 10×T<sub>4</sub> ligase buffer | 1 μl |
| T<sub>4</sub> ligase | 1 μl |
22℃连接过夜，转化到大肠杆菌DH5α，挑取转化子测序验证。测序验证正确的转化子转接到LB+Amp液体培养基（0.5%酵母提取物，1%蛋白胨，1%NaCl，100μg/mL氨苄青霉素，pH7.0）中，37℃过夜培养，提质粒，即为重组酵母表达质粒pPIC9K-NJU。
2、转化与筛选
将重组酵母表达质粒pPIC9K-NJU用Sal I进行线性化，线性化产物用柱纯化试剂盒纯化后，通过电穿孔法转化毕赤酵母GS115，涂布MD平板（1.34% YNB，4×10-5%生物素，1%甘油、2%琼脂糖）。在MD平板上生长出的菌落即为毕赤酵母工程菌株，然后涂布含不同浓度遗传霉素G418的YPD平板（1%酵母提取物，2%蛋白胨，2%葡萄糖）上筛选多拷贝的转化子。
3、摇瓶发酵验证
挑取单个多拷贝转化子分别接入BMGY培养基（2%蛋白胨，1%酵母提取物，100 mM磷酸钾缓冲液(pH6.0)，1.34% YNB，4×10-5%生物素，1%甘油）中，30℃、220rpm振荡培养24小时后，再转入BMMY培养基（2%蛋白胨，1%酵母提取物，100 mM磷酸钾缓冲液(pH6.0)，1.34%YNB，4×10-5%生物素，0.5%甲醇）中，30℃、220rpm振荡培养，每24小时添加0.5%的甲醇。诱导表达4d后，离心去除菌体，将上清液进行海藻酸裂解酶酶活力测定。
结果显示，本发明构建的毕赤酵母工程菌摇瓶发酵上清液中海藻酸裂解酶酶活最高达到1915U/ml。将该转化子命名为毕赤酵母NJU（Pichia pastoris NJU）。
实施例3、紫外诱变筛选
诱变育种的基本原理是基因突变，主要包括染色体畸变和基因突变。诱变育种是利用各种被称为诱变剂的物理因素和化学试剂处理微生物细胞，提高基因突变频率，再通过适当的筛选方法获得所需要的高产优质菌种的育种方法。
紫外诱变导致的突变随机性很强，突变产生的效果也是随机的，难以预测。因此，为了获得有效的正突变，技术人员通常需要进行多轮紫外诱变，筛选的工作量较大，而且存在无法获得有效正突变的可能性。但因为紫外诱变所需设备简单、费用少，并且可在短时间内获得大量突变体，因此，它现在仍是一种常用的诱变选育方法。
申请人以毕赤酵母NJU为出发菌株，通过紫外诱变方法对其进行遗传学改造，进一步提高其海藻酸裂解酶的产量。
将工程菌NJU接种于YPD平板，30℃培养2-3天，使用无菌水洗菌体，制成悬浮液，稀释至1×106个/mL，紫外灯（40W）照射2-10min，距离约22cm，致死率达到90%以上，涂布平板，30℃培养48h。
第一轮紫外诱变共获得了约100个突变菌单菌落，将各个单菌落分别接种于装有200ul BMGY液体培养基的96孔板，30℃、250rpm振荡培养1d后，离心去掉上层培养基，再加入200ul BMMY培养基，30℃、250rpm振荡培养2天，每天添加0.5%的甲醇。诱导表达2d后，离心去除菌体，得到含海藻酸裂解酶的上清液，测定海藻酸裂解酶的活力，筛选出酶活力得到显著提高的突变菌株。
结果显示，第一轮紫外诱变筛选获得的突变菌中，没有一株突变菌发酵上清液中海藻酸裂解酶酶活高于出发菌。申请人又按照上述方法继续进行了12轮诱变筛选，最终获得1株海藻酸裂解酶产量显著高于出发菌的突变菌株，命名毕赤酵母NJU-12（Pichia pastoris NJU-12）。
所述突变菌株NJU-12在摇瓶发酵条件下其发酵上清液中海藻酸裂解酶酶活力高达3006U/ml，比出发菌提高了57%，取得了意料不到的技术效果。
申请人已于2022年5月30日将毕赤酵母NJU-12（Pichia pastoris NJU-12）保藏于中国武汉 武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2022758。
海藻酸裂解酶检测方法
（1）海藻酸裂解酶酶活单位的定义
在40℃、pH7.5的条件下，每分钟降解底物海藻酸钠产生不饱和键，在235nm下，吸光度每增加0.1，为一个酶活单位U。
（2）试剂
磷酸缓冲液（0.05M，pH7.5）
1）配制0.05M磷酸二氢钠溶液
称取3.9g二水合磷酸二氢钠，去离子水溶解后，定容至500ml。
2）配制0.05m磷酸氢二钠溶液
称取17.91g十二水合磷酸氢二钠，去离子水溶解后，定容至1000ml。
3）混合配制0.05M磷酸缓冲液
将2中的磷酸氢二钠溶液放置于2L烧杯中，用1中的磷酸二氢钠溶液调节其pH至7.5。
底物：0.3%海藻酸钠溶液
取100ml小烧杯，加入约80ml磷酸缓冲液；称取0.3g海藻酸钠，磁力搅拌条件下均匀加入到小烧杯中，搅拌至溶解；用磷酸缓冲液定容至100ml，配制当天使用。
终止液：0.06mol/L的磷酸终止液。根据不同的磷酸浓度进行计算。
（3）海藻酸裂解酶酶活测定步骤
取三支15mm×150mm试管，加入1.8ml底物，40℃水浴预热5min，加入准备好的酶液0.2ml，准确计时，涡旋振荡，40℃保温10min，将试管从水浴中取出并立即加入2ml磷酸终止液，涡旋振荡，将试管放置在水浴锅外的试管架上。
空白：取15mm*150mm试管，加入1.8ml底物，40℃水浴预热5min，加入0.2ml缓冲液，准确计时，涡旋振荡，40℃保温10min，将试管从水浴中取出并立即加入2ml磷酸终止液，涡旋振荡，将试管放置在水浴锅外的试管架上。
比色：每个样品的空白和酶反应终止后，立即在235nm比色，并记录吸光值A0和A样。
计算：X=（A0-A样）×2×N/（t×0.1）。
X——酶活力，U/ml或U/g；
2——加入2ml磷酸终止液的体积系数；
t（min）——酶促反应时间（在酶反应的线性范围内）；
0.1——体系系数，即将吸光值增长单位换算为0.1；
N——稀释倍速；
经简化：酶活力（U/ml）=吸光值差值×2×N。
序列表
<110> 青岛蔚蓝生物集团有限公司
<120> 一种毕赤酵母突变菌株及其在海藻酸裂解酶生产中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1278
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcggaagtga atctggtgaa tccgagcttt gaacaggatt ttagcggctg gaccgaagtg 60
gacccgaccg cggtaagtgg tgtggcgtat gatggtgcga aaagcgcgaa atttagcggc 120
aatggcgcac gcctggaaca gagcgttcca gttaccagca ataccgaata taccctgagc 180
gcgtatgtgc tggcggatgc gaatattggc gtggaagtgg gcagcgatac ctttagcaaa 240
accgcgagca atagcgattg ggcgcagacc accattacct ttaatagcgg cgatgcgacc 300
gaaattacca tttttggcga atatagcggc gcggaaggcc gcgtggatct gtttaaactg 360
accagcagcg aaattattga tccgccgacc accagcctgc cggtgtttga tttagatccg 420
gcgctgccgc caagcggcaa ttttgatctg ctggattgga aactggatct gccggtggat 480
gataatggca atgcgagcgg cgatgcgcag gaagtgaaag aaggcgaact gagcagcggc 540
tttgaaaata gcgaattttt ctataccggc gacgacggcg gcctggtgtt tattagcccg 600
gtggaaggcg cgaccaccag cgcaaatacc aaatataccc gcagcgaaat gcgcgaaatg 660
ctgcgccgcg gtgataccag cattagcacc accggtatta ccaaaaataa ttgggtgttt 720
gcgtcggcgc cgagcgatga tcagaataat agcggcggcg tggatggcgt gctggaagcg 780
actttagcgg tgaatgcggt tacgaccacc ggcgatagca gccaggttgg ccgtgttatt 840
gtgggccaga ttcatgcgaa taatgatgaa ccgattcgcc tgtattatcg cctgctgccg 900
ggccatacca aaggcagctt atattttgcg catgaaccga atgaagatgc gagcagcgat 960
ccggaacagt ttattaatct gattggcagc agcgcgagca atgcgagcga accggaagat 1020
ggcattgcgc tgaatgaact gttcttttat cgcattgaag tgcagggcaa tcagctgatt 1080
gtgaccatta aacgcgatga tcatgaagat gtgaccgaaa ccgtggatat gaccaccagc 1140
ggctatgatg tgagcggcca gtatatgtat tttaaagcgg gcgtgtataa tcagaataac 1200
agcggcgatc cgaccgatta tgtgcaggcg accttttatt atctgaccaa tagccatgat 1260
ggctatgaat ttccgtaa 1278
<210> 2
<211> 425
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ala Glu Val Asn Leu Val Asn Pro Ser Phe Glu Gln Asp Phe Ser Gly
1 5 10 15
Trp Thr Glu Val Asp Pro Thr Ala Val Ser Gly Val Ala Tyr Asp Gly
20 25 30
Ala Lys Ser Ala Lys Phe Ser Gly Asn Gly Ala Arg Leu Glu Gln Ser
35 40 45
Val Pro Val Thr Ser Asn Thr Glu Tyr Thr Leu Ser Ala Tyr Val Leu
50 55 60
Ala Asp Ala Asn Ile Gly Val Glu Val Gly Ser Asp Thr Phe Ser Lys
65 70 75 80
Thr Ala Ser Asn Ser Asp Trp Ala Gln Thr Thr Ile Thr Phe Asn Ser
85 90 95
Gly Asp Ala Thr Glu Ile Thr Ile Phe Gly Glu Tyr Ser Gly Ala Glu
100 105 110
Gly Arg Val Asp Leu Phe Lys Leu Thr Ser Ser Glu Ile Ile Asp Pro
115 120 125
Pro Thr Thr Ser Leu Pro Val Phe Asp Leu Asp Pro Ala Leu Pro Pro
130 135 140
Ser Gly Asn Phe Asp Leu Leu Asp Trp Lys Leu Asp Leu Pro Val Asp
145 150 155 160
Asp Asn Gly Asn Ala Ser Gly Asp Ala Gln Glu Val Lys Glu Gly Glu
165 170 175
Leu Ser Ser Gly Phe Glu Asn Ser Glu Phe Phe Tyr Thr Gly Asp Asp
180 185 190
Gly Gly Leu Val Phe Ile Ser Pro Val Glu Gly Ala Thr Thr Ser Ala
195 200 205
Asn Thr Lys Tyr Thr Arg Ser Glu Met Arg Glu Met Leu Arg Arg Gly
210 215 220
Asp Thr Ser Ile Ser Thr Thr Gly Ile Thr Lys Asn Asn Trp Val Phe
225 230 235 240
Ala Ser Ala Pro Ser Asp Asp Gln Asn Asn Ser Gly Gly Val Asp Gly
245 250 255
Val Leu Glu Ala Thr Leu Ala Val Asn Ala Val Thr Thr Thr Gly Asp
260 265 270
Ser Ser Gln Val Gly Arg Val Ile Val Gly Gln Ile His Ala Asn Asn
275 280 285
Asp Glu Pro Ile Arg Leu Tyr Tyr Arg Leu Leu Pro Gly His Thr Lys
290 295 300
Gly Ser Leu Tyr Phe Ala His Glu Pro Asn Glu Asp Ala Ser Ser Asp
305 310 315 320
Pro Glu Gln Phe Ile Asn Leu Ile Gly Ser Ser Ala Ser Asn Ala Ser
325 330 335
Glu Pro Glu Asp Gly Ile Ala Leu Asn Glu Leu Phe Phe Tyr Arg Ile
340 345 350
Glu Val Gln Gly Asn Gln Leu Ile Val Thr Ile Lys Arg Asp Asp His
355 360 365
Glu Asp Val Thr Glu Thr Val Asp Met Thr Thr Ser Gly Tyr Asp Val
370 375 380
Ser Gly Gln Tyr Met Tyr Phe Lys Ala Gly Val Tyr Asn Gln Asn Asn
385 390 395 400
Ser Gly Asp Pro Thr Asp Tyr Val Gln Ala Thr Phe Tyr Tyr Leu Thr
405 410 415
Asn Ser His Asp Gly Tyr Glu Phe Pro
420 425
Claims (6)
1.一种重组质粒，其特征在于，所述的重组质粒携带有海藻酸裂解酶基因。
2.如权利要求1所述的重组质粒，其特征在于，所述海藻酸裂解酶基因的核苷酸序列为SEQ ID NO：1，编码的氨基酸序列为SEQ ID NO：2。
3.一种毕赤酵母工程菌，其特征在于，所述的毕赤酵母工程菌携带有权利要求1或2所述的重组质粒。
4.一种毕赤酵母突变菌，其特征在于，所述的突变菌是以权利要求3所述的毕赤酵母工程菌为出发菌，通过紫外诱变的方法获得的。
5.如权利要求4所述的毕赤酵母突变菌，其特征在于，所述突变菌为毕赤酵母NJU-12（Pichia pastoris NJU-12），已于2022年5月30日保藏于中国武汉 武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2022758。
6.一种生产海藻酸裂解酶的方法，其特征在于，所述方法是以权利要求4或5所述的毕赤酵母突变菌为发酵菌株。
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| CN115161210A (zh) * | 2022-08-03 | 2022-10-11 | 青岛蔚蓝生物集团有限公司 | 一株毕赤酵母突变菌及其在海藻酸裂解酶生产中的应用 |
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