CN113430188A - 一种新型壳聚糖酶及其制备方法 - Google Patents
一种新型壳聚糖酶及其制备方法 Download PDFInfo
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- CN113430188A CN113430188A CN202110960970.6A CN202110960970A CN113430188A CN 113430188 A CN113430188 A CN 113430188A CN 202110960970 A CN202110960970 A CN 202110960970A CN 113430188 A CN113430188 A CN 113430188A
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- chitosanase
- csna
- chitosan
- gly
- asp
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Abstract
本发明公开一种新型壳聚糖酶及其制备方法，属于基因工程技术领域。本发明壳聚糖酶基因CsnA的核苷酸序列如SEQID NO:1所示，其是通过对来源于天蓝色链霉菌的壳聚糖酶的序列根据毕赤酵母密码子的偏好性进行综合优化得到。本发明壳聚糖酶CsnA的氨基酸序列如SEQ ID NO:2所示，是一种真菌壳聚糖酶，不仅丰富了壳聚糖酶的种类和来源途径，且自身具有较高的酶活性，在较宽的温度和pH范围内均表现出良好的稳定性，具有良好的应用前景和产业价值。
Description
技术领域
本发明属于基因工程技术领域，特别涉及一种新型壳聚糖酶基因、新型壳聚糖酶及其制备方法。
背景技术
壳聚糖酶是一种生物酶，作为专一性的水解酶，由于水解壳聚糖是生产不同分子量的壳聚糖产物的关键酶，其可特异性地使糖苷键断裂，作为一种极为重要的壳聚糖的制备方法，其生产形成分子量较低的壳寡糖。壳寡糖具有提高免疫能力、抑制肿瘤细胞生长、降低胆固醇和抗氧化等方面具有较强活性,在功能食品、医药、化妆品及农作物生物制剂领域具有独特而重要的应用。
壳聚糖酶广泛地存在于多种生物中，在细菌、真菌、病毒以及植物中均发现了壳聚糖酶。目前壳聚糖酶的研究主要集中在细菌壳聚糖酶，主要分为链霉菌壳聚糖酶和芽孢杆菌壳聚糖酶。其中，芽孢杆菌壳聚糖酶能够有效分解壳聚糖并且水解产物具有很好的生物活性。但细菌壳聚糖酶价格昂贵、活力较低、温度范围较窄，且稳定性较差，严重限制了壳聚糖酶的应用前景。
发明内容
本发明针对现有技术的不足，提供一种新型壳聚糖酶及其制备方法，解决了目前壳聚糖酶来源途径、活力低、温度范围窄、pH稳定性差的问题。本发明通过天蓝色链霉菌壳聚糖酶的序列根据毕赤酵母密码子的偏好性进行综合优化得到，丰富了壳聚糖酶的种类和来源途径，且其自身具有较高的酶活性，在较宽的温度和pH范围内均表现出良好的稳定性。为实现本发明目的所使用的技术方案为：
一种新型壳聚糖酶CsnA，其核苷酸序列如SEQ ID NO：1所示。
优选地，其氨基酸序列如SEQ ID NO：2所示。
本发明还提供一种新型壳聚糖酶CsnA在降解壳聚糖/制备壳聚糖中的应用；在制备降解壳聚糖/制备壳聚糖的酶制剂的应用。
另外地，本发明提供一种重组表达载体，包括以上所述新型壳聚糖酶CsnA。
优选地，一种新型壳聚糖酶CsnA的制备方法如下，提供壳聚糖酶CsnA基因、构建载体质粒和毕赤酵母转化；
对所述载体质粒进行转化；
对所述壳聚糖酶CsnA基因进行扩增；
其中，所述壳聚糖酶CsnA的核苷酸序列如SEQ ID NO:1所示。
优选地，将所述壳聚糖酶CsnA基因进行扩增的步骤中，用于扩增的引物包括正向引物和反向引物，所述正向引物的核苷酸序列如SEQ ID NO:3所示，所述反向引物的核苷酸序列如SEQ ID NO:4所示。
优选地，所述载体为毕赤酵母表达载体。
优选地，所述表达菌株为毕赤酵母表达菌株。
本发明提供一种酶制剂，包含有以上所述的新型壳聚糖酶CsnA。
优选地，如以上所述酶制剂在降解壳聚糖/制备壳寡糖中的应用。
本发明的有益效果是：
本申请的壳聚糖酶CsnA不仅丰富了壳聚糖酶的种类和来源途径，由于该壳聚糖酶CsnA在较宽的温度和pH范围内均表现出良好的稳定性，因此用于水解壳聚糖时，可使水解反应高效、稳定地进行，有利于提升水解产物壳寡糖的产量。通过实验发现，本发明提供的壳聚糖酶CsnA水解壳聚糖，所得水解产物主要为壳二糖和壳三糖，该产物作为壳寡糖的主要活性成分，且其自身具有较高的酶活性，具有良好的应用前景和产业价值。
附图说明
图1是pPIC9K-CsnA Mut+His+表型筛选结果图。
图2是壳聚糖酶基因组PCR验证结果图。
图3是pPIC9K-CsnA多拷贝筛选结果图。
图4是壳聚糖酶SDS-PAGE检测结果图。
图5是壳聚糖酶酶活测定结果图。
图6是氨基葡萄糖标准曲线图。
图7是壳聚糖酶的最适反应温度（A）和温度稳定性（B）结果图。
图8是壳聚糖酶的最适作用pH（A）和酸碱稳定性（B）结果图。
图9是酶解产物1H NMR(a) 及13C NMR(b) 分析结果图。
图10是壳聚糖酶降解壳聚糖2h的薄层层析结果图。
具体实施方式
下面结合实施例对本发明方案做进一步详细描述，下述说明仅是为了解释本发明，并不对其内容进行限定。
本发明提供的壳聚糖酶基因CsnA的核苷酸序列如SEQ ID NO:1所示，其是通过对来源于天蓝色链霉菌的壳聚糖酶的序列根据毕赤酵母密码子的偏好性进行综合优化得到。本发明壳聚糖酶CsnA的氨基酸序列如SEQ ID NO:2所示，为真菌壳聚糖酶，其酶解产物特征与Ⅲ型壳聚糖酶底物识别特性相符。本专利不仅丰富了壳聚糖酶的种类和来源途径，且其自身具有较高的酶活性，在较宽的温度和pH范围内均表现出良好的稳定性，具有良好的应用前景和产业价值。
SEQID NO:1（密码子优化前序列）
GCGACGGGACTCGACGACCCGGCGAAGAAGGAGATCGCCATGCAACTGGTGTCGAGCGCCGAGAACTCCTCGCTCGACTGGAAGGCCCAGTACCGGTACATCGAGGACATCGGTGACGGTCGCGGCTACACCGCGGGCATCATCGGCTTCTGTTCCGGCACCGGCGACATGCTCGACCTGGTGGAGCTGTACGGCGAGCGCAGCCCCGGGAACGTCCTCGCGCCCTATCTGCCGGCGCTGCGCCGGGTGGACGGCTCCGACTCGCACGAGGGCCTCGACCCCGGCTTCCCGGACGACTGGCGCCGGGCCGCCGACCAGGACCCGCAGTTCCGCCGGGCCCAGGACGACGAGCGGGACCGCGTGTACTTCGACCCGGCCGTACGGCGCGGCAAGGAGGACGGGCTGCGGACGCTGGGGCAGTTCGCGTACTACGACGCGATGGTGATGCACGGCGACGGCGGCGGCCTCGGCTTCGGCAGTATCCGCGAGCGCGCCCTCGGCCGGGCCCGCCCGCCGGCCCAGGGCGGTGACGAGGTCGCCTACCTGCACGCCTTCCTCGACGAGCGGGTGTGGGCCATGAAGCAGGAGCAGGCGCACAGCGACACCAGCCGGGTCGACACGGCGCAGCGCGTCTTCCTGAACGAGGGCAACCTGGACCTCGAGCCGCCGCTGGACTGGCACGTGTACGGGGACGCCTACCACATCGGC
根据毕赤酵母密码子偏好性进行密码子优化后序列
GCCACTGGTTTGGATGATCCAGCTAAGAAGGAAATTGCTATGCAATTGGTTTCTTCTGCTGAAAACTCTTCTTTGGATTGGAAGGCTCAATACAGATATATTGAAGATATTGGTGACGGTAGAGGTTACACTGCTGGTATTATTGGTTTCTGTTCTGGTACTGGAGATATGCTGGATTTGGTTGAATTGTACGGTGAAAGATCTCCAGGTAACGTTTTGGCTCCATACTTGCCAGCTTTGAGAAGAGTTGATGGTTCTGATTCTCATGAAGGTTTGGATCCAGGTTTTCCAGATGATTGGAGAAGAGCTGCTGATCAAGATCCTCAATTCAGAAGAGCCCAAGATGATGAAAGAGATAGAGTTTACTTTGATCCAGCTGTTAGAAGAGGAAAGGAAGATGGTTTGAGAACTTTGGGTCAATTTGCTTACTACGATGCTATGGTTATGCATGGTGACGGTGGTGGTTTGGGTTTCGGTTCTATTAGAGAAAGAGCCTTGGGTAGAGCTAGACCTCCAGCTCAAGGTGGAGATGAAGTTGCTTACTTGCATGCTTTCTTGGATGAAAGAGTTTGGGCTATGAAGCAAGAACAAGCTCATTCTGATACTTCTAGAGTTGATACTGCTCAAAGAGTTTTCTTGAACGAAGGTAACTTGGATTTGGAACCACCATTGGATTGGCATGTTTACGGAGATGCTTACCATATTGGT
SEQ ID NO:2 （CsnA氨基酸序列）
Ala Thr Gly Leu Asp Asp Pro Ala Lys Lys Glu Ile Ala Met Gln Leu ValSer Ser Ala Glu Asn Ser Ser Leu Asp Trp Lys Ala Gln Tyr Arg Tyr Ile Glu AspIle Gly Asp Gly Arg Gly Tyr Thr Ala Gly Ile Ile Gly Phe Cys Ser Gly Thr GlyAsp Met Leu Asp Leu Val Glu Leu Tyr Gly Glu Arg Ser Pro Gly Asn Val Leu AlaPro Tyr Leu Pro Ala Leu Arg Arg Val Asp Gly Ser Asp Ser His Glu Gly Leu AspPro Gly Phe Pro Asp Asp Trp Arg Arg Ala Ala Asp Gln Asp Pro Gln Phe Arg ArgAla Gln Asp Asp Glu Arg Asp Arg Val Tyr Phe Asp Pro Ala Val Arg Arg Gly LysGlu Asp Gly Leu Arg Thr Leu Gly Gln Phe Ala Tyr Tyr Asp Ala Met Val Met HisGly Asp Gly Gly Gly Leu Gly Phe Gly Ser Ile Arg Glu Arg Ala Leu Gly Arg AlaArg Pro Pro Ala Gln Gly Gly Asp Glu Val Ala Tyr Leu His Ala Phe Leu Asp GluArg Val Trp Ala Met Lys Gln Glu Gln Ala His Ser Asp Thr Ser Arg Val Asp ThrAla Gln Arg Val Phe Leu Asn Glu Gly Asn Leu Asp Leu Glu Pro Pro Leu Asp TrpHis Val Tyr Gly Asp Ala Tyr His Ile Gly
实例1：壳聚糖酶CsnA基因密码子优化及表达载体制备
获得天蓝色链霉菌CsnA（SCO0677，GenBank：NC_003888.3）基因序列，根据相关文献去除信号肽，保留酶的成熟区，构建pPIC9K载体质粒（小写为载体序列，大写为CsnA成熟区序列）。
pPIC9K质粒载体的构建方法：
tatctctcgagaaaagagaggctgaagctGCCACTGGTTTGGATGATCCAGCTAAGAAGGAAATTGCTATGCAATTGGTTTCTTCTGCTGAAAACTCTTCTTTGGATTGGAAGGCTCAATACAGATATATTGAAGATATTGGTGACGGTAGAGGTTACACTGCTGGTATTATTGGTTTCTGTTCTGGTACTGGAGATATGCTGGATTTGGTTGAATTGTACGGTGAAAGATCTCCAGGTAACGTTTTGGCTCCATACTTGCCAGCTTTGAGAAGAGTTGATGGTTCTGATTCTCATGAAGGTTTGGATCCAGGTTTTCCAGATGATTGGAGAAGAGCTGCTGATCAAGATCCTCAATTCAGAAGAGCCCAAGATGATGAAAGAGATAGAGTTTACTTTGATCCAGCTGTTAGAAGAGGAAAGGAAGATGGTTTGAGAACTTTGGGTCAATTTGCTTACTACGATGCTATGGTTATGCATGGTGACGGTGGTGGTTTGGGTTTCGGTTCTATTAGAGAAAGAGCCTTGGGTAGAGCTAGACCTCCAGCTCAAGGTGGAGATGAAGTTGCTTACTTGCATGCTTTCTTGGATGAAAGAGTTTGGGCTATGAAGCAAGAACAAGCTCATTCTGATACTTCTAGAGTTGATACTGCTCAAAGAGTTTTCTTGAACGAAGGTAACTTGGATTTGGAACCACCATTGGATTGGCATGTTTACGGAGATGCTTACCATATTGGTCATCATCATCATCATCATTAAttcgccttagacatgactgttcc
实例2：毕赤酵母转化
毕赤酵母转化使用氯化锂法：（1）挑取毕赤酵母GS115于20mL YPD培养基中，30℃震荡培养过夜，次日以1%接种量接种至50 mL YPD培养基中，30℃振荡培养至OD600=0.8~1.0。（2）室温下5000 rpm收集细胞，以25ml灭菌去离子水洗涤一次，室温下5000rpm离心10min。（3）重悬细胞于1mL浓度为 100 mmol/L的氯化锂溶液中，转移至1.5mL离心管。（4）室温下12000 rpm离心1min，弃上清，重悬于400μL浓度为100mmol/L的氯化锂溶液中，按50μL分装，制得感受态细胞，立即进行转化。
转化过程：（1）将质粒pPIC9K-CsnA经SacI内切酶线性化后，胶回收纯化。（2）煮沸鲑鱼精DNA 5min，迅速置于冰上。（3）离心感受态细胞，弃上清，加入240μL PEG3350、1mol/L氯化锂36 μL和2mg/mL鲑鱼精DNA，50 μL胶回收纯化质粒，剧烈旋涡震荡混匀后置于30℃孵育30 min。（4）42℃热休克25 min，室温下8000 rpm，离心10 min。（5）pPICZαA-CsnA质粒转化加入1mL 的YPD培养基，30℃震荡培养2h，以产生博来霉素抗性，培养结束后涂布于含100μg/ml的YPD平板中，pPIC9K-CsnA质粒转化用1ml灭菌去离子水悬浮后涂布于MD平板，30℃培养2~4d。
实例3：毕赤酵母重组子表型筛选
pPIC9K-CsnA筛选：将正常生长于MD平板上的单菌落用消毒牙签蘸取后分别点接于MM与MD平板中，先点MM平板，于30℃培养2~4 d，若在两个平板都正常生长，表型即为Mut+His+（如图1）。
实例4：毕赤酵母重组子PCR鉴定
将pPIC9K-CsnA重组菌置于20mL的 YPD培养基中，30℃震荡培养过夜，提取基因组后，利用下表引物CsnA-F，CsnA-R引物进行PCR扩增鉴定，PCR反应程序为94℃预变性5 min，94℃变性30s，55℃退火45 s，72℃延伸45 s，72 ℃补充延伸10 min，PCR产物经1%琼脂糖凝胶电泳检测，得到708 bp的CsnA基因片段（如图2）。
表1. CsnA基因的引物信息
| 引物名称 | 引物序列 |
| CsnA-F | GCCACTGGTTTGGATGATCCA |
| CsnA-R | ACCAATATGGTAAGCATCTCCGT |
实例5：毕赤酵母重组子多拷贝筛选
将PCR鉴定正确的阳性菌于YPD培养基划线纯化，用消毒牙签蘸取单菌落分别点接于含1mg/ml，2mg/ml，3mg/ml，4mg/ml抗生素的YPD平板上，pPIC9K-CsnA使用遗传霉素G418，选取各个浓度平板上都可以正常生长的菌株，于YPD平板划线纯化后，可用于表达（如图3）。
实例6：壳聚糖酶诱导表达
挑取纯化好的单菌落于25mL的 BMGY培养基中，30℃，250 rpm振荡培养至OD600为2~6（约16~18h），室温5000 rpm离心收集菌体，弃上清，用一定量BMMY培养基重悬菌体，接种于100mL的 BMMY培养基中，使初始OD600为1~2，30 ℃，250 rpm振荡诱导表达168 h，每24 h添加100%甲醇使培养基中甲醇终浓度为0.5%，168 h后4℃，5000 rpm离心，上清则为粗酶液。经SDS-PAGE电泳检测，可见壳聚糖酶蛋白条带，大小为26.2kDa（如图4）。
实例7：壳聚糖酶酶活测定
在离心管中加入1%壳聚糖350 μL 和50 μL粗酶液，50℃水浴10 min，加入600 μLDNS试剂终止反应，沸水浴10 min，冷却后，室温12000 rpm离心5min，于紫外分光光度计测定OD540，以氨基葡萄糖作为参照，酶活定义：每分钟生成1 μmol氨基葡萄糖为一个酶活单位，毕赤酵母高拷贝菌株在适宜的发酵条件下，酶活力可达10.3 U/mL。利用硫酸铵盐析提取酶蛋白，当沉淀饱和度为75%时，蛋白溶液酶活最高达到 78.5 U/mL，此时溶液的比酶活力为545.1 U/mg（根据溶液中的蛋白含量计算），纯化倍数为1.85（如图5和图6所示）。
实例8：壳聚糖酶的最适反应温度和温度稳定性。
利用Q-Sepharose Fast Flow column离子交换柱层析纯化壳聚糖酶，将纯化的酶液分别于等体积柠檬酸缓冲液（pH 4.0, 5.0, 6.0,7.0）和甘氨酸-氢氧化钠缓冲液（pH8.0，9.0）混合，测定不同pH值下的酶活力。分别设定不同温度（20，30，40，50，60，70和80℃），测定不同酶活力。计算相对酶活力，确定最适反应pH值和温度。
壳聚糖酶的最适反应温度和温度稳定性如图 7所示，反应温度在 20℃～70℃范围内，酶活力变化较大；在50℃时，该壳聚糖酶的相对酶活力最高 99% ；而大于50℃，酶活力下降。因此，该壳聚糖酶的最适反应温度为 50℃ 。该酶在 35℃～50℃范围内稳定性较好，其中50℃保存 60 min 时相对酶活力仍残留85%以上，但当温度上升到60℃，酶活力显著下降。此条件下保温30 min和60 min 时，酶活力分别为57%和50%。因此，该壳聚糖酶在温度为35℃～60℃具有一定的稳定性。
实例9：壳聚糖酶的最适反应pH值和酸碱稳定性。
如图8所示，壳聚糖酶的最适作用pH值呈现先升高后降低的趋势，在pH 值为5.5和6.0时，该壳聚糖酶的相对酶活力最高，达97%。因此该壳聚糖酶的最适反应pH值为5.5-6.0。壳聚糖酶在不同pH值条件下保温60 min 后，酶活力都有所下降。如图8所示，在 pH 4.0-8.0 时，相对比较稳定；而当pH 值＜4.0或＞8.0时，酶活力显著下降。因此，认为该酶在pH4.0-8.0范围内，具有一定的稳定性。
实例10：壳聚糖酶的产物结构特征。
为获得壳聚糖酶对底物的位点识别特性及产物的结构特征，进一步使用1H NMR及13C NMR对水解产物进行鉴定，结果见图9。由图 9可知：水解产物的还原末端主要由氨基葡萄糖组成(图9(a))，而非还原末端则同时含有氨基葡萄糖及N乙酰氨基葡萄糖(图9(b))。上述结果说明该酶可同时水解糖苷键GlcN-GlcN及GlcN-Glc NAc，与Ⅲ型壳聚糖酶底物识别特性相符。通过薄层层析分析发现，壳聚糖酶降解壳聚糖2h的产物主要为二糖、三糖和少量四糖，结果见图10。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均应包含在本发明的包含范围之内。
序列表
<110> 南宁新科健生物技术有限公司
<120> 一种新型壳聚糖酶及其制备方法
<141> 2021-08-20
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 708
<212> DNA
<213> 天蓝色链霉菌(Streptomyces coeruleus)
<400> 1
gccactggtt tggatgatcc agctaagaag gaaattgcta tgcaattggt ttcttctgct 60
gaaaactctt ctttggattg gaaggctcaa tacagatata ttgaagatat tggtgacggt 120
agaggttaca ctgctggtat tattggtttc tgttctggta ctggagatat gctggatttg 180
gttgaattgt acggtgaaag atctccaggt aacgttttgg ctccatactt gccagctttg 240
agaagagttg atggttctga ttctcatgaa ggtttggatc caggttttcc agatgattgg 300
agaagagctg ctgatcaaga tcctcaattc agaagagccc aagatgatga aagagataga 360
gtttactttg atccagctgt tagaagagga aaggaagatg gtttgagaac tttgggtcaa 420
tttgcttact acgatgctat ggttatgcat ggtgacggtg gtggtttggg tttcggttct 480
attagagaaa gagccttggg tagagctaga cctccagctc aaggtggaga tgaagttgct 540
tacttgcatg ctttcttgga tgaaagagtt tgggctatga agcaagaaca agctcattct 600
gatacttcta gagttgatac tgctcaaaga gttttcttga acgaaggtaa cttggatttg 660
gaaccaccat tggattggca tgtttacgga gatgcttacc atattggt 708
<210> 2
<211> 236
<212> PRT
<213> 天蓝色链霉菌(Streptomyces coeruleus)
<400> 2
Ala Thr Gly Leu Asp Asp Pro Ala Lys Lys Glu Ile Ala Met Gln Leu
1 5 10 15
Val Ser Ser Ala Glu Asn Ser Ser Leu Asp Trp Lys Ala Gln Tyr Arg
20 25 30
Tyr Ile Glu Asp Ile Gly Asp Gly Arg Gly Tyr Thr Ala Gly Ile Ile
35 40 45
Gly Phe Cys Ser Gly Thr Gly Asp Met Leu Asp Leu Val Glu Leu Tyr
50 55 60
Gly Glu Arg Ser Pro Gly Asn Val Leu Ala Pro Tyr Leu Pro Ala Leu
65 70 75 80
Arg Arg Val Asp Gly Ser Asp Ser His Glu Gly Leu Asp Pro Gly Phe
85 90 95
Pro Asp Asp Trp Arg Arg Ala Ala Asp Gln Asp Pro Gln Phe Arg Arg
100 105 110
Ala Gln Asp Asp Glu Arg Asp Arg Val Tyr Phe Asp Pro Ala Val Arg
115 120 125
Arg Gly Lys Glu Asp Gly Leu Arg Thr Leu Gly Gln Phe Ala Tyr Tyr
130 135 140
Asp Ala Met Val Met His Gly Asp Gly Gly Gly Leu Gly Phe Gly Ser
145 150 155 160
Ile Arg Glu Arg Ala Leu Gly Arg Ala Arg Pro Pro Ala Gln Gly Gly
165 170 175
Asp Glu Val Ala Tyr Leu His Ala Phe Leu Asp Glu Arg Val Trp Ala
180 185 190
Met Lys Gln Glu Gln Ala His Ser Asp Thr Ser Arg Val Asp Thr Ala
195 200 205
Gln Arg Val Phe Leu Asn Glu Gly Asn Leu Asp Leu Glu Pro Pro Leu
210 215 220
Asp Trp His Val Tyr Gly Asp Ala Tyr His Ile Gly
225 230 235
<210> 3
<211> 21
<212> DNA
<213> 引物(Primer)
<400> 3
gccactggtt tggatgatcc a 21
<210> 4
<211> 23
<212> DNA
<213> 引物(Primer)
<400> 4
accaatatgg taagcatctc cgt 23
Claims (10)
1.一种新型壳聚糖酶CsnA，其特征在于，其核苷酸序列如SEQ ID NO：1所示。
2.如权利要求1所述的新型壳聚糖酶CsnA，其特征在于，其氨基酸序列如SEQ ID NO：2所示。
3.如权利要求1或2所述的新型壳聚糖酶CsnA在降解壳聚糖/制备壳聚糖中的应用；在制备降解壳聚糖/制备壳聚糖的酶制剂的应用。
4.一种重组表达载体，其特征在于，包括权利要求1所述新型壳聚糖酶CsnA。
5.如权利要求1所述新型壳聚糖酶CsnA的制备方法，其特征在于，
提供壳聚糖酶CsnA基因、构建载体质粒和毕赤酵母转化；
对所述载体质粒进行转化；
对所述壳聚糖酶CsnA基因进行扩增；
其中，所述壳聚糖酶CsnA的核苷酸序列如SEQ ID NO:1所示。
6.根据权利要求5所述新型壳聚糖酶CsnA的制备方法，其特征在于，将所述壳聚糖酶CsnA基因进行扩增的步骤中，用于扩增的引物包括正向引物和反向引物，所述正向引物的核苷酸序列如SEQ ID NO:3所示，所述反向引物的核苷酸序列如SEQ ID NO:4所示。
7.根据权利要求5所述新型壳聚糖酶CsnA的制备方法，其特征在于，所述载体为毕赤酵母表达载体。
8.根据权利要求5所述新型壳聚糖酶CsnA的制备方法，其特征在于，所述表达菌株为毕赤酵
母表达菌株。
9.一种酶制剂，其特征在于：包含有权利要求1所述的新型壳聚糖酶CsnA。
10.如权利要求9所述的酶制剂在降解壳聚糖/制备壳寡糖中的应用。
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| CN110819611A (zh) * | 2020-01-10 | 2020-02-21 | 中国农业科学院生物技术研究所 | 壳聚糖酶突变体及其编码基因和应用 |
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