CN105483102A - 耐产物抑制的β-N-乙酰葡糖胺酶HJ5nag及其制备方法 - Google Patents
耐产物抑制的β-N-乙酰葡糖胺酶HJ5nag及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种耐产物抑制的β-N-乙酰葡糖胺酶及其制备方法,所述β-N-乙酰葡糖胺酶的氨基酸序列如SEQ?ID?NO.1所示;所述方法包括:首先用包含β-N-乙酰葡糖胺酶基因hJ5nag的重组载体,重组载体转化宿主细胞,得重组菌株;然后培养重组菌株,诱导重组β-N-乙酰葡糖胺酶HJ5nag表达;最后回收所表达的β-N-乙酰葡糖胺酶HJ5nag。本发明的β-N-乙酰葡糖胺酶,最适pH6;最适温度为45℃;可催化水解几丁寡糖;在反应体系加入终浓度20mM?N-乙酰-D-胺基葡萄糖时,该酶仍保留约69.5%活性;可与内切几丁质酶协同降解几丁质,可应用于海产品加工、医学、功能性食品等行业。
Description
技术领域
本发明属于生物工程技术领域,尤其涉及一种耐产物抑制的β-N-乙酰葡糖胺酶及其制备方法。
背景技术
几丁质是由N-乙酰-D-胺基葡萄糖单体通过β-1,4键组成的多糖,广泛存在于真菌、昆虫细胞壁以及甲壳类动物外骨骼中,其含量仅次于纤维素,因此几丁质的水解具有重要的意义。内切几丁质酶可以把几丁质降解为几丁寡糖,而β-N-乙酰葡糖胺酶可催化几丁寡糖降解为N-乙酰-D-胺基葡萄糖,因而β-N-乙酰葡糖胺酶对几丁质的完全水解起到关键性的作用;同时,由于内切几丁质酶会被几丁寡糖抑制,所以β-N-乙酰葡糖胺酶可解除几丁寡糖对内切几丁质酶的抑制作用,从而与内切几丁质酶产生协同作用(Yangetal.,JournalofAgriculturalandFoodChemistry,2014,62:5181–5190)。但是,大多数β-N-乙酰葡糖胺酶易受到水解产物即N-乙酰-D-胺基葡萄糖的抑制,需要耐产物抑制的β-N-乙酰葡糖胺酶才能使几丁质更有效地彻底水解(Yangetal.,JournalofAgriculturalandFoodChemistry,2014,62:5181–5190)。根据氨基酸序列同源性,β-N-乙酰葡糖胺酶主要归类于糖苷水解酶第3、20、73和84家族(Cantareletal.,Nucleicacidsresearch,2009,37:D233–D238)。β-N-乙酰葡糖胺酶被广泛应用于功能性食品、保健品、化妆品、制药、饲料添加剂和生物杀虫剂等领域(Yangetal.,JournalofAgriculturalandFoodChemistry,2014,62:5181–5190)。例如,β-N-乙酰葡糖胺酶水解几丁寡糖的产物N-乙酰-D-胺基葡萄糖单体具有抗炎作用,对于溃疡结肠炎和其他的胃肠炎症的治疗有很好的效果(Salvatoreetal.,Alimentarypharmacology&therapeutics,2000,14:1567–1579),N-乙酰-D-胺基葡萄糖也应用于治疗儿童慢性肠炎疾病以及骨关节炎(Shikhmanetal.,Annalsoftherheumaticdiseases,2005,64:89–94)。
发明内容
本发明的目的在于提供一种耐产物抑制的β-N-乙酰葡糖胺酶及其制备方法,旨在解决现有的β-N-乙酰葡糖胺酶易受到水解产物即N-乙酰-D-胺基葡萄糖的抑制从而导致几丁质水解效率较低的问题。
本发明是这样实现的,一种耐产物抑制的β-N-乙酰葡糖胺酶,所述耐产物抑制的β-N-乙酰葡糖胺酶的氨基酸序列如SEQIDNO.1所示。
进一步,所述耐产物抑制的β-N-乙酰葡糖胺酶总共含535个氨基酸,理论分子量为55.89kDa,其中N端有25个氨基酸为预测信号肽序列MNRRRGRAIAAATVLAASLAGCSPA,成熟的β-N-乙酰葡糖胺酶HJ5nag含510个氨基酸。
本发明的另一目的在于提供一种所述耐产物抑制的β-N-乙酰葡糖胺酶的制备方法,所述耐产物抑制的β-N-乙酰葡糖胺酶的制备方法包括以下步骤:
首先用包含β-N-乙酰葡糖胺酶基因hJ5nag的重组载体,重组载体为pEasy-E2-hJ5nag,将乙酰葡糖胺酶基因插入到表达载体中,使核苷酸序列与表达调控序列相连接,重组载体转化宿主细胞,得重组菌株;
然后培养重组菌株,诱导重组β-N-乙酰葡糖胺酶HJ5nag表达;
最后回收所表达的β-N-乙酰葡糖胺酶HJ5nag。
进一步,所述宿主细胞为大肠杆菌细胞,将重组大肠杆菌表达质粒转化大肠杆菌细胞BL21(DE3),得到重组菌株BL21(DE3)/hJ5nag。
本发明的另一目的在于提供一种包含所述耐产物抑制的β-N-乙酰葡糖胺酶的重组载体。
进一步,所述重组载体是将β-N-乙酰葡糖胺酶的基因插入到表达载体中,使核苷酸序列与表达调控序列相连接。
本发明的另一目的在于提供一种包含所述耐产物抑制的β-N-乙酰葡糖胺酶的重组菌株。
进一步,所述重组菌株为大肠杆菌、酵母菌、芽孢杆菌或乳酸杆菌。
本发明提供的耐产物抑制的β-N-乙酰葡糖胺酶及其制备方法,采用新活性的β-N-乙酰葡糖胺酶:最适pH6;最适温度为45℃;可催化水解几丁寡糖;在反应体系加入终浓度20mMN-乙酰-D-胺基葡萄糖时,该酶仍保留约69.5%活性;该酶可与内切几丁质酶协同降解几丁质。本发明的β-N-乙酰葡糖胺酶可应用于海产品加工、医学、功能性食品等行业。
附图说明
图1是本发明实施例提供的耐产物抑制的β-N-乙酰葡糖胺酶的制备方法流程图。
图2是本发明实施例提供的在大肠杆菌中表达的重组β-N-乙酰葡糖胺酶HJ5nag的SDS-PAGE分析;
图中:M:蛋白质Marker;CK:含载体pEasy-E2大肠杆菌菌体破碎上清液;S1:含有重组载体pEasy-E2-hJ5nag的大肠杆菌菌体破碎上清液;S2:纯化的重组β-N-乙酰葡糖胺酶HJ5nag。
图3是本发明实施例提供的纯化的重组β-N-乙酰葡糖胺酶HJ5nag的pH活性。
图4是本发明实施例提供的纯化的重组β-N-乙酰葡糖胺酶HJ5nag的pH稳定性。
图5是本发明实施例提供的纯化的重组β-N-乙酰葡糖胺酶HJ5nag的热活性。
图6是本发明实施例提供的纯化的重组β-N-乙酰葡糖胺酶HJ5nag的热稳定性。
图7是本发明实施例提供的纯化的重组β-N-乙酰葡糖胺酶HJ5nag水解二乙酰壳二糖及四乙酰壳四糖的产物分析;
图中:M:N-乙酰-D-胺基葡萄糖;CK1:二乙酰壳二糖与灭活的HJ5nag;S1:二乙酰壳二糖与有活性的HJ5nag,CK2:四乙酰壳四糖与灭活的HJ5nag;S2:四乙酰壳四糖与有活性的HJ5nag。
图8是本发明实施例提供的纯化的重组β-N-乙酰葡糖胺酶HJ5nag在不同浓度N-乙酰-D-胺基葡萄糖中的活性。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
下面结合附图对本发明的应用原理作详细的描述。
本发明所述β-N-乙酰葡糖胺酶HJ5nag可得自微杆菌(Microbacteriumsp.)。HJ5nag的氨基酸序列如SEQIDNO.1所示。
微杆菌(Microbacteriumsp.),遗传资源属于微生物,获取方式为自行采集;于2010年10月,由周峻沛在中国、云南省、楚雄彝族自治州采集。
本发明的β-N-乙酰葡糖胺酶HJ5nag总共含535个氨基酸,理论分子量为55.89kDa,其中N端有25个氨基酸为预测信号肽序列MNRRRGRAIAAATVLAASLAGCSPA,成熟的β-N-乙酰葡糖胺酶HJ5nag含510个氨基酸。该酶最适pH6;最适温度为45℃;可催化水解几丁寡糖;在反应体系加入终浓度20mMN-乙酰-D-胺基葡萄糖时,该酶仍保留约69.5%活性;该酶可与内切几丁质酶协同降解几丁质。
本发明提供了编码上述β-N-乙酰葡糖胺酶的基因hJ5nag,该基因序列如SEQIDNO.2所示。
本发明通过基因组测序的方法获得β-N-乙酰葡糖胺酶HJ5nag的编码基因hJ5nag,其全长1608bp,起始密码为ATG,终止密码为TGA。
经BLAST比对,该β-N-乙酰葡糖胺酶HJ5nag为糖苷水解酶第20家族序列,其全序列与GenBank中的Microbacteriumsp.Root180来源的β-N-乙酰葡糖胺酶序列(WP_056119055)具有最高的氨基酸序列一致性,为76.3%。该Microbacteriumsp.Root180来源的蛋白只是通过序列相似性判断为β-N-乙酰葡糖胺酶,其活性还未研究,无法得知该蛋白的pH活性范围、热活性范围及N-乙酰-D-胺基葡萄糖的抑制程度等酶学性质。
本发明还提供了包含上述β-N-乙酰葡糖胺酶基因hJ5nag的重组载体,优选为pEasy-E2-hJ5nag。将本发明的乙酰葡糖胺酶基因插入到表达载体中,使其核苷酸序列与表达调控序列相连接,作为本发明的一个最优选的实施方案,将本发明的β-N-乙酰葡糖胺酶基因和表达载体pEasy-E2通过T-A方式相连接,得到重组大肠杆菌表达质粒pEasy-E2-hJ5nag。
本发明还提供了包含上述β-N-乙酰葡糖胺酶基因hJ5nag的重组菌株,优选所述菌株为大肠杆菌、酵母菌、芽孢杆菌或乳酸杆菌,优选为重组菌株BL21(DE3)/hJ5nag。
如图1所示,本发明实施例的耐产物抑制的β-N-乙酰葡糖胺酶及其制备方法包括以下步骤:
S101:用包含β-N-乙酰葡糖胺酶基因hJ5nag的重组载体,重组载体为pEasy-E2-hJ5nag,将乙酰葡糖胺酶基因插入到表达载体中,使核苷酸序列与表达调控序列相连接,重组载体转化宿主细胞,得重组菌株;
S102:培养重组菌株,诱导重组β-N-乙酰葡糖胺酶HJ5nag表达;
S103:回收所表达的β-N-乙酰葡糖胺酶HJ5nag。
其中,优选所述宿主细胞为大肠杆菌细胞,优选将重组大肠杆菌表达质粒转化大肠杆菌细胞BL21(DE3),得到重组菌株BL21(DE3)/hJ5nag。
下面结合试验对本发明的应用原理作进一步的描述。
试验材料和试剂
1、菌株及载体:微杆菌(Microbacteriumsp.)同文献报道菌种性质,如中国普通微生物菌种保藏管理中心菌株MicrobacteriumtestaceumCGMCC1.10357;大肠杆菌EscherichiacoliBL21(DE3)和表达载体pEasy-E2购于Novagen公司。
2、酶类及其它生化试剂:DNA聚合酶和dNTP购自TaKaRa公司;pNP(p-nitrophenol)、pNP-GlcNAc(p-nitrophenylβ-N-acetylglucosaminide)、p-nitrophenyl-α-D-galactopyranoside、p-nitrophenylβ-D-glucopyranoside购自Sigma公司;pNP-GalNAc(p-nitrophenylβ-N-acetylgalactosaminide)、二乙酰壳二糖及四乙酰壳四糖购自百灵威科技公司;GenomicDNAClean&Concentration试剂盒购自ZymoResearch公司;TureseqTMDNASamplePreparationKit购自Illumima公司;商业内切几丁质酶(来源于Streptomycesgriseus)购于上海源叶生物科技有限公司;其它都为国产试剂(均可从普通生化试剂公司购买得到)。
3、培养基:
LB培养基:Peptone10g,Yeastextract5g,NaCl10g,加蒸馏水至1000ml,pH自然(约为7)。固体培养基在此基础上加2.0%(w/v)琼脂。
说明:以下实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
3β-N-乙酰葡糖胺酶基因HJ5nag的克隆
提取微杆菌基因组DNA:将培养2d的液体菌液离心取菌体,加入1mL溶菌酶,37℃处理60min,再加入裂解液,裂解液组成为:50mMTris,20mMEDTA,NaCl500mM,2%SDS(w/v),pH8.0,70℃水浴裂解60min,每隔10min混匀一次,在4℃下10000rpm离心5min。取上清于酚/氯仿中抽提除去杂蛋白,再取上清加入等体积异丙醇,于室温静置5min后,4℃下10000rpm离心10min。弃上清,沉淀用70%的乙醇洗涤两次,真空干燥,加入适量TE溶解,置于-20℃备用。
用超声打断仪Biorupter将5μg的微杆菌基因组打断为400–600bp的片段,用GenomicDNAClean&Concentration试剂盒对打断的DNA片段进行纯化,纯化后用TureseqTMDNASamplePreparationKit进行DNA片段的末端补平、3'端加A碱基和加接头、及DNA片段的PCR扩增(操作按试剂盒说明书进行)。用MiSeq基因组测序仪(Illumima公司)对上述制备好的文库进行基因组测序。
基因组测序得到的数据经读码框预测和本地BLAST比对,得到β-N-乙酰葡糖胺酶基因hJ5nag,该基因序列如SEQIDNO.2所示。
4重组β-N-乙酰葡糖胺酶HJ5nag的制备
以5'GGGTGCAGCCCCGCCGC3'和5'CTCGGTGGCCCAGTCGATCTCGC3'为引物对,微杆菌基因组DNA为模板,进行PCR扩增。PCR反应参数为:94℃变性5min;然后94℃变性30sec,64℃退火30sec,72℃延伸2min,30个循环后72℃保温10min。PCR结果得到β-N-乙酰葡糖胺酶基因hJ5nag,并将该酶基因与表达载体pEasy-E2相连接,获得含有β-N-乙酰葡糖胺酶基因hJ5nag的重组质粒pEasy-E2-hJ5nag,将pEasy-E2-hJ5nag转化大肠杆菌BL21(DE3),获得重组大肠杆菌菌株BL21(DE3)/hJ5nag。
取含有重组质粒pEasy-E2-hJ5nag的重组大肠杆菌菌株BL21(DE3)/hJ5nag,以0.1%的接种量接种于LB(含50μgmL-1Amp)培养液中,37℃快速振荡16h。然后将此活化的菌液以1%接种量接种到新鲜的LB(含50μgmL-1Amp)培养液中,快速振荡培养约2–3h(OD600达到0.6–1.0)后,加入终浓度0.7mM的IPTG进行诱导,于20℃继续振荡培养约20h。12000rpm离心5min,收集菌体。用适量的pH7.0Tris-HCl缓冲液悬浮菌体后,于低温水浴下超声波破碎菌体,破碎后在4℃下13,000rpm离心10min,吸取上清进行SDS-PAGE分析。SDS-PAGE结果(图2)表明,重组β-N-乙酰葡糖胺酶HJ5nag在大肠杆菌中得到了表达,经纯化后,产物为单一条带。
下面对纯化的重组β-N-乙酰葡糖胺酶HJ5nag的性质及应用效果作详细的描述。
1、纯化的重组β-N-乙酰葡糖胺酶HJ5nag的活性分析
纯化的重组β-N-乙酰葡糖胺酶HJ5nag的活性测定方法采用pNP法:将底物溶于0.1M缓冲液中,使其终浓度为2mM;反应体系含50μL适量酶液,450μL底物;底物在反应温度下预热5min后,加入酶液后再反应10min,然后加2mL1MNa2CO3终止反应,冷却至室温后在405nm波长下测定OD值。1个酶活单位(U)定义为在给定的条件下每分钟分解pNP类化合物产生1μmolpNP所需的酶量。对底物二乙酰壳二糖、四乙酰壳四糖、几丁质的活性测定方法采用DNS法:将底物溶于0.1M缓冲液中,使其终浓度为0.5%;反应体系含100μL适量酶液,900μL底物;底物在反应温度下预热5min后,加入酶液后再反应适当时间,然后加1.5mLDNS终止反应,沸水煮5min,冷却至室温后在540nm波长下测定OD值;1个酶活单位(U)定义为在给定的条件下每分钟分解底物产生1μmol还原糖(以乙酰葡糖胺计)所需的酶量。
2、纯化的重组β-N-乙酰葡糖胺酶HJ5nag的pH活性和pH稳定性测定:
酶的最适pH测定:将β-N-乙酰葡糖胺酶HJ5nag在37℃下和0.1MpH5.0–9.0的缓冲液中进行酶促反应。酶的pH稳定性测定:将酶液置于0.1MpH5.0–12.0的缓冲液中,在37℃下处理1h,然后在pH6及37℃下进行酶促反应,以未处理的酶液作为对照。缓冲液为:0.1MMcIlvainebuffer(pH5.0–8.0)和0.1Mglycine-NaOH(pH9.0–12.0)。以pNP-GlcNAc为底物,反应10min,测定HJ5nag的酶学性质。结果表明:HJ5nag的最适pH为6,在pH5.5–8.0的范围内维持62%以上的酶活性(图3);经pH6.0–10.0的缓冲液处理1h,该酶剩余酶活约55%以上(图4)。
3、纯化的重组β-N-乙酰葡糖胺酶HJ5nag的热活性及热稳定性测定:
酶的最适温度测定:在pH6的缓冲液中,于0–60℃下进行酶促反应。酶的热稳定性测定:将同样酶量的酶液置于30℃、37℃、45℃和50℃中,处理0–60min后,在pH6及45℃下进行酶促反应,以未处理的酶液作为对照。以pNP-GlcNAc为底物,反应10min,测定HJ5nag的酶学性质。结果表明:HJ5nag的最适温度为45℃(图5);该酶在30℃下处理1h,酶保持稳定(图6)。
4、不同金属离子及化学试剂对纯化的重组β-N-乙酰葡糖胺酶HJ5nag活力的影响:
在酶促反应体系中加入1mM和10mM或0.5%(v/v)和1%(v/v)的金属离子及化学试剂,研究其对酶活性的影响。在45℃及pH6条件下,以pNP-GlcNAc为底物测定酶活性。结果(表1)表明,1mM和10mM的SDS、AgNO3和HgCl2可完全抑制HJ5nag;10mM的FeSO4对HJ5nag的抑制较强,10mM的ZnSO4对HJ5nag的抑制较弱;而0.5%(v/v)的Tween-80可激活HJ5nag;其余金属离子和化学试剂对HJ5nag基本无影响。
表1.金属离子及化学试剂对重组β-N-乙酰葡糖胺酶HJ5nag活力的影响
5、纯化的重组β-N-乙酰葡糖胺酶HJ5nag对底物的降解:
在pH6及45℃下,重组β-N-乙酰葡糖胺酶HJ5nag对pNP-GlcNAc、pNP-GalNAc、二乙酰壳二糖及四乙酰壳四糖的酶活分别为1773.1Umg-1、146.0Umg-1、481.4Umg-1、445.0Umg-1,但该酶对几丁质、p-nitrophenyl-α-D-galactopyranoside及p-nitrophenylβ-D-glucopyranoside均无酶活。
6、纯化的重组β-N-乙酰葡糖胺酶HJ5nag水解几丁寡糖的产物分析:
产物分析反应体系含80μL0.5%(w/v)的几丁寡糖和80μL纯酶液,在pH6及37℃下,反应6h。采用薄层层析法进行产物分析,薄层层析法步骤如下:
(1)配制展开剂(冰醋酸、双蒸水、正丁醇体积比为1:1:2,配制适量)倒入展开槽,静置约30min;
(2)将硅胶板放于110℃烘箱活化30min,冷却后划线,点样(每次0.5μL,吹干,共点3次);
(3)将点样的一端硅胶板朝下放入展开槽,点样点不可没入展开剂;
(4)待展开剂到硅胶板上沿1.5cm时,取出硅胶板,吹干,再展一次;
(5)第二次展开结束后,硅胶板直接浸入适量显色剂(1g二苯胺溶入50ml丙酮,溶解后加1ml苯胺及5ml85%的磷酸,混匀,现用现配);
(6)几秒后,立即取出硅胶板并放入90℃烘箱10-15min,使斑点显色
结果表明,HJ5nag可将二乙酰壳二糖及四乙酰壳四糖水解为N-乙酰-D-胺基葡萄糖单糖(图7)。
7、N-乙酰-D-胺基葡萄糖对重组β-N-乙酰葡糖胺酶HJ5nag活性的影响
在酶促反应体系中加入终浓度为0–20mMN-乙酰-D-胺基葡萄糖,于pH6.0及45℃下进行酶促反应。以pNP-GlcNAc为底物,反应10min,测定纯化的HJ5nag的酶学性质。结果表明:当反应体系加入终浓度20mMN-乙酰-D-胺基葡萄糖时,该酶仍保留约69.5%的活性,表明N-乙酰-D-胺基葡萄糖对HJ5nag抑制作用较低(图8)。
8、β-N-乙酰葡糖胺酶HJ5nag与几丁质酶协同作用水解几丁质
几丁质预处理:几丁质用85%磷酸溶解后,再用双蒸水一直洗涤至pH呈中性,成为胶体几丁质。在pH6.0及25℃条件下,采用DNS法,分别用海源叶生物科技有限公司的商业内切几丁质酶和HJ5nag水解胶体几丁质,反应2h;同时添加内切几丁质酶和HJ5nag水解胶体几丁质;内切几丁质酶和HJ5nag在不同时间条件下水解胶体几丁质。结果(表2)表明:β-N-乙酰葡糖胺酶HJ5nag与商业内切几丁质酶可协同降解胶体几丁质,同时添加内切几丁质酶和HJ5nag或添加内切几丁质酶作用30min后加入HJ5nag再作用1h30min,还原糖生成量都可提高大约1倍。
表2.β-N-乙酰葡糖胺酶HJ5nag与商业内切几丁质酶协同降解几丁质
注:CK:内切几丁质酶与底物作用2h;S1:HJ5nag与底物作用2h;S2:内切几丁质酶与HJ5nag共同作用2h;S3:内切几丁质酶作用30min后加入HJ5nag再作用1h30min;S4:内切几丁质酶作用1h后加入HJ5nag再作用1h;S5:内切几丁质酶作用1h30min后加入HJ5nag再作用30min。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种耐产物抑制的β-N-乙酰葡糖胺酶,其特征在于,所述耐产物抑制的β-N-乙酰葡糖胺酶的氨基酸序列如SEQIDNO.1所示。
2.如权利要求1所述的耐产物抑制的β-N-乙酰葡糖胺酶,其特征在于,所述耐产物抑制的β-N-乙酰葡糖胺酶总共含535个氨基酸,理论分子量为55.89kDa,其中N端有25个氨基酸为预测信号肽序列MNRRRGRAIAAATVLAASLAGCSPA,成熟的β-N-乙酰葡糖胺酶HJ5nag含510个氨基酸。
3.一种编码权利要求1所述的β-N-乙酰葡糖胺酶HJ5nag的β-N-乙酰葡糖胺酶基因hJ5nag,其特征在于其核苷酸序列如SEQIDNO.2所示。
4.一种如权利要求1所述耐产物抑制的β-N-乙酰葡糖胺酶的制备方法,其特征在于,所述耐产物抑制的β-N-乙酰葡糖胺酶的制备方法包括以下步骤:
首先用包含β-N-乙酰葡糖胺酶基因hJ5nag的重组载体,重组载体为pEasy-E2-hJ5nag,将乙酰葡糖胺酶基因插入到表达载体中,使核苷酸序列与表达调控序列相连接,重组载体转化宿主细胞,得重组菌株;
然后培养重组菌株,诱导重组β-N-乙酰葡糖胺酶HJ5nag表达;
最后回收所表达的β-N-乙酰葡糖胺酶HJ5nag。
5.如权利要求4所述耐产物抑制的β-N-乙酰葡糖胺酶的制备方法,其特征在于,所述宿主细胞为大肠杆菌细胞,将重组大肠杆菌表达质粒转化大肠杆菌细胞BL21(DE3),得到重组菌株BL21(DE3)/hJ5nag。
6.一种包含权利要求1所述耐产物抑制的β-N-乙酰葡糖胺酶的重组载体。
7.如权利要求6所述的重组载体,其特征在于,所述重组载体是将β-N-乙酰葡糖胺酶的基因插入到表达载体中,使核苷酸序列与表达调控序列相连接。
8.一种包含权利要求1所述耐产物抑制的β-N-乙酰葡糖胺酶的重组菌株。
9.如权利要求8所述的重组菌株,其特征在于,所述重组菌株为大肠杆菌、酵母菌、芽孢杆菌或乳酸杆菌。
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