CN115232805A - 一种硫酸软骨素裂解酶及其重组菌株和应用 - Google Patents
一种硫酸软骨素裂解酶及其重组菌株和应用 Download PDFInfo
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Abstract
本发明公开了一种硫酸软骨素裂解酶及其重组菌株和应用。所述硫酸软骨素裂解酶的氨基酸序列如SEQ ID NO.3所示,编码氨基酸序列的核苷酸序列如SEQ ID NO.1所示。所述硫酸软骨素裂解酶可快速降解透明质酸,硫酸软骨素(A、B、C和D)为不饱和二糖。本发明还构建了含所述硫酸软骨素裂解酶编码基因的重组表达载体和重组菌株。通过诱导和补料同时进行的工艺,在吨级水平实现了重组大肠杆菌菌株的高密度发酵及所述硫酸软骨素裂解酶的水溶性高效表达,发酵液酶活达到390.8 U/mL。重组枯草芽孢杆菌的发酵液酶活达到30.9 U/mL。本发明还提供了利用所述硫酸软骨素裂解酶制备特定聚合物的生物活性硫酸软骨素寡糖的方法,该方法酶用量少且产物结构可控。
Description
技术领域
本发明涉及一种高表达水溶性硫酸软骨素裂解酶及其重组菌株和应用,属于生物工程技术领域。
背景技术
糖胺聚糖(GAG),根据其单糖组成、糖苷键连接形式以及硫酸化等差异,分为透明质酸、肝素、硫酸乙酰肝素、硫酸软骨素、硫酸皮肤素及硫酸角质素等,在细胞识别、神经细胞发育等多方面发挥重要作用。低分子量GAG因水溶性、人体吸收率和生物活性均高于多糖,在医药、食品、保健品等大健康领域具有广泛的市场需求。基于糖胺聚糖降解酶的生物酶法制备低分子量GAG相对高效、温和,产物结构明确,应用前景好。而我国目前能供应的糖胺聚糖降解酶酶制剂,量少且价格昂贵,完全不能满足生产需要。因此,迫切需要挖掘具有较高酶活力、表达量高且制备工艺简便的糖胺聚糖降解酶。
目前已报道有来源于肝素黄杆菌、芽抱杆菌属、链霉菌属、粘质沙雷氏菌、肝素黄杆菌、温和气单胞菌、少动鞘氨醇单胞菌、普通变形杆菌、多形拟杆菌(CN 102277345 A)、不动杆菌(CN105802875A)、弧菌FC509(CN 103923899 A和CN 105713890 A)、微杆菌WS15(CN106906161 A)等多种微生物的硫酸软骨素裂解酶。但野生菌株发酵周期较长、工艺复杂、纯化步骤多、酶活及收率较低,不利于工业放大。如专利申请CN 105802875 A公开了一种来源于潮间带污泥的不动杆菌(Acinetobacter sp. C26)及其产生的硫酸软骨素酶ABC。但其中的菌体发酵液需经过硫酸铵沉淀、超滤或透析、离子交换层析、凝胶过滤层析等系列处理才能获得电泳纯硫酸软骨素裂解酶ABC,工艺步骤非常复杂。
异源重组表达是一种简捷高效的获取高表达硫酸软骨素裂解酶的手段。但要实现硫酸软骨素裂解酶产量的大幅提升,不仅需要具有高比活力的酶编码基因,还需要获取能高效稳定表达酶的工程菌株并实现工程菌株的高密度发酵。目前虽有少量具有较高比活力的硫酸软骨素裂解酶的编码基因被挖掘出来,但这些酶的表达量均非常低。如Han et al.(J Biol Chem. 2014)报道了一种高酶活的硫酸软骨素裂解酶HCLase,该酶对透明质酸和硫酸软骨素的比活分别达到450,000 U/mg和330,000 U/mg,但以此构建的含有该酶表达质粒的E. coli BL21(DE3)细胞的产酶量仅为0.02~0.1 mg/L(发酵液)。Wang et al.(Biotechnol J. 2020)报道了Proteus vulgaris chondroitinase ABC I基因的NΔ5/E694P突变体在大肠杆菌中表达,3L发酵罐发酵可获得1.7 g/L的表达量,但总酶活仅为1.0×105 U/L。尤其值得注意的是,目前硫酸软骨素裂解酶工程菌株的发酵规模小,多停留在实验室水平的摇瓶和小体积发酵罐发酵,难以实现硫酸软骨素裂解酶的规模化量产。高密度发酵是获得高外源性蛋白产率的重要策略,通常会在生长阶段获得较大量菌体,进而在生产阶段诱导外源基因表达获得表达产物,但在实际应用中经常发生菌体过早衰老、外源蛋白表达量低,或者菌量很高但不表达目标产物等问题。
枯草芽孢杆菌(Bacillus subtilis)不分泌外毒素,也不含内毒素,蛋白分泌能力强,遗传背景清晰,被美国食品药品监督管理局认定为生物安全菌株(Generally Regardedas Safe,GRAS),是生产异源蛋白的理想宿主,在食品和药品的发酵应用上,较大肠杆菌更有安全性上的优势。但硫酸软骨素裂解酶在枯草芽孢杆菌中的异源表达面临着表达量不高的问题,如专利申请CN106148265 A公开了源自普通变形杆菌的硫酸软骨素裂解酶在枯草芽孢杆菌中的进行异源表达,但其最高酶活仅有21.4 U/mL。
发明内容
针对现有技术的不足,本发明的第一个目的是提供一种可高表达的水溶性硫酸软骨素裂解酶,可快速降解透明质酸以及不同结构硫酸软骨素(A、B、C和D)为不饱和二糖,底物适应性广泛并且具有较高的降解活性。
本发明的第二个目的是提供生产上述硫酸软骨素裂解酶的重组菌株及规模化发酵工艺,该重组菌株可稳定、高效地表达水溶性的硫酸软骨素裂解酶;规模化发酵工艺简单易控,可实现高纯度硫酸软骨素裂解酶的批量化生产,满足工业需求。
本发明的第三个目的是提供利用所述硫酸软骨素裂解酶高效制备可在食品领域应用的生物活性硫酸软骨素寡糖。
为实现上述为实现上述发明目的,本发明采用以下技术方案:
一种硫酸软骨素裂解酶(HClyM),其氨基酸序列如SEQ ID NO. 3(a)所示,或在(a)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有硫酸软骨素裂解酶活性的由(a)衍生的氨基酸序列。
进一步的,编码所述硫酸软骨素裂解酶的氨基酸序列的核苷酸序列如SEQ IDNO.1(i)所示,或对(i)所述的核苷酸序列进行优化表达,包括密码子优化、核糖体结合位点优化或启动子优化后的核苷酸序列。
进一步的,所述硫酸软骨素裂解酶的最适反应温度为50 oC,最适反应pH为7.3。
进一步的,所述的硫酸软骨素裂解酶可快速降解透明质酸,硫酸软骨素(A、B、C和D)为不饱和二糖,降解方式为内切型。
本发明还提供了包含所述硫酸软骨素裂解酶基因的重组表达载体。
进一步的,所述重组表达载体为pET-24a(+)、pET-22b(+)或pMAL-p5X等,所述重组菌株为大肠杆菌BL21(DE3)。
进一步的,所述重组表达载体为pHT43、pP43NMK、pSTOP1622等,所述重组菌株为枯草芽孢杆菌Bacillus. subtilis WB800。
本发明提供了一种上述硫酸软骨素裂解酶的规模化生产方法,包括如下步骤:
1)将制备的重组大肠杆菌的种子液按体积比2~10%的接种量,接种至含有600 L基础发酵培养基的1000L发酵罐中发酵培养,发酵温度为30~38 oC,搅拌速度为200~300 rpm,通气量为10~25 m3/h,pH为6.8~7.4,溶氧为0~30%,发酵时间为5~15 h。
2)待细胞增殖至OD600为15~20时,调整发酵温度为15~30 oC,向发酵液中加入IPTG诱导产酶,IPTG终浓度为0.05~0.25 mM;同时向发酵液补加补料培养基,补料速度为1.0~3.5 L/h,搅拌速度为250~350 rpm,通气量为10~30 m3/h,溶氧量为0~30%;通过向发酵液补加25%氨水控制发酵液的pH值为6.5~8.0,发酵时间为20~40 h。
3)将规模化发酵结束后的培养液经管式离心机离心获得菌体,菌体重悬后破碎,再次离心收集上清液并经镍离子亲和柱分离纯化,获得纯度大于96%、回收率高于76%的所述硫酸软骨素裂解酶。
本发明还提供了一种上述硫酸软骨素裂解酶的制备方法,其特征在于,将重组枯草芽孢杆菌的种子液以1~5%的接种量接种于含有10~50 μg/mL四环素抗性TB液体培养基中,30~38 oC下振荡培养2~8 h后,调节温度为15~30 oC,加入0.2~1.5%(w/v)木糖诱导培养10~20 h;离心收集菌体,菌体破碎后取上清,经固定化金属螯合层析一步纯化获得纯度大于96%、回收率高于85%的硫酸软骨素裂解酶。
本发明还提供了一种利用重组枯草芽孢杆菌所产硫酸软骨素裂解酶制备硫酸软骨素寡糖的方法,包括如下步骤:
将硫酸软骨素裂解酶加入到硫酸软骨素的水溶液中进行酶解反应,所述硫酸软骨素水溶液的浓度为5%~20%,硫酸软骨素裂解酶的用量为1×103~2×104 U/L,反应时间为0.5~ 6 h,反应温度为30 ~50 oC;反应液经超滤分离获得聚合度为2-10的硫酸软骨素寡糖。所述超滤膜包的截留分子量为3~5 kDa。
与现有技术相比,本发明有益效果主要体现在:
1、本发明提供的硫酸软骨素裂解酶,可快速降解透明质酸和不同结构硫酸软骨素(A、B、C和D)为不饱和二糖;以透明质酸和硫酸软骨素A为底物时,比活力分别为216 U/mg和242 U/mg,降解方式为内切型。
2、本发明提供了可稳定高效表达水溶性的重组硫酸软骨素裂解酶的重组菌株。采用诱导与补料同时进行的策略,重组大肠杆菌在吨级发酵水平发酵33 h,发酵液酶活达到390.8 U/mL,为目前报道的最高水平。重组枯草芽孢杆菌在摇瓶水平发酵22小时,发酵液酶活达到30.9 U/mL。
3、本发明还提供了一种高效规模化制备硫酸软骨素裂解酶的方法,将产硫酸软骨素裂解酶重组工程菌发酵后所获菌体高压破碎后,经镍柱一步纯化最终获得的电泳纯酶纯度大于96%,回收率超过76%,实现了水溶性硫酸软骨素裂解酶的批量化生产,经济效益可观。
4、本发明还提供了利用所述硫酸软骨素裂解酶制备特定聚合物的生物活性硫酸软骨素寡糖的应用,将所述硫酸软骨素裂解酶与硫酸软骨素反应的产物超滤,即可获得聚合度为2~10的硫酸软骨素寡糖,酶用量少,反应条件温和,反应迅速,产物结构可控,可实现具有特殊生物学功能的硫酸软骨素寡糖的高效制备,应用前景广阔。
附图说明
图1是重组大肠杆菌的吨级发酵生长及产酶曲线图。
图2是源自重组大肠杆菌的HClyM的聚丙烯酰胺凝胶电泳图。
其中,M-marker;1-HClyM。
图3为重组枯草芽孢杆菌的生长及产酶曲线图。
图4 是源自重组枯草芽孢杆菌的HClyM的聚丙烯酰胺凝胶电泳图。
其中,M-marker;1-HClyM。
图5 是pH对HClyM活性的影响图。
图6是反应温度对HClyM活性的影响图。
图7是金属离子、EDTA及SDS对HClyM活性的影响图。
图8是HClyM的底物特异性图。
图9是HClyM降解硫酸软骨素A不同时间的产物的TLC图。
图10是硫酸软骨素经HClyM酶解0.5 h的产物超滤后获得的滤过液的TLC图。
具体实施方式
下面结合附图并通过具体实施例来进一步详细说明本发明。
以下实施例的阐述,是为了全面公开本发明如何实施的一些常用技术,而不是为了限制本发明的保护范围。发明人已经尽最大努力确保实施例中各参数的准确性,但是一些实验误差和偏差也应该予以考虑。除非另有说明,本发明中分子量是指重均分子量,温度是摄氏度。
实施例1:硫酸软骨素裂解酶(HClyM)的获得
挑取微杆菌菌株接种到100 mL液体培养基中,在30℃、160rpm下摇床培养48 h,离心收集培养液上清并用饱和度为80%的硫酸铵盐析,收集沉淀透析,获得硫酸软骨素裂解酶。
上述液体培养基组成如下:酵母粉10 g/L,CS 5 g/L,NaCl 5 g/L,MgSO4·7H2O 5g/L及KH2PO4 0.3 g/L,最适起始pH 8.5。
上述微杆菌(Microbacteriumsp. M28)是从青岛海域海泥浸出液经常规稀释法和划线法接种涂布于唯一碳源固体培养基上逐步筛选得到。微杆菌M28菌株外观呈黄色,革兰氏染色阳性,无芽胞和荚膜。于2022年4月22日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO: M2022457,保藏地址为湖北省武汉市武汉大学。
实施例2:微杆菌基因组的提取
将微杆菌菌株接种至液体培养基(同实施例1)中,30 ℃、160 rpm下振荡培养至OD600 为0.8,取培养液1mL,按试剂盒提取,得到基因组DNA。
实施例3:微杆菌菌株基因组扫描及其序列分析
将实施例2制得的大分子量基因组DNA进行测序。用NCBI等软件对测序结果进行分析,分析结果显示菌株基因组上携带一个硫酸软骨素裂解酶基因,基因编码区长2415 bp,其核苷酸序列如SEQ ID NO.1所示。信号肽分析结果显示N端第1~18个氨基酸是信号肽序列。去除信号肽后的核苷酸序列如SEQ ID NO.2所示。该基因编码的硫酸软骨素裂解酶由804个氨基酸组成,其氨基酸序列如SEQ ID NO.3所示,该序列与Chondroitin AC lyase(PDB ID:1RWA,分辨率为1.30Å)的氨基酸序列有45.56%的一致性。
实施例4:HClyM的大肠杆菌重组表达载体的构建
以实施例2所述的基因组为模板,进行PCR扩增,使用的扩增引物如下:
F:GGAGATATACATATGTCAGCTCTGCTGATG(NdeI)
R: GTGGTGGTGCTCGAGGCGGTGCAGCGAGAA (Xho I)
进行PCR扩增条件为:94 ℃预变性3 min;94 ℃变性30 s,55 ℃退火30 s,72 ℃延伸1 min,共30个循环;72 ℃延伸2min;4 ℃稳定15 min。琼脂糖凝胶电泳回收PCR产物。
将pET-24a(+)载体用Xho I和Nde I双酶切,琼脂糖凝胶电泳回收酶切后的载体大片段。
将回收的PCR产物与双酶切的pET-24a(+)载体连接,连接产物转入大肠杆菌DH5a感受态细胞,涂布于含卡那霉素(50 μg/mL)的LB固体平板,37 ℃培养过夜。挑取阳性克隆测序,获得在pET-24a (+)载体的Xho I和Nde I酶切位点之间正确插入所述硫酸软骨素裂解酶核苷酸序列的重组质粒。该核苷酸序列如SEQ ID NO.2所示,其编码的氨基酸序列如SEQ ID NO.4所示。使用质粒提取试剂盒提取质粒。热激法将重组表达质粒转化大肠杆菌菌株BL21(DE3)的感受态(购自美国Novagen公司),获得含有目的基因序列的重组大肠杆菌菌株BL21(DE3)/pET-24a (+)-HClyM。
实施例5:HClyM的枯草芽孢杆菌重组表达载体的构建
以实施例2所述的基因组为模板,进行PCR扩增,使用的扩增引物如下:
F:GTCCAAACTAGTTCGAAGATCTTGTCAGCTCTGCTGATGGACGC (BgIII)
R:CATGCGGCCGGTACCGGATCCTTAGTGATGATGATGATGATGGCGGTG (BamHI)
进行PCR扩增条件为:98 ℃预变性5min;98 ℃变性10 s,58 ℃退火5 s,72 ℃延伸25 s,共35个循环;72 ℃延伸10 min。琼脂糖凝胶电泳回收PCR产物。
将pSTOP1622载体用BgIII和BamHI双酶切后琼脂糖凝胶电泳回收线性载体片段。将回收的PCR产物与线性pSTOP1622载体连接,连接产物转入大肠杆菌DH5α感受态细胞,涂布于含氨苄青霉素(50 μg/mL)的LB固体平板,37 ℃培养过夜。挑取阳性克隆测序,获得在pSTOP1622载体的BgIII和BamHI酶切位点之间正确插入所述硫酸软骨素裂解酶核苷酸序列的重组质粒。该核苷酸序列如SEQ ID NO.2所示,其编码的氨基酸序列如SEQ ID NO.4所示。使用质粒提取试剂盒提取质粒。将重组表达质粒转化入枯草芽孢杆菌B.subtilisWB800的感受态细胞中,获得含有目的基因序列的枯草芽孢杆菌B.subtilisWB800重组菌株B. subtilis WB800/pSTOP1622-HClyM。
实施例6:酶活力测定
酶活力(单位为U/L)的测定方法如下:取硫酸软骨素A 4.0 mL (3 mg/mL, 20 mMTris-HCl, pH 7.4) ),加入3.75 mL 20 mM Tris-HCl (pH 7.4)以及50 μL适当稀释的酶液(或者煮沸灭活的),混匀后37 ℃反应20 min,以加入50 μL煮沸灭活的酶液的为参照,测定样品在232 nm的吸光度,取消光系数ε=5.1 mM-1。
酶活力单位定义为37℃每分钟催化糖胺聚糖产生1μmoL不饱和双键所需要的酶量。
用BCA蛋白质定量试剂盒测定溶液中的蛋白含量,计算得重组硫酸软骨素裂解酶HClyM的比活力(U/mg)。
实施例7:HClyM的吨级发酵及制备
挑取平板上重组大肠杆菌BL21(DE3)/pET-24a (+)-HClyM的单菌落,接种到5 mLLB液体培养基中37 ℃下过夜培养,再转接到100 mL LB液体培养基中,37℃下振荡培养12小时后,再一次转接到3 L LB液体培养基中37℃下振荡培养12小时,获得一级种子液。于100L种子罐中配制60L LB培养基,121 ℃灭菌20 min,接种前加入卡那霉素至浓度为50ug/mL。将一级种子液接入种子罐,37 ℃,200 rpm,通气量为10 m3/h,发酵10 h,获得二级种子液。于1000L发酵罐中配制600L发酵培养基,121 ℃灭菌20 min,接种前加入卡那霉素至浓度为50 ug/mL。将二级种子液接种至发酵罐中发酵培养,控制发酵温度为30~37 oC,搅拌速度为200 rpm,通气量为 30 m3/h,发酵12h后向发酵液中加入0.1mM IPTG诱导产酶,控制发酵温度为25 oC。同时向发酵液补加补料培养基(组成为:60 g/L甘油,12 g/L蛋白胨,12 g/L酵母粉)来控制发酵液的溶氧量为0~30 %,通过向发酵液补加25%氨水控制发酵液的发酵液的pH值为6.8~7.4,发酵33 h。图1为重组大肠杆菌的吨级发酵曲线图。在此培养条件下,发酵液的酶活达到390.8 U/mL,菌量达到(OD600)52.9。
发酵结束后离心收集菌体。菌体重悬于10 mL的缓冲液A(20 mM PB,0.5 M NaCl,pH 7.3)中,低温高压破碎后于8000 rpm、4 ℃离心10min,上清液(即粗酶液)经0.45 μm滤膜过滤后加到已经用缓冲液A平衡的Ni Sepharose FF柱(CV 50 mL)中,用缓冲液A淋洗至基线水平后,用添加0.2 M Imidazole的缓冲液A进一步除去杂蛋白,最后用添加0.3 MImidazole的缓冲液A洗脱目的组分,收集目的组分并置于透析袋中透析,透析液为20 mMPB(pH 7.3)。用SDS-PAGE检测重组硫酸软骨素裂解酶的纯化情况(图2),结果显示单一条带,分子量为 86.0 kDa,与理论分子量相符。
以硫酸软骨素A为底物,测得透析后的重组硫酸软骨素裂解酶的酶活力为242U/mg。将透析后的重组硫酸软骨素裂解酶-20 ℃存放备用。
实施例8:重组枯草芽孢杆菌的发酵培养及HClyM的制备
将重组菌B.subtilis WB800/pSTOP1622-HClyM的种子液以1%的接种量接种于50mL 含有四环素抗性(10 μg/mL)TB液体培养基中,37 oC,160 rpm振荡培养。当OD600达到0.7左右时,加入0.5%(w/v)木糖诱导培养,定时收集发酵液检测菌量及酶活。图3为重组枯草芽孢杆菌B.subtilis WB800/pSTOP1622-HClyM的生长及产酶曲线。在此条件下培养22h,酶活达到30.9 U/mL。发酵结束后离心收集菌体。菌体重悬于50 mL的缓冲液A(20 mM PB,0.5 M NaCl,pH 7.3)中,超声破碎后离心,进一步经Ni Sepharose FF柱(CV 5 mL)纯化、透析,获得电泳纯的重组硫酸软骨素裂解酶(图4),并用于特定聚合度硫酸软骨素寡糖的制备。
实施例9:HClyM的酶学性质测定
1. pH和温度对酶活性的影响
配制不同pH的20 mM Na2HPO4- Citric acid缓冲液、Na2HPO4-NaH2PO4缓冲液、Tris-HCl缓冲液和Glycine-NaOH缓冲液。再用这些缓冲液分别配制浓度为0.3% (w/v)的硫酸软骨素溶液作为酶解反应的底物。分别取上述硫酸软骨素底物900 μL和100 μL酶液混合后,在50 ℃反应10 min,按前述的紫外法测定酶活力。结果显示,HClyM在使用pH 7.3的20mM Tris-HCl缓冲液时,重组酶的酶活最高(如图5)。
在最适pH下,以质量浓度为0.3% (w/v)的硫酸软骨素A溶液作为酶解反应的底物,取900μL底物和100 μL酶液混合,分别在不同温度(0-60 ℃)下反应10 min,按前述的紫外法测定酶活力。结果显示,HClyM在50 ℃时达到最大活力(如图6)。
2. 金属离子及EDTA、SDS对HClyM酶活的影响
将各种金属离子(Al3+、Mn2+、Na+、Mg2+、Fe2+、Zn2+、Ba2+、Hg2+、K+、Li+、Cu2+、Fe3+、Ca2+、Ni2+)、EDTA和SDS分别加入到0.3% (w/v)硫酸软骨素A底物溶液中使其终浓度分别为1 mM、5mM和10 mM,取900 μL底物与100 μL酶液混合后在最适温度和最适pH下测定剩余酶活,以未加入金属离子、EDTA、SDS时的酶活定义为100%相对活力。结果如图7所示,1 mM Ni2+、Zn2+对酶活有轻微的抑制作用,1 mM Fe2+、Hg+、Cu2+、Fe3+则对酶活有明显的抑制作用。5 mM的EDTA对酶活有轻微的抑制作用,10 mM的SDS则对酶活有明显的抑制作用。
3. HClyM的底物特异性
以20 mM Tris-HCl (pH 7.3)为溶剂配制浓度为0.3%的硫酸软骨素A、硫酸软骨素B、硫酸软骨素C、硫酸软骨素D、透明质酸、肝素以及海藻酸钠溶液作为底物。取900 μL底物和100 μL酶液混合37 ℃下反应10 min后,按前述的紫外法测定酶活力。以最高酶活力为100%计算其他底物的相对酶活力。结果显示,HClyM能降解硫酸软骨素A、硫酸软骨素B、硫酸软骨素C、硫酸软骨素D和透明质酸(图8),且对硫酸软骨素A和透明质酸具有同等的酶降解活力。
4. HClyM降解硫酸软骨素A的方式
将纯化的HClyM加入到10 mL 0.3%的硫酸软骨素A溶液中,40 ℃下分别反应不同时间,取出反应产物于沸水浴中10 min后,点样于TLC层析板,随后置于有展开剂(正丁醇∶冰乙酸∶水=2∶2∶1)的展缸中2 h,吹干层析板,浸入显色剂 (苯胺二苯胺)中2 s,取出吹干,110 oC烘10 min即可见产物斑点。TLC检测结果如图9所示。可以看出,HClyM对硫酸软骨素A和透明质酸的降解非常迅速,在反应0.5min即可观察到聚合度为2~10的系列寡糖出现,随着反应时间延长,降解产物逐渐集中于二糖。表明HClyM是以内切酶的酶切方式降解硫酸软骨素A。
实施例10:利用HClyM制备特定聚合度硫酸软骨素寡糖
蒸馏水配制浓度为10%的硫酸软骨素溶液1L,加入透析后HClyM溶液30 mL,37 ℃下搅拌反应0.5 h,煮沸,9000 rpm下将酶解溶液离心20 min,离心液上清经超滤膜包(MWCO5kD)超滤,滤过液通过TLC进行检测(图10),获得聚合度为2~10 的硫酸软骨素寡糖,寡糖收率为58%。图9为硫酸软骨素经HClyM酶解0.5 h后超滤获得的滤过液的TLC谱图。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
序列表
<110> 中国海洋大学
<120> 一种硫酸软骨素裂解酶及其重组菌株和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2415
<212> DNA
<213> 微杆菌(Microbacterium sp.)
<400> 1
atgttcaccc cctctcgccg caatgtcctg cagctgggcg gcttcgcgct ggcctcagct 60
ctgctgatgg acgccggccg tccgctgacg gcattggcgt cgctgcagcc gctgagcgtc 120
ggcgttcccg agatcgaggc gctgcgcacc cgctgggtcg agacgctgac cggccgcggc 180
atcatcagcg gctctcccgc gaccttcgcc tcggcgatcg cccgacagga tcggggcacc 240
gacgctctgc tcgcgaagat cagcccgacc gccacccgct acttctcgga tcaggactgg 300
tcgatcggcg ccaccgagat cgcgaagtcc aacgccatgc gcatgaacta ctcgggcatc 360
cagagtctcg ccgtctcgtg gtcgacccct ggctcgaagc acgagggatc ggccgttgtg 420
ctcgacgccg cgctgcgcgg gctggctcac atgcacgagc gggtctacaa cccgaacacg 480
cagtggtggg gcaactggtg gtcgtggaac atcggtgccg cccagcccct ggcgaacacg 540
atggcgctgc tgcacgacga gctcgaccag accgagatcg acgcgtactg cgcatccatc 600
gaccacttca tccccggtcg agacccacgc atgcagctgc atccgaccgg accgcaggag 660
tccgacggcg cgaaccgcgt cgacatctgc caggcgatca tcgtgcgggc gatcgcacag 720
cccgatcccg acctgctgcg cgccgcggtg gccgcgctga gcgacacctg gcagtacatc 780
accgagggca acgggttctt ccgcgacggc tcattcgtgc agcactccac gatcggctac 840
accggaacct acgggctggt gctgctgggc gggctggcga agctcttcgc cctgctcgcc 900
ggcaccccgt tcgacgtcac cgaccccagc cggtccaacg tcagcggatt cgtggagagc 960
tcgttcgcgc cgctgatgtt ccgcggccag atgatggacg ccgtgcgcgg gcgagcggtc 1020
gcccggtacg ccgagcgcag catcagcaac ggaaacgacc tcatcgagca cacgctgcgc 1080
ctcgcccagt ccgccgacgc tgcgacggct gcacgctggc gggggctctg ccggcagtgg 1140
atcgagagca attccgcggc gaacatcacc acgaccacga gcatcgtgcg cctgtcgctg 1200
gtcaccgagc tgctgagctc ggacgcggtg gccgtggcgg acccgaccgg cccccggctg 1260
ttcccggcga tggaccggct cgtgcaccgt gccgccgacg gctcatgggc cctcgccgtc 1320
gcgatgtgct cgaaccggat cgcgtggcac gagggcaccg aagccgagaa cttcgagggc 1380
gtgaagacca gtcagggaat gacctatctg tacctgcgcg gcgatgagga tcacttcgac 1440
gacgagttct ggtcgacctc cgacctcgcg tcccctccgg gcaccacggt cgacctcacc 1500
ccgctgccac gcaacccgga gggcgagtgg ggcgagcgca caccggccaa cgagtggacc 1560
ggcggtgtca ccttcgaaga caccgcgctc gccgccatgc acctggtcgc tccgggaggg 1620
acgggtctgg tcgcgcgcaa ggtgtggttc acgcttccgg atgcctatgt cgcgctgggc 1680
tcggacatct ccaccgcgag tgacggcgag gtgcgcacga cgatcgagca tcgcaacctg 1740
ggcacctcgg cgcgccgact ggtcgtcgac ggcgctgccg tgaccacgcc gcagaccgtg 1800
tccggggcac ggtgggcgca tctggagggc gtcggcggct acgtgcttct cgacggtccg 1860
gtcgatctcc tcgccggggt cgacgagcgc gagggcacct ggcgccgcaa cagcaccaac 1920
gccgcggccg gcaccgaggt gctgcgccgc cggtggtacg gcaccctgag cctgtcgcac 1980
ggcgtgggag ggcaggcacg cggcggatac gcctacgccg tgcatccggg tgccgacgcc 2040
gagacgactg cagcagccgc ccgctccggt cgcttccgcg tgctgcgcaa cgacgaggtg 2100
gcacaggcga tccagcattc cggccacgtg accgccgccg cgttctggaa ggcaggcgca 2160
gtcggcgccc tcaccgccga ccgtgccgcc tgcgtgatcg cccgcatcac gcccggcatg 2220
gtgcgcatgt cggtgagcga cccgacgcac agcgccacga cactggtgat cgatgtcgcc 2280
cagaccgcgg tcacccgggt gaagggagcc gacgccgccc gcgtcgcact cgcccgccgc 2340
ggcgacggca tccggctcac catcgacacc gccgggacgg cgggaacgac gctggagttc 2400
tcgctgcacc gctga 2415
<210> 2
<211> 2358
<212> DNA
<213> 微杆菌(Microbacterium sp.)
<400> 2
tcagctctgc tgatggacgc cggccgtccg ctgacggcat tggcgtcgct gcagccgctg 60
agcgtcggcg ttcccgagat cgaggcgctg cgcacccgct gggtcgagac gctgaccggc 120
cgcggcatca tcagcggctc tcccgcgacc ttcgcctcgg cgatcgcccg acaggatcgg 180
ggcaccgacg ctctgctcgc gaagatcagc ccgaccgcca cccgctactt ctcggatcag 240
gactggtcga tcggcgccac cgagatcgcg aagtccaacg ccatgcgcat gaactactcg 300
ggcatccaga gtctcgccgt ctcgtggtcg acccctggct cgaagcacga gggatcggcc 360
gttgtgctcg acgccgcgct gcgcgggctg gctcacatgc acgagcgggt ctacaacccg 420
aacacgcagt ggtggggcaa ctggtggtcg tggaacatcg gtgccgccca gcccctggcg 480
aacacgatgg cgctgctgca cgacgagctc gaccagaccg agatcgacgc gtactgcgca 540
tccatcgacc acttcatccc cggtcgagac ccacgcatgc agctgcatcc gaccggaccg 600
caggagtccg acggcgcgaa ccgcgtcgac atctgccagg cgatcatcgt gcgggcgatc 660
gcacagcccg atcccgacct gctgcgcgcc gcggtggccg cgctgagcga cacctggcag 720
tacatcaccg agggcaacgg gttcttccgc gacggctcat tcgtgcagca ctccacgatc 780
ggctacaccg gaacctacgg gctggtgctg ctgggcgggc tggcgaagct cttcgccctg 840
ctcgccggca ccccgttcga cgtcaccgac cccagccggt ccaacgtcag cggattcgtg 900
gagagctcgt tcgcgccgct gatgttccgc ggccagatga tggacgccgt gcgcgggcga 960
gcggtcgccc ggtacgccga gcgcagcatc agcaacggaa acgacctcat cgagcacacg 1020
ctgcgcctcg cccagtccgc cgacgctgcg acggctgcac gctggcgggg gctctgccgg 1080
cagtggatcg agagcaattc cgcggcgaac atcaccacga ccacgagcat cgtgcgcctg 1140
tcgctggtca ccgagctgct gagctcggac gcggtggccg tggcggaccc gaccggcccc 1200
cggctgttcc cggcgatgga ccggctcgtg caccgtgccg ccgacggctc atgggccctc 1260
gccgtcgcga tgtgctcgaa ccggatcgcg tggcacgagg gcaccgaagc cgagaacttc 1320
gagggcgtga agaccagtca gggaatgacc tatctgtacc tgcgcggcga tgaggatcac 1380
ttcgacgacg agttctggtc gacctccgac ctcgcgtccc ctccgggcac cacggtcgac 1440
ctcaccccgc tgccacgcaa cccggagggc gagtggggcg agcgcacacc ggccaacgag 1500
tggaccggcg gtgtcacctt cgaagacacc gcgctcgccg ccatgcacct ggtcgctccg 1560
ggagggacgg gtctggtcgc gcgcaaggtg tggttcacgc ttccggatgc ctatgtcgcg 1620
ctgggctcgg acatctccac cgcgagtgac ggcgaggtgc gcacgacgat cgagcatcgc 1680
aacctgggca cctcggcgcg ccgactggtc gtcgacggcg ctgccgtgac cacgccgcag 1740
accgtgtccg gggcacggtg ggcgcatctg gagggcgtcg gcggctacgt gcttctcgac 1800
ggtccggtcg atctcctcgc cggggtcgac gagcgcgagg gcacctggcg ccgcaacagc 1860
accaacgccg cggccggcac cgaggtgctg cgccgccggt ggtacggcac cctgagcctg 1920
tcgcacggcg tgggagggca ggcacgcggc ggatacgcct acgccgtgca tccgggtgcc 1980
gacgccgaga cgactgcagc agccgcccgc tccggtcgct tccgcgtgct gcgcaacgac 2040
gaggtggcac aggcgatcca gcattccggc cacgtgaccg ccgccgcgtt ctggaaggca 2100
ggcgcagtcg gcgccctcac cgccgaccgt gccgcctgcg tgatcgcccg catcacgccc 2160
ggcatggtgc gcatgtcggt gagcgacccg acgcacagcg ccacgacact ggtgatcgat 2220
gtcgcccaga ccgcggtcac ccgggtgaag ggagccgacg ccgcccgcgt cgcactcgcc 2280
cgccgcggcg acggcatccg gctcaccatc gacaccgccg ggacggcggg aacgacgctg 2340
gagttctcgc tgcaccgc 2358
<210> 3
<211> 804
<212> PRT
<213> 人工序列(Microbacterium sp.)
<400> 3
Met Phe Thr Pro Ser Arg Arg Asn Val Leu Gln Leu Gly Gly Phe Ala
1 5 10 15
Leu Ala Ser Ala Leu Leu Met Asp Ala Gly Arg Pro Leu Thr Ala Leu
20 25 30
Ala Ser Leu Gln Pro Leu Ser Val Gly Val Pro Glu Ile Glu Ala Leu
35 40 45
Arg Thr Arg Trp Val Glu Thr Leu Thr Gly Arg Gly Ile Ile Ser Gly
50 55 60
Ser Pro Ala Thr Phe Ala Ser Ala Ile Ala Arg Gln Asp Arg Gly Thr
65 70 75 80
Asp Ala Leu Leu Ala Lys Ile Ser Pro Thr Ala Thr Arg Tyr Phe Ser
85 90 95
Asp Gln Asp Trp Ser Ile Gly Ala Thr Glu Ile Ala Lys Ser Asn Ala
100 105 110
Met Arg Met Asn Tyr Ser Gly Ile Gln Ser Leu Ala Val Ser Trp Ser
115 120 125
Thr Pro Gly Ser Lys His Glu Gly Ser Ala Val Val Leu Asp Ala Ala
130 135 140
Leu Arg Gly Leu Ala His Met His Glu Arg Val Tyr Asn Pro Asn Thr
145 150 155 160
Gln Trp Trp Gly Asn Trp Trp Ser Trp Asn Ile Gly Ala Ala Gln Pro
165 170 175
Leu Ala Asn Thr Met Ala Leu Leu His Asp Glu Leu Asp Gln Thr Glu
180 185 190
Ile Asp Ala Tyr Cys Ala Ser Ile Asp His Phe Ile Pro Gly Arg Asp
195 200 205
Pro Arg Met Gln Leu His Pro Thr Gly Pro Gln Glu Ser Asp Gly Ala
210 215 220
Asn Arg Val Asp Ile Cys Gln Ala Ile Ile Val Arg Ala Ile Ala Gln
225 230 235 240
Pro Asp Pro Asp Leu Leu Arg Ala Ala Val Ala Ala Leu Ser Asp Thr
245 250 255
Trp Gln Tyr Ile Thr Glu Gly Asn Gly Phe Phe Arg Asp Gly Ser Phe
260 265 270
Val Gln His Ser Thr Ile Gly Tyr Thr Gly Thr Tyr Gly Leu Val Leu
275 280 285
Leu Gly Gly Leu Ala Lys Leu Phe Ala Leu Leu Ala Gly Thr Pro Phe
290 295 300
Asp Val Thr Asp Pro Ser Arg Ser Asn Val Ser Gly Phe Val Glu Ser
305 310 315 320
Ser Phe Ala Pro Leu Met Phe Arg Gly Gln Met Met Asp Ala Val Arg
325 330 335
Gly Arg Ala Val Ala Arg Tyr Ala Glu Arg Ser Ile Ser Asn Gly Asn
340 345 350
Asp Leu Ile Glu His Thr Leu Arg Leu Ala Gln Ser Ala Asp Ala Ala
355 360 365
Thr Ala Ala Arg Trp Arg Gly Leu Cys Arg Gln Trp Ile Glu Ser Asn
370 375 380
Ser Ala Ala Asn Ile Thr Thr Thr Thr Ser Ile Val Arg Leu Ser Leu
385 390 395 400
Val Thr Glu Leu Leu Ser Ser Asp Ala Val Ala Val Ala Asp Pro Thr
405 410 415
Gly Pro Arg Leu Phe Pro Ala Met Asp Arg Leu Val His Arg Ala Ala
420 425 430
Asp Gly Ser Trp Ala Leu Ala Val Ala Met Cys Ser Asn Arg Ile Ala
435 440 445
Trp His Glu Gly Thr Glu Ala Glu Asn Phe Glu Gly Val Lys Thr Ser
450 455 460
Gln Gly Met Thr Tyr Leu Tyr Leu Arg Gly Asp Glu Asp His Phe Asp
465 470 475 480
Asp Glu Phe Trp Ser Thr Ser Asp Leu Ala Ser Pro Pro Gly Thr Thr
485 490 495
Val Asp Leu Thr Pro Leu Pro Arg Asn Pro Glu Gly Glu Trp Gly Glu
500 505 510
Arg Thr Pro Ala Asn Glu Trp Thr Gly Gly Val Thr Phe Glu Asp Thr
515 520 525
Ala Leu Ala Ala Met His Leu Val Ala Pro Gly Gly Thr Gly Leu Val
530 535 540
Ala Arg Lys Val Trp Phe Thr Leu Pro Asp Ala Tyr Val Ala Leu Gly
545 550 555 560
Ser Asp Ile Ser Thr Ala Ser Asp Gly Glu Val Arg Thr Thr Ile Glu
565 570 575
His Arg Asn Leu Gly Thr Ser Ala Arg Arg Leu Val Val Asp Gly Ala
580 585 590
Ala Val Thr Thr Pro Gln Thr Val Ser Gly Ala Arg Trp Ala His Leu
595 600 605
Glu Gly Val Gly Gly Tyr Val Leu Leu Asp Gly Pro Val Asp Leu Leu
610 615 620
Ala Gly Val Asp Glu Arg Glu Gly Thr Trp Arg Arg Asn Ser Thr Asn
625 630 635 640
Ala Ala Ala Gly Thr Glu Val Leu Arg Arg Arg Trp Tyr Gly Thr Leu
645 650 655
Ser Leu Ser His Gly Val Gly Gly Gln Ala Arg Gly Gly Tyr Ala Tyr
660 665 670
Ala Val His Pro Gly Ala Asp Ala Glu Thr Thr Ala Ala Ala Ala Arg
675 680 685
Ser Gly Arg Phe Arg Val Leu Arg Asn Asp Glu Val Ala Gln Ala Ile
690 695 700
Gln His Ser Gly His Val Thr Ala Ala Ala Phe Trp Lys Ala Gly Ala
705 710 715 720
Val Gly Ala Leu Thr Ala Asp Arg Ala Ala Cys Val Ile Ala Arg Ile
725 730 735
Thr Pro Gly Met Val Arg Met Ser Val Ser Asp Pro Thr His Ser Ala
740 745 750
Thr Thr Leu Val Ile Asp Val Ala Gln Thr Ala Val Thr Arg Val Lys
755 760 765
Gly Ala Asp Ala Ala Arg Val Ala Leu Ala Arg Arg Gly Asp Gly Ile
770 775 780
Arg Leu Thr Ile Asp Thr Ala Gly Thr Ala Gly Thr Thr Leu Glu Phe
785 790 795 800
Ser Leu His Arg
<210> 4
<211> 786
<212> PRT
<213> 人工序列(Microbacterium sp.)
<400> 4
Ser Ala Leu Leu Met Asp Ala Gly Arg Pro Leu Thr Ala Leu Ala Ser
1 5 10 15
Leu Gln Pro Leu Ser Val Gly Val Pro Glu Ile Glu Ala Leu Arg Thr
20 25 30
Arg Trp Val Glu Thr Leu Thr Gly Arg Gly Ile Ile Ser Gly Ser Pro
35 40 45
Ala Thr Phe Ala Ser Ala Ile Ala Arg Gln Asp Arg Gly Thr Asp Ala
50 55 60
Leu Leu Ala Lys Ile Ser Pro Thr Ala Thr Arg Tyr Phe Ser Asp Gln
65 70 75 80
Asp Trp Ser Ile Gly Ala Thr Glu Ile Ala Lys Ser Asn Ala Met Arg
85 90 95
Met Asn Tyr Ser Gly Ile Gln Ser Leu Ala Val Ser Trp Ser Thr Pro
100 105 110
Gly Ser Lys His Glu Gly Ser Ala Val Val Leu Asp Ala Ala Leu Arg
115 120 125
Gly Leu Ala His Met His Glu Arg Val Tyr Asn Pro Asn Thr Gln Trp
130 135 140
Trp Gly Asn Trp Trp Ser Trp Asn Ile Gly Ala Ala Gln Pro Leu Ala
145 150 155 160
Asn Thr Met Ala Leu Leu His Asp Glu Leu Asp Gln Thr Glu Ile Asp
165 170 175
Ala Tyr Cys Ala Ser Ile Asp His Phe Ile Pro Gly Arg Asp Pro Arg
180 185 190
Met Gln Leu His Pro Thr Gly Pro Gln Glu Ser Asp Gly Ala Asn Arg
195 200 205
Val Asp Ile Cys Gln Ala Ile Ile Val Arg Ala Ile Ala Gln Pro Asp
210 215 220
Pro Asp Leu Leu Arg Ala Ala Val Ala Ala Leu Ser Asp Thr Trp Gln
225 230 235 240
Tyr Ile Thr Glu Gly Asn Gly Phe Phe Arg Asp Gly Ser Phe Val Gln
245 250 255
His Ser Thr Ile Gly Tyr Thr Gly Thr Tyr Gly Leu Val Leu Leu Gly
260 265 270
Gly Leu Ala Lys Leu Phe Ala Leu Leu Ala Gly Thr Pro Phe Asp Val
275 280 285
Thr Asp Pro Ser Arg Ser Asn Val Ser Gly Phe Val Glu Ser Ser Phe
290 295 300
Ala Pro Leu Met Phe Arg Gly Gln Met Met Asp Ala Val Arg Gly Arg
305 310 315 320
Ala Val Ala Arg Tyr Ala Glu Arg Ser Ile Ser Asn Gly Asn Asp Leu
325 330 335
Ile Glu His Thr Leu Arg Leu Ala Gln Ser Ala Asp Ala Ala Thr Ala
340 345 350
Ala Arg Trp Arg Gly Leu Cys Arg Gln Trp Ile Glu Ser Asn Ser Ala
355 360 365
Ala Asn Ile Thr Thr Thr Thr Ser Ile Val Arg Leu Ser Leu Val Thr
370 375 380
Glu Leu Leu Ser Ser Asp Ala Val Ala Val Ala Asp Pro Thr Gly Pro
385 390 395 400
Arg Leu Phe Pro Ala Met Asp Arg Leu Val His Arg Ala Ala Asp Gly
405 410 415
Ser Trp Ala Leu Ala Val Ala Met Cys Ser Asn Arg Ile Ala Trp His
420 425 430
Glu Gly Thr Glu Ala Glu Asn Phe Glu Gly Val Lys Thr Ser Gln Gly
435 440 445
Met Thr Tyr Leu Tyr Leu Arg Gly Asp Glu Asp His Phe Asp Asp Glu
450 455 460
Phe Trp Ser Thr Ser Asp Leu Ala Ser Pro Pro Gly Thr Thr Val Asp
465 470 475 480
Leu Thr Pro Leu Pro Arg Asn Pro Glu Gly Glu Trp Gly Glu Arg Thr
485 490 495
Pro Ala Asn Glu Trp Thr Gly Gly Val Thr Phe Glu Asp Thr Ala Leu
500 505 510
Ala Ala Met His Leu Val Ala Pro Gly Gly Thr Gly Leu Val Ala Arg
515 520 525
Lys Val Trp Phe Thr Leu Pro Asp Ala Tyr Val Ala Leu Gly Ser Asp
530 535 540
Ile Ser Thr Ala Ser Asp Gly Glu Val Arg Thr Thr Ile Glu His Arg
545 550 555 560
Asn Leu Gly Thr Ser Ala Arg Arg Leu Val Val Asp Gly Ala Ala Val
565 570 575
Thr Thr Pro Gln Thr Val Ser Gly Ala Arg Trp Ala His Leu Glu Gly
580 585 590
Val Gly Gly Tyr Val Leu Leu Asp Gly Pro Val Asp Leu Leu Ala Gly
595 600 605
Val Asp Glu Arg Glu Gly Thr Trp Arg Arg Asn Ser Thr Asn Ala Ala
610 615 620
Ala Gly Thr Glu Val Leu Arg Arg Arg Trp Tyr Gly Thr Leu Ser Leu
625 630 635 640
Ser His Gly Val Gly Gly Gln Ala Arg Gly Gly Tyr Ala Tyr Ala Val
645 650 655
His Pro Gly Ala Asp Ala Glu Thr Thr Ala Ala Ala Ala Arg Ser Gly
660 665 670
Arg Phe Arg Val Leu Arg Asn Asp Glu Val Ala Gln Ala Ile Gln His
675 680 685
Ser Gly His Val Thr Ala Ala Ala Phe Trp Lys Ala Gly Ala Val Gly
690 695 700
Ala Leu Thr Ala Asp Arg Ala Ala Cys Val Ile Ala Arg Ile Thr Pro
705 710 715 720
Gly Met Val Arg Met Ser Val Ser Asp Pro Thr His Ser Ala Thr Thr
725 730 735
Leu Val Ile Asp Val Ala Gln Thr Ala Val Thr Arg Val Lys Gly Ala
740 745 750
Asp Ala Ala Arg Val Ala Leu Ala Arg Arg Gly Asp Gly Ile Arg Leu
755 760 765
Thr Ile Asp Thr Ala Gly Thr Ala Gly Thr Thr Leu Glu Phe Ser Leu
770 775 780
His Arg
785
Claims (9)
1.一种硫酸软骨素裂解酶,其特征在于,其氨基酸序列如(a)或(b)所示:
(a)氨基酸序列如SEQ ID NO. 3所示;
(b)在(a)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有硫酸软骨素裂解酶活性的由(a)衍生的氨基酸序列。
2.根据权利要求1所述的硫酸软骨素裂解酶,其特征在于,编码所述硫酸软骨素裂解酶氨基酸序列的核苷酸序列如(i)或(ii)所示:
(i)核苷酸序列如SEQ ID NO. 1所示;
(ii)对(i)所述的核苷酸序列进行优化表达,包括密码子优化、核糖体结合位点优化或启动子优化后的核苷酸序列。
3.一种重组表达载体,其特征在于,在表达载体中插入了权利要求2所述的硫酸软骨素裂解酶编码基因。
4.如权利要求3所述的重组表达载体,其特征在于,所述表达载体选自大肠杆菌表达载体或枯草杆菌表达载体。
5.一种重组菌或转基因细胞系,在其特征在于,宿主细胞或细胞系中插入了权利要求2所述的硫酸软骨素裂解酶编码基因。
6.如权利要求5所述的重组菌或转基因细胞系,其特征在于,所述宿主细胞或细胞系选自大肠杆菌或枯草杆菌宿主细胞。
7.一种如权利要求1所述硫酸软骨素裂解酶的规模化生产方法,其特征在于,包括如下步骤:将重组大肠杆菌的种子液以2~10%的接种量接至含有600 L 基础发酵培养基的1000L发酵罐中,控制发酵温度为30~38 oC,搅拌速度为200~300 rpm,通气量为10~25 m3/h,pH为6.8~7.4,溶氧为0~30%,发酵时间为5~15 h后,调节温度至15~30 oC,加入0.05~0.5 M IPTG诱导培养,同时开始补料;所述补料培养基由甘油、酵母粉和蛋白胨组成,补料速度为1.0~3.5 L/h,诱导培养25~40 h;将发酵液离心,菌体破碎后取上清,通过固定化金属螯合层析一步纯化获得纯度大于96%、回收率高于76%的硫酸软骨素裂解酶。
8.一种如权利要求1所述硫酸软骨素裂解酶的制备方法,其特征在于,将重组枯草芽孢杆菌的种子液以1~5%的接种量接种于含有10-50 μg/mL四环素抗性TB液体培养基中,30~38 oC下振荡培养2~8 h后,调节温度为15~30 oC,加入0.2~1.5%(w/v)木糖诱导培养10~20 h;离心收集菌体,菌体破碎后取上清,经固定化金属螯合层析一步纯化获得纯度大于96%、回收率高于85%的硫酸软骨素裂解酶。
9.一种权利要求1所述硫酸软骨素裂解酶用于制备聚合度为2-10的硫酸软骨素寡糖以及解析糖胺聚糖结构的应用。
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