一种类芽孢杆菌甲壳素酶及其应用的制作方法

本发明属于生物
技术领域：
，具体涉及一种类芽孢杆菌甲壳素酶及其应用。
背景技术：
：甲壳素酶（chitinase,ec.3.2.1.12）是一类可水解甲壳素中β-1,4糖苷键的水解酶，主要存在于细菌、真菌及植物中。根据其结构特征，可分为糖苷水解酶（glycosidehydrolase,gh）18和19两个家族，大部分细菌来源的甲壳素酶属于gh18家族，而gh19家族甲壳素酶主要为植物来源。甲壳寡糖是甲壳素经甲壳素酶水解得到的低聚糖，在医药、食品保健和植物防御等方面的应用已经被发掘，并已成为甲壳素研究的一个热点。在工业上，常用化学法（强酸、强碱）和物理法对甲壳素类生物大分子进行水解以制备甲壳低聚糖，但这两种方法制备的壳寡糖产品性质不稳定、降解条件难以控制，且化学法对环境污染较严重。酶法转化甲壳素为下游壳聚糖及壳寡糖类产品，是降低传统化学法对环境造成污染的一个有效的解决途径。蛋白酶及纤维素酶等非特异性酶的使用，虽然能在一定程度上缓解以上状况，但专一性的不足限制了此类酶制剂的使用。因此，研发高活性的甲壳素酶对壳寡糖类产品的工业生产具有重要意义。自然界中，多菌种混合发酵在纤维素类生物质的水解中发挥重要作用，而在此过程中，产生多种生物酶可实现对纤维素的高效利用。多酶协同催化可弥补单一酶制剂催化过程中的不足，同时可能存在的互相促进作用可进一步实现底物水解效率的最大化。本发明研究人员前期从本课题组筛选的一株产甲壳素酶菌株中克隆得到一个甲壳素酶ppchi1，在前期工作基础上，本发明从该菌株中克隆的到另一个甲壳素酶，实现其可溶性表达，对其进行表征，并考察了两种甲壳素酶协同水解甲壳素制备甲壳寡糖的过程。技术实现要素：本发明的目的是提供一种类芽孢杆菌甲壳素酶及其应用，旨在提供一种高效降解甲壳素制备壳寡糖的方法，克服目前现有技术的不足。本发明为实现上述目的所采用的技术方案如下。一种类芽孢杆菌甲壳素酶，所述类芽孢杆菌甲壳素酶的氨基酸序列如seqidno.1所示。优选的，编码该类芽孢杆菌甲壳素酶的基因的核酸序列如seqidno.2所示。优选的，制备该类芽孢杆菌甲壳素酶的方法包括以下步骤：将seqidno.2所示的类芽孢杆菌甲壳素酶基因(ppchi2)与表达载体连接，构建重组载体，然后将重组载体转入表达菌株中，诱导并获得可胞内可溶性表达的类芽孢杆菌甲壳素酶。优选的，所述类芽孢杆菌甲壳素酶基因来源于类芽孢杆菌菌株cs0611(分类命名为paenibacilluspasadenensiscs0611，保藏单位为中国典型培养物保藏中心，保藏地址为中国.武汉.武汉大学，保藏编号为cctccno：m2014458，保藏日期为2014年10月8日)，为本实验室筛选得到，其全基因组测序由深圳华大基因完成。进一步优选的，所述表达载体为pet-22b,pet-28a及pet-30a中的一种。进一步优选的，所述表达菌株为大肠杆菌bl21(de3)。进一步优选的，所述诱导是在添加了0.1-1.0mmiptg的lb液体培养基中，温度为16-30℃下诱导16-24h。进一步优选的，所述类芽孢杆菌甲壳素酶制备的具体步骤包括：（1）将甲壳素酶ppchi2氨基酸序列seqidno.1提交至生物信息学数据库signalp4.1server(http://www.cbs.dtu.dk/services/signalp/)，对其进行信号肽分析；（2）利用pcr扩增出无信号肽的甲壳素酶目的基因，并将目的基因与表达载体pet-28a连接，构建重组质粒并对其进行测序以验证序列正确性；（3）将上述重组质粒转化至表达菌株大肠杆菌e.colibl21(de3),经iptg诱导培养获得甲壳素酶；（4）对胞内表达甲壳素酶ppchi2的大肠杆菌进行破碎，取上清经亲和层析柱纯化，获得可溶性表达的ppchi2；（5）对上述纯化后的甲壳素酶进行活力测定，并用sds-page对其纯度进行分析。以上所述的一种类芽孢杆菌甲壳素酶的应用，包括以下步骤：以两种甲壳素酶混合成的酶液为催化剂，以预处理后的蟹壳粉为底物，双酶协同水解蟹壳粉制备可溶性的甲壳寡糖；其中，两种甲壳素酶包括类芽孢杆菌甲壳素酶ppchi1和类芽孢杆菌甲壳素酶ppchi2，所述类芽孢杆菌甲壳素酶ppchi1的氨基酸序列如seqidno.3所示，所述类芽孢杆菌甲壳素酶ppchi2的氨基酸序列如seqidno.1所示。优选的，所述类芽孢杆菌甲壳素酶ppchi1和类芽孢杆菌甲壳素酶ppchi2的体积比为1:4-4:1。优选的，所述甲壳素酶总用量为0.1-0.2mg，预处理后的蟹壳粉的用量为10-30mg。优选的，所述酶解的时间为1-24h，酶解的温度为30-50℃。与现有技术相比，本发明具有如下有益效果：（1）本发明的甲壳素酶来源于类芽孢杆菌paenibacilluspasadenensiscs0611，使用大肠杆菌bl21(de3)为表达菌株。此前未有paenibacilluspasadenensis来源的甲壳素酶被报道，且所用质粒pet-28a为高表达载体，大肠杆菌bl21(de3)为工业化生产生物催化酶制剂常用菌株。因此，本发明所述甲壳素酶不仅有一定的学术价值，而且可大量生产，作为农业级甲壳寡糖的工业化生产用酶制剂。（2）本发明所用的蟹壳粉底物为离子液体预处理后的底物，结晶度大大降低，水解效率大大提高。两种甲壳素酶协同降解甲壳素，其催化效率同单一酶催化相比，均有提高。因此，本发明所述的双酶协同降解甲壳素制备甲壳寡糖的体系具有巨大的应用潜力，对虾蟹壳的高值化应用具有重要意义。附图说明图1是本发明所述甲壳素酶ppchi2纯化后的sds-page电泳图。图2是温度对甲壳素酶ppch2相对酶活影响曲线图，最适温度处的活力定义为100%。图3是ph对甲壳素酶ppchi2相对酶活影响曲线图，最适ph处的活力定义为100%。图4是不同比例ppchi1和ppchi2对其协同水解预处理后蟹壳粉的影响图。图5是特定比例ppchi1和ppchi2协同水解预处理后蟹壳粉的时间曲线图。具体实施方式下面结合实施例对本发明的技术方案做进一步详细的描述，但本发明的实施方式不限于此。实施例1类芽孢杆菌甲壳素酶ppchi2的克隆表达与分离纯化（1）ppchi2的克隆根据基因组测序结果，选取含有信号肽序列的ppchi2的基因序列（genebankno.：kx431049.1）为研究对象，结合信号肽序列预测结果，设计正向引物chip1_f:5’-ggaattccatatggcggcctggacgcccggc-3’，反向引物chip1_r：5’-gaccgctcgaggccgaagtagcccttcatcgcgt-3’，以paenibacilluspasadenensiscs0611的基因组为模板，扩增ppchi2。pcr反应体系为：10μl5xq5reactionbuffer，1μl10mmdntps，2.5μl10μmchip2_f和chip2_r，1μl基因组dna,0.5μlq5高保真dna聚合酶，10μl5xq5highgcenhancer,23μlddh2o。pcr反应条件为：98℃预变性30s，98℃变性10s，72℃退火10s，72℃延伸30s，反应30个循环，72℃继续延伸2min。将目的基因与表达载体pet-28a分别用ndei和xhoi进行双酶切，回收后进行连接反应，并将连接产物转化到大肠杆菌dh5α感受态细胞中，涂布于带有抗性的lb平板上，37℃培养过夜（8h，下同），挑取单菌落再次过夜培养，提取质粒并测序。测序结果与seqidno.2一致，表明重组质粒pet-28a-chip2构建成功。（2）ppchi2的表达与分离纯化利用热激法将重组质粒pet-28a-chip2转化至大肠杆菌bl21(de3)超级感受态细胞中。挑取单菌落至50mllb液体培养基（含50μg/ml卡那霉素），37℃培养过夜，按1%的比例接种至100mllb液体培养基中（含50μg/ml卡那霉素）中，37℃培养至od=0.6,继续将培养瓶置于17℃培养50min，加入终浓度0.15mm的iptg，继续于17℃培养20h。4℃，5000g离心5min,收集菌体，并用生理盐水洗去残余培养液，至于-80℃保存备用。ppchi2的分离纯化采用hitrapffni亲和柱。首先，用bindingbuffer(ph7.4，50mmnah2po4,500mmnacl,40mmimidazole）重悬细胞，超声波破碎菌体（300w,开1s，关2s）。在4℃，10000g条件下离心30min，收集上清粗酶液。将粗酶液上样至柱子，并用bindingbuffer洗脱以除去结合力弱的蛋白质；然后用washbuffer(ph7.4，50mmnah2po4,500mmnacl,250mmimidazole）梯度洗脱，收集含有高甲壳素酶活性的组分，并用透析袋过夜透析除盐，所得酶液即为ppchi2。通过sds-page电泳鉴定，确认该酶表观分子量为48.0kda，如图1所示。ppchi1的制备方法与ppchi2一致，经sds-page电泳显示，该酶分子量为70.2kda。实施例2温度和ph对ppchi2酶活力的影响甲壳素酶活力测定：甲壳素酶酶活力的测定采用dns测定反应产物中还原糖的方法，底物为2wt%的胶体甲壳素溶液。1ml反应产物中加入2mldns试剂，沸水浴5min，冷却，补水至10ml，测定其在540nm处的吸光值。标准曲线的绘制以n-乙酰葡萄糖胺为标准底物，酶活力单位定义为每分钟产物1μmn-乙酰葡萄糖胺为一个单位。温度的影响：分别将0.5mlppchi2溶液（0.1mg/ml，5mmph7.0pbs）和0.5ml2wt%胶体甲壳素溶液（ph5.00.2m柠檬酸缓冲液）加入10ml离心管中，然后将反应体系置于不同温度（30,35,40,45,50,55,60,65,70,80℃）下，磁力搅拌反应30min,加入2mldns试剂终止反应，沸水浴5min，冷却，补水至10ml,测定540nm处吸光度值。如图2所示，在55℃时，ppchi2的相对活力最高，60℃时的酶相对活力可达90%，而70℃时的酶活力降低至40%，30-70℃时酶活力均在40%以上。因此，ppchi2具有相对较大的反应温度范围，最适温度为55℃。ph的影响：分别将0.5mlppchi2溶液（0.1mg/ml，5mmph7.0pbs）和0.5ml2wt%胶体甲壳素溶液加入10ml离心管中。胶体甲壳素溶液由0.2m不同ph（ph3-11）的缓冲液配置而成。将反应体系置于55℃的水浴锅中，磁力搅拌30min，加入2mldns试剂终止反应，沸水浴5min，冷却，补水至10ml,测定540nm处吸光度值。如图3所示，在ph5.0时，ppchi2的相对活力最高。在ph4-8的缓冲液中，ppchi2可保持40%以上的相对活性。因此，ppchi2的最适ph为5.0实施例3不同比例ppchi1和ppchi2协同水解预处理后蟹壳粉ppchi1是和ppchi2性质不同的、来源于同一目标菌株的另一个甲壳素酶，本实施例考察不同质量比例的ppchi1和ppchi2协同水解经离子液体预处理的蟹壳粉。ppchi1和ppchi2由实施例1中的方法制备，ppchi1的浓度为3.3mg/ml，ppchi2的浓度为2.9mg/ml。所用底物为经离子液体（1-丁基-3-甲基咪唑醋酸盐）预处理后的蟹壳粉(treated-csp)，其预处理步骤为，将60mg过100目筛的蟹壳粉加入2ml1-丁基-3-甲基咪唑醋酸盐中，100℃条件下孵育1h，加水使甲壳素析出，抽滤分离沉淀物。所得沉淀物加入20ml去离子水，超声5min,抽滤分离沉淀物，上述过程连续操作5次，以除去残余离子液体。最后将干沉淀物冷冻干燥，即为预处理后的蟹壳粉。酶解体系为0.8ml1%1-丁基-3-甲基咪唑醋酸盐预处理的蟹壳粉溶液（w/v，ph5.0，0.2m柠檬酸缓冲液）和0.2ml不同体积比（如下表1）的双酶混合酶液，磁力搅拌，40℃反应4h，按酶活测定方法测定产生的还原糖。结果如图4所示，由图可看出混合酶液的酶解效率比ppchi1和ppchi2单独使用时提高了1.3-3.2倍。当双酶体积比为1:1时，和ppchi1单独使用相比，产物中还原糖浓度由2.5mg/ml提高至3.4mg/ml。因此，双酶水解效率较高。表1组合12345ppchi1（μl）200140100600ppchi2（μl）060100140200实施例4特定比例ppchi1和ppchi2协同水解预处理后蟹壳粉由实施例3中实验结果证实双酶水解效率比单酶水解效率高，因此本实施例考察特定比例的ppchi1和ppchi2协同水解预处理后蟹壳粉的水解过程。酶解体系为0.8ml1%1-丁基-3-甲基咪唑醋酸盐预处理的蟹壳粉溶液（w/v，ph5.0，0.2m柠檬酸缓冲液）和0.2ml按体积比1:1混合的双酶混合酶液，磁力搅拌，40℃反应，于不同时间取出1支反应管，测定反应液中还原糖的浓度。过程曲线如图5所示，反应6h后，反应液中还原糖浓度达到最大。同未处理的蟹壳粉相比，预处理的蟹壳粉水解效率提高了2倍，水解效率可达40%。序列表<110>华南理工大学<120>一种类芽孢杆菌甲壳素酶及其应用<160>3<170>siposequencelisting1.0<210>1<211>437<212>prt<213>类芽孢杆菌甲壳素酶(paenibacilluspasadenensis)<400>1alaalatrpthrproglythralatyrlysalaglyaspvalvalthr151015tyralaglylysasptyrargcysvalglnserhisthralaleuthr202530glytrpgluproproasnvalproalaleutrpthrprothrglygly354045thrthrprothrprothrproseralathrprothralathrprothr505560prothralathrprothrprolysprothrvalthrprothrprothr65707580alaprovalthrprothrprothrlysthrprovalproseralathr859095prothrprothrvalalaprothralathrproileproseralathr100105110proserthrglylysvalvalvalglytyrtrphisasnpheaspasn115120125glyserglyvalileproleuargaspvalserproasnpheasnval130135140ileasnvalserphealagluthrleuserasphisalathrleuthr145150155160phevalpropheglnthrthrproalaglnphelysalaaspvalala165170175tyrleuglnserglnglyargargvalhisileserileglyglyala180185190asnalathrvalgluleuasnthralaalaalalysalaserpheval195200205serserleulysasnileileaspthrtyrglypheaspglyileasp210215220ileaspleugluglyserserleuserleuasnglyglyaspthrasp225230235240phelysasnprothrthrprolysvalvalasnleuileseralaile245250255lysgluilemetalathrtyrglyserglypheaspleuthrmetala260265270progluthralatyrvalglnglyglyglnilealatyrglyglypro275280285trpglyalatyrleuprovalilehisalavalargaspglnleuasp290295300tyrilehisvalglnhistyrasnserglysermetglnglyleuasp305310315320glylyssertyralaglnglythralaasppheglnvalalametala325330335gluvalleuleulysglypheproilealaglyasnalaserasnleu340345350pheproalaleuargalaaspglnvalalaileglyleuproserser355360365proalaalaalaglyglyglytyrthrglnproalaaspilevallys370375380alaleuasntyrleuthrlysglyglnserpheglyglyglntyrthr385390395400leuglnthrproglyglytyrserglypheargglyvalmetthrtrp405410415serileasntrpasplysthrthrsertyrvaltrpserasnalamet420425430lysglytyrphegly435<210>2<211>1314<212>prt<213>类芽孢杆菌甲壳素酶(paenibacilluspasadenensis)<400>2glycysglyglycyscysthrglyglyalacysglycyscyscysgly151015glycysalacyscysglycyscysthralacysalaalaglyglycys202530glyglyglycysglyalacysglythrcysglythrglyalacysgly354045thralacysglycyscysglyglycysalaalaglyglyalacysthr505560alacyscysglycysthrglycysglythrglycysalaglythrcys65707580cyscysalathralacysglyglycysglycysthrglyalacyscys859095glyglycysthrglyglyglyalaglycyscysglycyscysglyala100105110alacysglythrglycyscyscysglycysglycysthrglythrgly115120125glyalacysglycyscysglyalacysglyglyglycysglyglycys130135140alacysglyalacysglycyscysglyalacysthrcyscysglyala145150155160cysglycyscysthrthrcyscysglycyscysalacysglycyscys165170175glyalacysalaglycyscysalacysthrcyscysglyalacysthr180185190cyscysglyalacyscysglycyscysalacysglycyscysglyala195200205cysthrcyscyscysalaalaglycyscysglyalacyscysglythr210215220glyalacysglycyscysglyalacysglycyscysthralacyscys225230235240glycysglycyscysglyglythrglyalacysglycyscysglyala245250255cysthrcyscysglyalacyscysalaalaalaalacysglycyscys260265270glyglythrthrcyscysglythrcyscysglycysthralacysthr275280285cyscysglyalacysglycyscysthralacysglyglythrcysgly290295300cysglycyscysglyalacyscysglycyscysalacysglycyscys305310315320glyalathrthrcyscysthrthrcysglyglycyscysalacysgly325330335cyscysthrthrcysglyalacyscysglyglycysalaalaglygly340345350thrcysglythrcysglythrcysglyglycysthralacysthrgly355360365glycysalathralaalacysthrthrcysglyalacysalaalacys370375380glyglycysthrcyscysglyglycysglythrcysalathrcyscys385390395400cysglycysthrglycysglycysglyalacysglythrglythrcys405410415glycyscysglyalaalacysthrthrcysalaalacysglythrcys420425430alathrcysalaalacysglythrcysalaglycysthrthrcysgly435440445cyscysglyalaglyalacysglycysthrglyalaglycysglyala450455460cyscysalathrglycysglyalacysglycysthrcysalacysgly465470475480thrthrcysglythrglycyscysglythrthrcyscysalaglyala485490495cysglyalacyscyscyscysglyglycyscyscysalaglythrthr500505510cysalaalaglyglycyscysglyalacysglythrcysglycyscys515520525thralacyscysthrglycysalaalaalaglycyscysalaglygly530535540glyalacysglycyscysglycysglythrglycysalacysalathr545550555560cysthrcyscysalathrcysglyglycysglyglycysglycyscys565570575alaalacysglycyscysalacyscysglythrcysglyalaglycys580585590thrglyalaalacysalacysglyglycysglyglycysalaglycys595600605cysalaalaglyglycyscysalaglycysthrthrcysglythrcys610615620alaglycysthrcysglycysthrglyalaalaglyalaalacysala625630635640thrcysalathrcysglyalathralacysglythralacysglygly645650655cysthrthrcysglyalacysglyglycysalathrcysglyalacys660665670alathrcysglyalacyscysthrcysglyalaalaglyglyalaala675680685glycysthrcysglycysthrglythrcysglycysthrglyalaala690695700cysglyglycysglyglycysglyalacysalacyscysglyalacys705710715720thrthrcysalaalaglyalaalacyscyscysglyalacysglyala725730735cysglycyscyscysalaalaglyglythrcysglythrcysalaala740745750cyscysthrglyalathrcysalaglycysglycyscysalathrcys755760765alaalaglyglyalaglyalathrcysalathrglyglycysglyala77077578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