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制备方法和应用。
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。
本发明研究人员前期从含甲壳素土壤中筛选出一株产甲壳素酶菌株,通过对其发酵培养基进行优化,使其甲壳素酶产量有了一定的提高。通过对其全基因组进行测序及分析,发现该菌株有多种甲壳素酶基因。因此,为获得高活性的甲壳素酶,我们选择选取其中一种甲壳素基因进行克隆并在大肠杆菌中进行表达。
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制备方法和应用,旨在提供一种经济高效的甲壳素酶,并将其应用于经离子液体预处理的蟹壳粉末的降解中,具有很好的工业化应前景。
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.2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.1所示。
以上所述的一种类芽孢杆菌甲壳素酶的制备方法,该方法包括以下步骤:
将核酸序列如seqidno.1所示的甲壳素酶基因与表达载体连接,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,所述甲壳素酶基因(ppchi1)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。通过illuminahiseq2500平台测序,样品共产出1,255mb数据。基因组组分分析后发现,样品paenibacillus的基因组含有5,468个基因,总长度为4,848,621bp,平均长度887bp,占基因组全长的85.63%。串联重复序列共964个,总长为73,335bp,占基因组全长的1.2952%。小卫星序列701个,微卫星序列52个,trna22个,rrna0个。
优选的,所述表达载体为pet-22b、pet-28a及pet-30a中的一种。
优选的,所述表达菌株为大肠杆菌bl21(de3)。
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。
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ppchi1氨基酸序列seqidno.2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ppchi1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ppchi1;
(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:
利用离子液体对蟹壳粉进行预处理,然后以预处理后的蟹壳粉为底物,纯化的类芽孢杆菌甲壳素酶为催化剂,酶解制备具有还原性的可溶性甲壳寡糖。
优选的,所述离子液体为1-烯丙基-3-乙基咪唑氯盐、1-丁基-3-甲基咪唑四氟氯盐、1-丁基-3-甲基咪唑醋酸盐和1-乙基-3-甲基咪唑醋酸盐中的一种以上。
优选的,所述预处理条件为:固液比为1-4wt%,预处理温度为80-140℃,时间为0.5-3h。
优选的,所述酶解的体系为:0.1-0.2mg类芽孢杆菌甲壳素酶,10-30mg预处理的蟹壳粉,酶解时间为1-24h。
优选的,所述构建甲壳寡糖制备体系的具体步骤包括:
(1)将一定量的蟹壳粉末与离子液体混合,在特定的温度和时间下进行预处理;
(2)上述预处理的蟹壳粉-离子液体胶状物,经加水沉淀甲壳素,然后对沉淀物进行抽滤分离离子液体溶液;
(3)上述沉淀物经冻干即为预处理后的甲壳素酶底物,回收的离子液体溶液经减压旋蒸浓缩后可再次用于预处理的溶剂;
(4)以上述预处理的蟹壳粉为底物,纯化的甲壳素酶为催化剂,在适宜的温度和缓冲液条件下进行水解反应,以dns法检测水解混合物中的寡糖含量。
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。未经处理的蟹壳粉为甲壳素、蛋白质和碳酸钙的混合物,结晶度较高,不易水解,经离子液体预处理后,水解效率提高了3倍。用离子液体直接对蟹壳粉进行预处理,未经过传统的强酸强碱操作过程,降低对环境的影响,且离子液体可回收再利用。因此,本发明所述的甲壳寡糖制备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ppchi1纯化后的sds-page结果图。
图2是甲壳素酶ppchi1在不同温度下的活力对比图,最适温度处的活力定义为100%。
图3是甲壳素酶ppchi1在不同ph下的活力对比图,最适ph处的活力定义为100%。
图4a、图4b分别是蟹壳粉末预处理前后的sem图。
图5是使用甲壳素酶ppchi1水解预处理后蟹壳粉的时间曲线图,图中横坐标为时间,纵坐标为反应产生的还原糖浓度。
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ppchi1的克隆表达和酶学性质表征
(1)ppchi1的克隆表达与分离纯化
根据基因组测序结果,选取含有信号肽序列的ppchi1的基因序列(genebankno.:kx431050.1)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’-gacaagcttgcgcatcaaactacaagatc-3’,反向引物chip1_r:5’-gtgctcgagtttcagcttccaaagagc-3’,以paenibacilluspasadenensiscs0611的基因组为模板,扩增ppchi,将其连接至表达载体pet-28a,酶切位点为hindiii和xhoi,构建重组质粒pet-28a-chip1,并将其测序以确定序列正确性。
利用热激法将重组质粒pet-28a-chip1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),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,5000g离心5min,收集菌体,并用生理盐水洗去残余培养液,置于-80℃保存备用。
采用超声破碎法破碎菌体,收集上清,利用hitrapffni亲和层析柱对甲壳素酶进行纯化。首先,用bindingbuffer(ph7.4,50mmnah2po4,500mmnacl,20mmimidazole)重悬细胞,超声波破碎菌体(300w,开3s,关5s)。在4℃,10000g条件下离心20min,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ppchi1。得到重组表达的ppchi1,其sds-page结果如图1所示。由图1可见重组蛋白的分子量为70.2kda。
(2)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。向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。
(3)温度和ph对酶活力的影响
温度的影响。分别将0.5mlppchi1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(30,35,40,45,50,55,60,70,80℃)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45℃时,ppchi1的相对活力最高,40℃时的相对活力可达82%,而55℃时的酶活力降低至40%。因此,ppchi1的最适温度为45℃。
ph的影响。分别将0.5mlppchi1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45℃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,ppchi1的相对活力最高。在ph4-8的缓冲液中,ppchi1可保持40%以上的相对活性。因此,ppchi1的最适ph为5.0
实施例2
ppchi1水解离子液体预处理后蟹壳粉的应用
(1)蟹壳粉的预处理
考察不同离子液体对预处理效果的影响:分别将90mg过100目筛的蟹壳粉加入3ml1-烯丙基-3-乙基咪唑氯盐、1-丁基-3-甲基咪唑四氟氯盐、1-丁基-3-甲基咪唑醋酸盐和1-乙基-3-甲基咪唑醋酸盐4种离子液体中,100℃条件下预处理1h,加水使甲壳素析出,分离固体甲壳素,并冻干沉淀物,即为预处理后的蟹壳粉(treated-csp1,treated-csp2,treated-csp3,treated-csp4)。取10mg预处理后蟹壳粉于0.8mlph5.00.2m柠檬酸缓冲液,加入0.2mlppchi1(0.2mg)溶液,在45℃及ph5.0条件下,反应30min,以未经处理的蟹壳粉为对照,比较不同离子液体预处理后蟹壳粉的水解效率。结果发现,treated-csp3的水解效率最好,同未处理蟹壳粉相比,水解效率提高了2倍。预处理前后的蟹壳粉的扫描电镜图如图4a、图4b所示。图4a为1-丁基-3-甲基咪唑醋酸盐预处理前的蟹壳粉,图4b为预处理后的蟹壳粉。由图可看出,预处理后蟹壳粉的形貌发生变化。
考察蟹壳粉添加量对预处理效果的影响:分别将30、60和90mg过100目筛的蟹壳粉加入3ml1-丁基-3-甲基咪唑醋酸盐离子液体中,100℃条件下预处理1h,加水使甲壳素析出,分离固体甲壳素,并冻干沉淀物,比较不同蟹壳粉添加量对预处理效果的影响。结果发现,固液比为3wt%时的水解效率最高,同未处理的蟹壳粉相比,预处理后的水解效率提高了2.3倍。
(2)ppchi1水解经离子液体预处理蟹壳粉的反应体系
取60mgtreated-csp3于4.8mlph5.00.2m柠檬酸缓冲液,并加入1.2mlppchi1(1.2mg)溶液,反应体系置于40℃水浴锅中,于不同时间取出1ml反应液,测定反应液中还原糖的浓度。同时,以未处理的蟹壳粉为对照。反应过程曲线如图5所示,反应8h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至3倍,水解效率可达31%。
序列表
<110>华南理工大学
<120>一种类芽孢杆菌甲壳素酶及其制备方法和应用
<160>2
<170>siposequencelisting1.0
<210>1
<211>1842
<212>prt
<213>类芽孢杆菌甲壳素酶(paenibacilluspasadenensis)
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alaglycysalaalaglycysthrcysglythrglycysthrcysgly
865870875880
glycysalathrglyglycyscysthrthrcysthralacysglygly
885890895
thrcysglyalaglyglycysthrglyglyglyglycysglyglycys
900905910
thrglythrcyscysglyalacysglyglycysalaglyglyalaala
915920925
alathrglyglyalacysalaglythralacyscysalaalaglythr
930935940
cysthrglycysglycysglyglyglycysalathrthrthrcyscys
945950955960
thrcyscysalacysglyglyglyalaalacysthrthrglyglygly
965970975
alaglyalaalaglyglyglycysalaglycysthralacysglyala
980985990
cysthrthrcysthralathrglyalathrcysthrglyglyalaala
99510001005
glycysglyalaalacysthralathralathrcysalaalacysala
101010151020
alaglyalaalacysglyglycysthralacysalacysglycysgly
1025103010351040
cysthralathrthrglyglyalaalathrglyalathrglycyscys
104510501055
glycysthralaalaglyglythrglycyscysthrthralacyscys
106010651070
thrglythralacysalaalathrcyscysglyalacyscysalaala
107510801085
cysglyglycysalacysglythralacysalathrcysalaglycys
109010951100
thralathrglyalacysglyalathrglythrthrcysalaalathr
1105111011151120
cyscysthrthrcysglyalacysthrthrcysalaalaglyalacys
112511301135
glyalaglycysthralacyscysthrglyalaalaalathrcyscys
114011451150
alaalaalaglyglythrcysthrcysglycyscysglyglycysgly
115511601165
cysglyalathrglythrthrcysthrglyglyglyalaglyalacys
117011751180
glyalaglycysglyglycysglyalacyscysglycysalaalacys
1185119011951200
alaalaglyalacysglycysthrglycysthrglyalaalacysala
120512101215
alaglycysthrglyglycyscysthrcyscysglyalathrcysthr
122012251230
cysglyglycysthralathrglycysglyglyglycysglycysala
123512401245
alacysglycyscysglyalacysglycyscysglyglythralacys
125012551260
cysglyalacysglyglycysthralacysglycyscysglyalagly
1265127012751280
cysglycysglyalacysglycyscysglyalacysglycyscysala
128512901295
alacyscysglycyscysalacysglycyscysglyalaglycysgly
130013051310
thrcysalacysglycyscysglyalacysalacyscysglyalacys
131513201325
glyalacysalaalaglycyscyscysalaalaglycysglycysala
133013351340
alacysglycyscysthralacysglycyscysalaalaglycysgly
1345135013551360
cysglyalacysglycyscysglyalacysthrcyscysglyglygly
136513701375
cysalacysglythrglycysalacysglyglythrglyalacysgly
138013851390
glycysalathrglyglyalaglycysthrcysglyalacysglygly
139514001405
cysglyglythrthrthralathralacysglyglyglycysglygly
141014151420
alacysalaalaalaalaglyglycysthrthrcyscysthralacys
1425143014351440
alaalacysglyglycysalacysglyalathrthrthralacysgly
144514501455
alaalaglycysglyalaalaglythrglyglythrglyglyalacys
146014651470
cyscysalaalaglyglycysglyalathrcysglythrcyscysgly
147514801485
glyalathrcysthrglyalaglycysglyglyalaglycysglyala
149014951500
alacysglyglythrcyscysthrthrglyglyalaalaglyglycys
1505151015151520
alaglycyscysglyglycysalacysthrthrglycysglyglyala
152515301535
alacysglyalacysalaalaglyglycyscysglyalacysglycys
154015451550
cysglyalaglycysglycysglyalacysglycyscysthralacys
155515601565
glycyscysalaalacysglycyscysglyalacyscysglycysgly
157015751580
alacysglycyscysthralacysglycyscysalaalaglycysgly
1585159015951600
cysalaalacysglycyscysglyalaalaalacyscysglyalagly
160516101615
cysglycysalaalacysglycyscysglyalacysglycyscysgly
162016251630
alacysglyglycysalaalacysglycyscysthralacysglycys
163516401645
cysalaalaglycysglycysglyalacysthrcyscysthralacys
165016551660
alacyscysthralacysglyglycysalaalacysglycyscysgly
1665167016751680
alacysglycyscysglythrcyscysglycysalaalacysglycys
168516901695
cysalaglyglycysglycyscysthrcysthrglycyscysthrgly
170017051710
glyglycysthrglycysalaglyglycysglythralaglycysgly
171517201725
thralathralaalaalaglycysglyglyglyalaglyalathrgly
173017351740
thrcysglythrglyalacysthrthralacysalaglycysglygly
1745175017551760
cysalaalaglyalacysglythralathrglycyscysthrglycys
176517701775
cysthrglycysalaglycyscysthrcysalacysalacyscysthr
178017851790
cysglycysthrcysglycyscysglyglycysthrglyglyglyala
179518001805
glycyscysglyglycysthralacysglyalacysthrcyscyscys
181018151820
glycysthrcysthrthrthrglyglyalaalaglycysthrglyala
1825183018351840
alaala
<210>2
<211>614
<212>prt
<213>类芽孢杆菌甲壳素酶(paenibacilluspasadenensis)
<400>2
alaserasntyrlysilevalglytyrtyralasertrpalaalatyr
151015
glyargalatyrasnvalthraspileaspalaserlysmetasnval
202530
ileasntyralaphealaaspilecystrpasnglylyshisglyasn
354045
proaspprothrglyproasnprothrthrtrpsercysglnasnglu
505560
alaglyglnalaileasnvalproasnglythrvalvalleuglyasp
65707580
protrpileaspalaglnlysserpheglyaspasplystrpaspasp
859095
proilelysglyasnleulysglnleutrplysleulysasplysasn
100105110
proaspleulysthrvalileserileglyglytrpthrtrpserasn
115120125
argpheseraspvalalaalathralaalathrarggluvalpheala
130135140
asnserservalasppheilearglystyrlysmetaspglyvalasp
145150155160
leuasptrpglutyrprovalserglyglyleuaspglyasnsertyr
165170175
argprogluasplysglnasntyrvalleuleuleuglnlysilearg
180185190
glulysleuaspalaalaglulysalaaspglylysasptyrleuleu
195200205
thrilealaserglyalaglyprothrtyrileglnasnasnaspleu
210215220
alaglyilealaserilevalasptrpileasnilemetthrtyrasp
225230235240
pheasnglysertrpasnlysthrserglyhisasnalaproleutyr
245250255
tyraspthralaalaalathrserglyleuthraspproglnasnphe
260265270
asnileasplysalavalthrthrtyrleualalysglyvalproala
275280285
serlysleuvalleuglymetalaphetyrglyargglytrpglygly
290295300
cysprothralaglyasnglyglntyrglnvalcysalaglyileser
305310315320
serthrglythrtrpglulysglysertyraspphetyraspleuglu
325330335
alaasntyrileasnlysasnglytyrthrargtyrtrpasnaspala
340345350
alalysvalprotyrleutyrasnprothrasnglythrtyrileser
355360365
tyraspaspvalglnserpheaspphelysthrsertyrleulysser
370375380
lysglyleualaglyalametphetrpgluthrserglyaspargasn
385390395400
lysthrleuleuasnlysleualaseraspleuglytyralaglyala
405410415
thrprothrprovalprothralathrproseralathrprothrpro
420425430
thralathrproservalthrprothrprothrthrserproserala
435440445
thrprothrproseralathrprothrproglythrcysthrvalthr
450455460
alatrpserserthralavaltyrthrglyglyglnlysalasertyr
465470475480
asnglythriletyrglualalystrptrpthrglnglyaspargpro
485490495
aspleuserglyalaasnglyprotrplysalaalaglythrcysgly
500505510
thrthrargprothrproseralathrprothrprothrprothrala
515520525
thrprothrproseralathrprolysproseralathrprothrpro
530535540
thralathrprothrproseralathrprothrprothralathrpro
545550555560
thrproseralathrproglyalaseralatrpalaalaglyvalala
565570575
tyrlysalaglyaspvalvalthrtyrserglylysthrtyralacys
580585590
leuglnprohisthrserleualaglytrpgluproalathrthrpro
595600605
alaleutrplysleulys
610