CN104762307A - 几丁质酶基因chiC及其编码蛋白和应用 - Google Patents
几丁质酶基因chiC及其编码蛋白和应用 Download PDFInfo
- Publication number
- CN104762307A CN104762307A CN201510219342.7A CN201510219342A CN104762307A CN 104762307 A CN104762307 A CN 104762307A CN 201510219342 A CN201510219342 A CN 201510219342A CN 104762307 A CN104762307 A CN 104762307A
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- CN
- China
- Prior art keywords
- chic
- chitinase
- gly
- ala
- chitin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明涉及几丁质酶基因chiC及其编码蛋白和应用,属于基因工程技术领域。本发明以假交替单胞菌(Pseudoalteromonas sp.DL-6)为研究对象,首次克隆出具有SEQ ID No.1所示核苷酸序列的几丁质酶基因chiC,并获得了高活性几丁质酶,其制备方法简单,生产成本低,产品易保存,适宜的酶解温度是30℃,酶解pH值为9.0,对胶体几丁质具有高降解活性可达1.569±0.017U/ml,几丁质酶chiC降解α几丁质和β几丁质生成几丁二糖,具有工业化生产前景。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种几丁质酶基因chiC,特别涉及一种来源于假交替单胞菌的新菌株(Pseudoalteromonas sp.DL-6)(CGMCC No.8580)的几丁质酶基因chiC及其编码蛋白和应用。
背景技术
几丁质(chitin),俗称甲壳素,是一种可从虾蟹壳等废弃物中提取的天然多糖,其结构为N-乙酰-D-氨基葡萄糖(50~100%)和少量D-氨基葡萄糖通过β-1,4-糖苷键(0~50%)连接而成,自然界中每年由生物体合成的甲壳素为100-1000亿吨,是海洋环境中含量最丰富,自然界中仅次于纤维素的第二大生物质来源。几丁质降解产物为高附加值的几丁寡糖、几丁单糖及其衍生物等,具有增强免疫力、抗肿瘤、抗菌、降血压和降低胆固醇等生物活性,已广泛应用于医药、农业、工业和食品等众多领域。
目前,几丁质制备几丁寡糖普遍采用的方法是化学法,即用强酸去除盐类、蛋白等,用强碱去除脂类或脱除乙酰基等。但是该工艺耗能大,反应过程及产物的分子量分布难以控制,最重要的问题是生产过程中排放出大量的废酸碱水,造成了严重的环境污染。因此,寻找一种绿色、高效高产高品质几丁寡糖的方法越来越受重视。几丁质酶(chitinase,chi,EC3.2.1.14)是指专一降解几丁质为几丁寡糖或单糖的一组酶的总称。自然界中,chi广泛存在于细菌、病毒、真菌、昆虫、植物以及动物中,该酶具有重要的生理功能,不同来源的chi,其催化机理和生理功能可能差别很大。细菌产生的几丁质酶可降解几丁质产生碳源和能源,获取自身生长、繁殖所需养料,同时保证自然界中几丁质的循环利用;真菌几丁质酶可实现自身细胞的***与形态构建;昆虫等节肢动物分泌几丁质酶满足蜕皮和生长发育的需要;病毒几丁质酶在发病机制中产生;植物分泌几丁质酶却是为了防御病源微生物的侵袭而进行的自我保护;一些原生动物需要几丁质酶消化昆虫的围食膜。人类产生的几丁质酶功能尚未确定,据推测可能与抵抗体内几丁质源的病原体或在碳水化合物代谢过程中发挥生理功能。
几丁质是海洋环境最丰富的资源,据估计10%的海洋细菌依赖几丁质作为碳源和氮源,海洋具有高盐(3.5%)、高压(﹥100Mpa)、低温、低光照、寡营养以及无光照、局部高温(400℃)等极端生态环境,海洋低温chi具有低温下高酶活力及高催化效率、结构高效柔顺性和热不稳定性等生物学特性。复杂多变的生态环境和遗传机制和遗传结构赋予海洋低温chi产生特有的具有不同的聚合度(degree of polymerization,DP)几丁寡糖。几丁质主要来源于海洋生物体,如虾、蟹和藻类和其他软体动物是,因此海洋中积聚着十分丰富的几丁质资源。几丁质资源如果处理不好,不仅造成浪费而且污染环境。海洋低温几丁质酶降解几丁质生产几丁寡糖及其衍生物解释了海洋环境几丁质物质循环的基本规律,不仅为新型海洋微生物资源的开发与利用、海洋环境的保护与治理提供重要的理论依据,而且对于开拓几丁质行业的市场具有应用价值。
发明内容
本发明解决的技术问题是提供一种具有SEQ ID No.1所示核苷酸序列的几丁质酶基因chiC,并构建了chiC原核表达载体,获得了高活性的几丁质酶。
为解决上述技术问题,本发明采用的构思是:以假交替单胞菌(Pseudoalteromonassp.DL-6)(CGMCC No.8580)菌株为研究对象,克隆和原核表达了几丁质酶chiC基因,为获得高活性几丁质酶及具有几丁质酶的功能的产品提供了新思路。
本发明采用的技术方案是:
一种几丁质酶基因chiC,为SEQ ID No.1所示的核苷酸序列。
上述几丁质酶基因chiC的扩增方法,以假交替单胞菌株(Pseudoalteromonas sp.DL-6)基因组DNA为模板,以引物对chiC-F和chiC-R进行PCR扩增;上游引物chiC-F:5’-CCCGGATCCGGCGCCTTCTACACCAAGCATTAATTGG-3’,下游引物chiC-R:5’-CCGCTCGAGAAGTGCTAACCATACATCGG-3’。
本发明的第二个目的是请求保护一种由上述几丁质酶基因chiC表达的几丁质酶。
所述的几丁质酶,具有如SEQ ID No.4所示的氨基酸序列。
本发明的第三个目的是请求保护一种几丁质酶chiC的表达与纯化方法,步骤如下:
(1)以PCR方法扩增如SEQ ID No.1所示的核苷酸序列;
(2)构建大肠杆菌克隆载体pMD19-T-chiC:将PCR扩增的DNA序列和pMD19-T simple载体用同样的限制性内切酶进行双酶切,酶切产物经回收纯化,用T4DNA连接酶连接纯化的酶切产物,得到克隆载体pMD19-T-chiC;
(3)构建大肠杆菌重组表达载体pET28a-chiC:将克隆载体pMD19-T-chiC质粒转化至E.coli DH5α,得到阳性转化子,用限制性内切酶BamHI和XhoI将chiC基因片段从pMDl9-T-chiC质粒上切下,连接到pET-28a表达载体上,转化至E.coli BL21(DE3),通过菌落PCR、酶切筛选鉴定阳性转化子,得到重组表达载体pET28a-chiC;
(4)构建大肠杆菌重组表达菌株BL21(DE3)-pET28a-chiC:将重组表达载体pET28a-chiC质粒转化至E.coli BL21(DE3),挑选阳性转化子克隆,得到重组表达菌株BL21(DE3)-pET28a-chiC;
(5)体外诱导表达:将重组表达菌株BL21(DE3)-pET28a-chiC接入含卡那霉素的LB培养基中,于37℃过夜培养14h后,按1%的接种量接种到含卡那霉素的LB培养基中,当OD600nm=0.6-0.8,加入终浓度为0.2mM的IPTG,于30℃,100rpm低温诱导6h;
(6)几丁质酶chiC的纯化:体外诱导表达结束后于4℃,5,000×g离心5min收集菌体,用PBS缓冲液洗涤菌体三次,再用PBS缓冲液重悬菌体,之后置于冰上超声破碎三次,每次30s,每次间隔1min,然后于4℃,12,000×g离心20min收集上清液,即为粗蛋白,粗蛋白用Ni2+-NTA柱进行亲和层析纯化,得到几丁质酶chiC。
本发明还请求保护上述的几丁质酶在降解含几丁质底物中的应用。
所述的几丁质酶chiC的应用,适宜的酶解条件为:酶解温度30℃,pH值为9.0。
所述的几丁质酶chiC对胶体几丁质具有高降解活性达1.569±0.017U/mL。
所述的几丁质酶chiC降解α几丁质和β几丁质生成几丁二糖。
一种具有几丁质酶的功能的产品,具有如SEQ ID No.4所示的氨基酸序列。
本发明的有益效果是:
1、本发明获得了几丁质酶基因chiC的序列,该序列首次在假交替单胞菌(Pseudoalteromonas sp.DL-6)中克隆;
2、本发明构建了chiC原核表达载体,成功获得分子量约92.659kD的酶蛋白,得到了具有高活性的酶,且制备过程简单,为chiC功能研究提供了简便方法;
3、本发明中几丁质酶chiC低温酶活高,酶解最适宜的反应温度为30℃,在pH=6.0~9.0酶活较高,当pH=9.0达到最高,且生产成本低,产品易保存;
4、本发明中几丁质酶chiC对胶体几丁质有较高的降解活性,对α几丁质和β几丁质降解生成几丁二糖,具有一定的工业化生产前景。
附图说明
图1为本发明chiC基因的PCR扩增产物琼脂糖凝胶电泳检测结果;
图2为本发明chiC蛋白的SDS-PGAE检测结果,图中M:蛋白标准分子量;1~3均为BL21/chiC-pET28aSDS-PGAE检测结果:1为破壁全菌;2为超声破碎上清液;3为破壁沉淀;4~6均为BL21/pET28a检测结果:4为破壁全菌;5为破壁后上清液;6为破壁后沉淀;
图3为本发明chiC蛋白纯化的SDS-PGAE检测结果,图中M:蛋白标准分子量;1:重组菌破壁后上清;2:重组菌破碎后上清经镍柱纯化的流出液;3:20mmol的咪唑洗脱收集液;4:150mmol的咪唑洗脱收集液;5:300mmol的咪唑洗脱收集液;1~5上样量均为10μL;
图4为chiC蛋白的标准曲线和标准曲线方程;
图5为酶活标准曲线和标准曲线方程;
图6为本发明中温度对几丁质酶chiC活性的影响;图中,A:几丁质酶chiC活性最适温度;B:几丁质酶chiC温度稳定性;
图7为本发明中pH对几丁质酶chiC活性的影响;图中,A:几丁质酶chiC最适pH;B:几丁质酶chiC pH稳定性;
图8为本发明chiC蛋白的动力学常数分析;
图9为本发明chiC蛋白的底物特异性;
图10为本发明chiC蛋白降解产物分析;图中,A:α几丁质;B:β几丁质。
具体实施方式
下面通过实施例对本发明做进一步描述,但不用于限制本发明的保护范围。实施例中的实验方法,如无特殊说明,均为常规方法;所用到实验原料主要包括:大肠杆菌感受态细胞E.coli DH5α、E.coli BL21(DE3),pMD19-T Simple购自TaKaRa公司;T4DNA连接酶、限制性内切酶BamHI和XhoI、蛋白质分子量标准购自TaKaRa公司;Ni2+-NTA树脂购自Novagen公司;虾壳α型几丁质、鱿鱼β型几丁质购自Sigma公司;IPTG、卡那霉素、丙烯酰胺十二烷基磺酸钠、甘氨酸购自生工生物工程(上海)股份有限公司,其它常用化学试剂均为市售分析纯。
实施例1 chiC基因的扩增
本发明根据NCBI上已报道的假交替单胞菌几丁质酶的基因序列设计引物,通过聚合酶链式反应(PCR)的方法获得了假交替单胞菌(Pseudoalteromonas sp.DL-6)几丁质酶chiC的保守序列。该菌株已在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏日期:2013年12月13日,其保藏编号为CGMCC No.8580。
利用PCR技术以假交替单胞菌(Pseudoalteromonas sp.DL-6)菌株的基因组DNA为模板,采用Primer5.0软件设计引物,分别为:上游引物chiC-F(SEQ ID No.2):5’-CCCGGATCCGGCGCCTTCTACACCAAGCATTAATTGG-3’设计有BamHI酶切位点;下游引物chiC-R(SEQ ID No.3):5’-CCGCTCGAGAAGTGCTAACCATACATCGG-3’设计有XhoI酶切位点。
PCR反应体系:
基因组DNA模板 | 2.5ng |
10×LA PCR Buffer(Mg2+plus) | 5μL |
dNTPMixture(各2.5mM) | 8μL |
上游引物(20μM) | 1μL |
下游引物(20μM) | 1μL |
TaKaRa LA Taq(5U/μL) | 0.5μL |
RNase Free dH2O up to | 50μL |
PCR反应条件:94℃ 2min,1个循环;94℃ 30s,55℃ 30s,68℃ 7min,30个循环;68℃ 15min,1个循环。PCR反应结束以1%琼脂糖凝胶电泳检测,如图1所示,在2631bp处有明亮单一条带,即为目的基因chiC。其核苷酸序列如SEQ ID No.1所示(Genbank登录号为:KF234017)。本发明所述的chiC编码876个氨基酸,分子量约92.659kD,具有如SEQID No.4所述的氨基酸序列。
实施例2 大肠杆菌克隆载体pMDl9-T-chiC的制备
1、chiC基因与pMDl9-T的连接
对实施例1得到的PCR产物进行纯化回收,使用TaKaRa公司试剂盒(TaKaRa DNALigation Kit<Mighty Mix>),将回收的chiC的PCR产物与pMD19-T simple克隆载体连接。
连接反应体系为:
chiC的PCR产物 | 2μL |
pMD19-TSimple(50ng/μL) | 1μL |
Ligation Mix(50ng/μL) | 5μL |
dH2O | 2μL |
连接反应的条件为:16℃过夜连接,构建克隆载体pMDl9-T-chiC。
2、克隆载体(pMDl9-T-chiC)的转化
(1)取冰上冻融的100μL大肠杆菌感受态细胞E.coli DH5α,于上述10μL的连接反应液中,轻柔混合均匀;
(2)冰中放置30分钟后,在42℃热激处理1min,然后立即转移冰中放置1min;
(3)加入900μL的SOC培养液,37℃振荡培养1h;
(4)取100μL涂LB/Kana培养基表面,37℃过夜培养,筛选阳性转化子。
LB/Kana培养基:在950mL去离子水中加入:胰蛋白胨10g,酵母提取物5g,NaCl 10g,摇动容器直至溶质溶解。用5N NaOH溶液(约0.2mL)调pH至7.0。用去离子水定容至1L,加入15g琼脂。高温高压灭菌冷却至室温。加入1mL卡那霉素(kanamycin,kana,50mg/mL)后混合均匀,4℃保存。
3、克隆载体(pMDl9-T-chiC)的提取
用TaKaRa公司试剂盒(TaKaRa MiniBEST Plasmid Purification),提取克隆载体(pMDl9-T-chiC)。
实施例3 大肠杆菌表达载体pET28a-chiC的制备
1、chiC基因的制备
已构建的重组质粒pMDl9-T-chiC用BamHI和XhoI两种限制性内切酶酶切鉴定,反应体系如下:
pMDl9-T-chiC(30ng/μL) | 10μL |
BamHI(5U/μL) | 2μL |
XhoI(5U/μL) | 2μL |
10×M buffer | 10μL |
dH2O up to | 100μL |
反应体系于37℃水浴中酶解2-3h,产物经1%琼脂糖凝胶电泳检测。酶解产物用TaKaRa公司试剂盒TaKaRa Agarose Gel DNA Purification Kit Ver.2.0,回收基因chiC。
2、表达载体pET-28a的制备
将表达载体pET-28a利用同样的限制性内切酶BamHI和XhoI进行双酶切鉴定,反应体系如下:
pET-28a(20ng/μL) | 20μL |
BamHI(5U/μL) | 2μL |
XhoI(5U/μL) | 2μL |
10×M buffer | 10μL |
dH2O up to | 100μL |
反应体系于37℃水浴中酶解2-3h,产物经1%琼脂糖凝胶电泳检测。酶解产物用TaKaRaAgarose Gel DNA Purification Kit Ver.2.0,回收表达载体pET-28a。
3、原核重组表达载体pET28a-chiC的构建
使用DNALigation Mix试剂盒(购自Takara公司),将BamHI和XhoI限制性内切酶酶切产生的chiC基因亚克隆至同样酶切处理的表达载体pET-28a中,反应体系:克隆载体(pMDl9-T-chiC)酶切产物chiC基因(30ng/μL)1μL,pET-28a(20ng/μL)2μL,5μL LigationMix,2μL dH2O。反应条件为:16℃过夜连接。
实施例4 构建大肠杆菌重组表达菌株
1、重组原核表达质粒pET28a-chiC的转化
(1)取冰上冻融的100μL大肠杆菌感受态细胞E.coli BL21(DE3),于上述10μL的连接反应液中,轻柔混合均匀;
(2)冰中放置30分钟后,在42℃热激处理1min,然后立即转移冰中放置1min;
(3)加入900μL的SOC培养液,37℃振荡培养1h;
(4)取100μL涂平板(LB/Kana),37℃过夜培养筛选阳性转化子;
(5)重组原核表达载体pET28a-chiC的提取,用TaKaRa公司试剂盒(TaKaRa MiniBESTPlasmid Purification)提取表达载体pET28a-chiC。
2、构建大肠杆菌重组表达菌株
(1)种培养:将含有重组原核表达载体pET-28a重组原核表达载体pET28a-chiC及空融合表达载体pET-28a的E.coli BL21(DE3)细胞挑1-2个单菌落接种到含卡那霉素终浓度为50μg/mL的2mL LB/Kana培养基中,37℃过夜培养。
(2)主培养:培养14小时后按1%接种量转移至100mL LB/Amp培养基中,37℃振荡培养至培养液的OD600nm达到0.6-0.8。
(3)诱导表达:加入终浓度为0.2mM的IPTG在30℃下100rpm低温诱导表达6h。
(4)收集全细胞:于4℃,5,000×g,离心10min收集菌体,并用300μL的PBS悬浮菌体细胞,混匀后取200μL菌悬液超声波处理10s/2~3次,直至液体透明,取出50μL作为全细胞样品。
(5)可溶蛋白的抽提:将上述菌液的残余部分于12,000×g,4℃离心5min,取50μL上清液即为可溶蛋白。
(6)不可溶蛋白的抽提:添加150μL PBS缓冲液于步骤(5)沉淀中,充分振荡混匀即为不可溶蛋白。
(7)SDS-PAGE电泳检测表达的目的蛋白,见附图2所示。
实施例5 重组几丁质酶chiC蛋白的纯化
1、chiC蛋白的大量诱导表达
(1)挑取一个重组大肠杆菌菌落接入50mL含卡那霉素(50μg/mL)的LB培养液,在250mL摇瓶中于37℃过夜培养;
(2)将50mL过夜培养物接入500mL含卡那霉素(50μg/mL)的LB培养液,在2L摇瓶中于37℃振荡过夜培养,至对数中期(OD600nm=0.6-0.8);
(3)加入终浓度为0.2mM的IPTG在30℃下,100rpm低温诱导靶蛋白6h;
(4)于4℃以5,000×g,离心5min收集菌体(约3.5g);
(5)将菌体沉淀重悬于8倍体积的PBS缓冲液中,4℃超声波破碎10s×3次,共直至液体透明;
(6)于4℃以12,000×g,离心20min收集上清液进行chiC蛋白纯化研究。
2、chiC蛋白通过Ni2+-NTA树脂进行大规模纯化
(1)用二次水清洗Ni2+-NTA树脂;
(2)用PBS清洗Ni2+-NTA树脂;
(3)将大量诱导表达的融合蛋白粗提取物上柱;
(4)用10倍体积20mM-300mM bufferA/咪唑洗脱(Elute)结合的重组蛋白,每份800μL分布收集,监测OD280,用20μL等份的蛋白进行10%SDS聚丙烯酰胺凝胶电泳,如图3所示,分析chiC蛋白的分布情况,获得重组几丁质酶chiC纯化蛋白。
实施例6 chiC对照蛋白溶液的制备
用质粒pET28a代替pET28a-chiC进行实施例1和实施例6的步骤制备对照蛋白溶液。
实施例7 chiC蛋白浓度的测定
一、蛋白标准曲线的测定
使用BCA-100蛋白浓度测定试剂盒(增强型)进行蛋白测定。首先根据待测样品数目,准备BCA A与BCA B的混合液(50份试剂A+1份试剂B),混匀后每个反应使用200μL。每个反应准备3个平行测定。按表1的次序加入0.5mg/mL BSA标准及BCA A+B混合物,混匀。
表1 BSA标准曲线试验
测试条件:37℃保温30分钟,冷却到室温,测定570nm的光吸收值,根据吸光度值,拟合出标准曲线,根据标准曲线计算待测样品的蛋白浓度。蛋白标准曲线及方程如附图4所示,
二、chiC蛋白浓度的测定
取适量浓度的chiC蛋白,加入一定体积的50mM Tris-HCl(pH9.0),总体积为20μL,加入Mix(A+B)200μL,37℃保温30分钟,冷却到室温,测定570nm的光吸收。根据图4方程计算得出chiC蛋白溶液的浓度为1.186mg/mL。
实施例8 chiC蛋白的几丁质酶酶活
一、胶体几丁质的配制:取10.0g粉状α几丁质置于研钵,于通风橱内缓慢加入100mL,85%的浓磷酸,同时不断的用玻璃棒充分搅拌。将研钵用保鲜膜封口,4℃下过夜。然后用大量的二次水冲洗,直到pH为5.0左右后离心,用缓冲液(如0.1mol/L磷酸缓冲液,pH7.0)定容为1L。配制成1%胶体几丁质溶液,避光保存于冰箱备用。
二、酶活测定标准曲线的建立
采用铁***法测定chiC酶活性,取1mL样品溶液,加入1mL铁***试剂(配制1L 0.5M碳酸钠溶液,加入0.5g铁***),在沸水中煮10min,然后用自来水冷却至室温,测量OD420nm,具体如下表2。以N-乙酰-D-氨基葡萄糖(mmol/L)为横坐标,OD420nm吸光度为纵坐标,计算出N-乙酰-D-氨基葡萄糖标准曲线。标准曲线及方程如图5所示。
表2铁***法测定还原糖含量
三、chiC蛋白的几丁质酶活性
取粗酶液0.1mL与0.9mL,1%胶体几丁质混合,20℃下保温30min后,将反应混合物在沸水浴中加热10分钟终止酶反应,15,000×g离心5min,取上清0.5mL,加入1mL,Schales试剂,沸水浴10min,冷却。以灭活的酶液作为对照组,以对照为参比,在420nm下测定吸光度。根据标准曲线计算出还原糖含量,从而计算出酶活。每个梯度设置3个重复,取平均值。
酶活力单位定义(U):在上述条件下,每分钟释放1μmol还原糖(以N-乙酰-D-氨基葡萄糖计)所需的酶量。根据图5方程计算chiC蛋白几丁质酶活性为6.947U/mL。
实施例9 chiC蛋白酶学性质的研究
对实施例5获得的几丁质酶chiC蛋白的酶学性质进行测定,包括最适温度、温度稳定性、最适pH、pH稳定性、金属离子和化学试剂对chiC蛋白酶活的影响,以胶体几丁质为底物进行动力学常数测定以及底物特异性研究。
(1)酶活测定
具体见实施例8。
(2)温度对几丁质酶chiC的影响
①最适反应温度:在pH值为8.0(50mmol/L Tris-HCl缓冲液)的条件下,以3.5mg/mL胶体几丁质为底物,在4℃-70℃的温度范围内测定酶活,根据酶在不同温度下的相对活性绘制曲线,确定酶的最适反应温度。结果如图6A所示,chiC的最适温度为30℃。
②chiC的热稳定性:将酶液分别置于不同温度(4℃-70℃)下保温1h,然后在最适反应pH和最适反应温度下检测残余酶活,并和未处理的酶活进行比较,计算相对活性。结果如图6B所示,chiC在低于30℃的温度下预孵育24h后,酶活保持在60%以上。
(3)pH对几丁质酶chiC的影响
①最适反应pH值:酶活反应温度为30℃,以3.5mg/mL胶体几丁质为底物,在pH值为3-12的50mmol/L缓冲液(pH 3.0~5.0磷酸-柠檬酸,pH 6.0~10.0Tris–HCl,pH 11.0~12.0Na2HPO4–NaOH)中测定酶活,根据酶在不同的pH值下的相对活性绘制曲线,确定酶的最适反应pH值。结果如图7A所示,chiC的最适pH是pH9.0。
②chiC的pH值稳定性:将酶液分别置于50mmol/L不同pH的缓冲液(pH 3.0~5.0phosphate-citrate,pH 6.0~10.0Tris–HCl,pH 11.0~12.0Na2HPO4–NaOH)中,于30℃下放置24h,然后在最适反应pH和最适反应温度下检测残余酶活,并和未处理的酶活进行比较,计算相对活性。结果如图7B所示,chiC在30℃,不同的pH预孵育24h后,在pH6以上活性开始提高,在pH>8.0酶活保持75%以上,表明chi C偏碱性。1M Tris-HCl(pH8.0):6.057g Tris,29.22g NaCl,定容到1L,HCl调到8.0,用时稀释至所需浓度。
(4)金属离子和化学试剂对几丁质酶chiC的影响
向反应体系中加入终浓度为1mmol/L或5mmol/L的KCl、NaCl、CaCl2、MgCl2、MnCl2、CuCl2、ZnCl2、CoCl2、FeSO4和FeCl3,然后在标准条件下测定酶活,并和不添加任何金属离子的酶活进行比较,计算相对活性。向反应体系中加入终浓度为1mmol/L或5mmol/L的EDTA、尿素、PMSF、DTT、SDS、Triton X-100和Tween-20,然后在标准条件下测定酶活,并和不添加任何化学试剂时的酶活进行比较,计算相对活性,结果见表3,从表3可知反应液中添加1mM和5mM的K+、Na+、Mg2+、Mn2+、Ca2+、1mM EDTA和PMSF对酶促反应有促进作用,1mM和5mM urea和DTT对酶促反应影响不大,而当添加1mM和5mM的Cu2+、Zn2+、Fe2+、Fe3+、Co2+、SDS、Triton X-100和Tween-20对酶促反应有抑制作用。
表3金属离子和化学试剂对chiC蛋白的影响
(5)酶动力学参数的测定
用一定摩尔浓度([E])的纯酶液,与不同浓度的底物反应,在标准分析条件下检测初始反应速度,因为kcat=Vmax/[E],其中[E]已知,可计算出kcat,最终得到Km和kcat。具体测定方法:用50mmol/L Tris-HCl缓冲液(pH8.0)稀释底物溶液胶体几丁质至一系列不同浓度(2~10mg/mL)的底物溶液;取0.9mL不同浓度的底物溶液与0.1mL适当浓度的chiC混合均匀,置于30℃水浴反应2h混匀。以煮沸灭活的酶液作为对照组,以对照为参比根据标准曲线计算出初始反应速度。结果见图8,动力学常数分析表明chiC的Km值为3.47mg/mL,Vmax为12.32μmol/min。
(6)chiC的底物特异性
1mL反应体系包含5μM的chiC酶,2mg/mL的α-几丁质、β-几丁质、胶体几丁质、β-几丁质纳米须、微晶纤维素和壳聚糖6种底物,在30℃反应24h,而后测还原糖含量(具体参照实施例8)。以灭活的酶液作为对照组,设置3个重复。结果见图9,由图可见chiC更容易降解胶体几丁质,酶活性达1.569±0.017U/mL,依次为α几丁质、β几丁质、壳聚糖、β几丁质纳米须和微晶纤维素,对应的酶活性分别为1.422±0.044U/mL、1.34±0.019U/mL、1.249±0.027U/mL、1.179±0.013U/mL和0.971±0.018U/mL。chiC偏好于胶体几丁质较高的降解活性说明由于酸水解处理后的胶体几丁质破坏了粉状几丁质的晶体结构,更利于几丁质酶活性位点的结合与酶催化反应发生。
实施例10 chiC蛋白的降解产物分析
利用基质辅助激光解析串联飞行时间质谱(MALDI-TOF/TOF 5800)检测chiC作用几丁质底物生成的产物。1mL反应体系:5mmol/L chiC,2mg/mLα几丁质和β几丁质,溶于50mmol/L Tris-HCl缓冲液(pH 9.0),30℃水浴锅中反应24h。取0.5μL处理好的上清液点加在MALDI TOF质谱仪专用的样品板上,在室温条件下自然晾干(约需3min),其上滴加0.5μL基质(2,4-二羟二苯甲酮,DHB)溶液覆盖,同法晾干后备用,每个样品重复3个孔。采用氮激光源,波长为337nm,线性阳离子检测模式;加速电压为15000V;延迟时间为300ns;激光强度为7000Hz;每个样品点设激光随机射击40个点、每次射击50次,m/z采集范围为1000D~20000D。采用仪器配置的Data Explorer 4.0软件对质谱图进行基线改正和默认平滑处理。如图10所示,chiC降解α几丁质和β几丁质主要生成几丁二糖为主要产物。
<110> 大连大学
<120> 几丁质酶基因chiC及其编码蛋白和应用
<130> 2015
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2631
<212> DNA
<213> Pseudoalteromonas sp. DL-6
<400> 1
atgaatatta aacaattaag cgcagctatg ggtgttgcac tatttgccgg ttctgtatcg 60
gcagcgcctt ctacaccaag cattaattgg gaaccgcagc aatattcgtt tgttgaggta 120
gatcttgagg gaaacggctc ttataagcag ctagttaccc gtgttgagca agttaatatc 180
aacattgagt ggagtgcatg gagtggtgat ggtggcgata gctacaaagt gtactttgat 240
gacatgctag tcaatgaagg cacgcttgct gctggttcta aaagtggcac tatcacattc 300
ccttatgata aagcgggccg ccatacaatg tatgttgagc tatgtgaagg tggcacaacg 360
tgtgctcgca gtgcaggcaa accaattgta attgctgaca ccgacggcgg acacctagca 420
ccattaccta tggatgttga cccaaataac cgtgacattg gtatcaaaca aggtttagtt 480
acaggcgctt actttgttga gtggggaatt tatggccgtg attatgatgt aacgaatatg 540
ccagcacaaa atctgagtca tattttgtat ggctttattc caatttgtgg tgaaaacgca 600
tcgttatcgg gtggtcctaa gcgtgcgctt gatactgcct gcgccggctc tgcagattac 660
gaagtggtta ttcacgaccc atgggcagca gtacaaaaag cactgccagg ggttgatgca 720
aaagatccta tccgtggtac ttactctcag ctaatggcgc ttaaacagcg ttacccagat 780
attaaaattt taccatctgt aggtggttgg acgttatcag acccattcgg tggctttacc 840
aataaagcta accgcgacac ctttgttgcc tcaatggaag aatttttaag aacatggaaa 900
ttctatgatg gtgtagatat tgactgggaa ttcccaggtg gtgacggccc caacccagac 960
attggcgatc caatcaacga tggcccagcc tatgtggcgc taatgcaaga gctacgcgct 1020
atgcttgata aacttgaagc agaaacgggc cgtacttacg agttaacctc agccattggt 1080
gcaggttacg ataaaattga agatgtagat taccaagccg caagccagta tatggattac 1140
atttttgcca tgacctatga cttttatggc gcatggagta acgtaaccgg acaccaaaca 1200
gcactttact gtggcgaaca tatgagtgtt ggtcaatgta atggtaccgg gcttgatgaa 1260
aatggcgaac ctcgtaaagg ccctgcttat accaccgata atgcagtgca gctgttactt 1320
gcgcaaaatg taccatcgaa aaaaatcgtc gtgggcacag ctatgtatgg ccgtggttgg 1380
gaaggtgttt acccgcaaaa tgcagcaatt gatggtaacc caatgaccgc ccctggtaat 1440
ggcccgttaa aaggctctac ggcacaaggc gtatgggaag atggggttat tgattacaaa 1500
ggcgttaaag caaatatgat tggtgcggcg ggcaccggta ttaatggttt tgaagtgggt 1560
tatgatgagc aagcgcaagc cgcttatgta tggaatcgtg caaccggtaa actaattact 1620
tatgatagcc gtaagtcagt actggctaag ggcgcgtatg ttaatcagta taatttaggt 1680
ggtttatttg catgggaaat agatgctgat aatggtgata ttctaaatgc tatgcatgat 1740
ggtttaggag gggtagttgc tccgccaaca aataaaaagc cagttgtttc tgtgtcttca 1800
tcagtgtctg ttaattcggg tgagagtatc acagtaacag cttctgcaac cgatgccgat 1860
aacgatccac ttagctttag ctggagtgct gataacgcac ttgtagtatc tggacaaaat 1920
agcgcatcat tagtcattac agcgccaaca gtgactgcag atacacagta tgtggcaacc 1980
gttgcggtat cagatggtga ggcaaccgtt aatcgtgatg ttgttgttaa tgttattgcg 2040
ccaacatcag gtggtgaaaa cacagctcct agcgttgatg ctatcgctaa tatcagcgtt 2100
gaagaaggcg catcaacatc agttgctgtt gtggcaagcg atgctcaaaa cgatgcagtt 2160
acatatacgt ggacagtacc agctggttta acgttagtgg gtagtggttc aaatgtaact 2220
attgaggctg gcgcagttga tgcagataca gatttcacag tgtctgttgc agttagtgat 2280
ggcgcattaa ccacaacgca aagctttagc gtaacagtta caaacgttga tactacaaat 2340
ccacctacag gaagctggga tgcgagtgtt gcatacgttg gtggtgatgt cgttacttat 2400
aacggcgttg aatataaagc aaagtggtgg actcaaggcg agcgtcctga tttaggtggt 2460
gcatgggaag caacaacaca acctacagat ggtacgggtg tcgcagtttg gcagccaacg 2520
gctatttata acagtggtga tgaagtttct tatcaaggta ataagtacca agctaaatgg 2580
tggactcaag ggaacgaacc tggttcaacc gatgtatggt tagcacttta a 2631
<210> 2
<211> 37
<212> DNA
<213> 人工合成
<400> 2
cccggatccg gcgccttcta caccaagcat taattgg 37
<210> 3
<211> 29
<212> DNA
<213> 人工合成
<400> 3
ccgctcgaga agtgctaacc atacatcgg 29
<210> 4
<211> 876
<212> PRT
<213> Pseudoalteromonas sp. DL-6
<400> 4
Met Asn Ile Lys Gln Leu Ser Ala Ala Met Gly Val Ala Leu Phe Ala
1 5 10 15
Gly Ser Val Ser Ala Ala Pro Ser Thr Pro Ser Ile Asn Trp Glu Pro
20 25 30
Gln Gln Tyr Ser Phe Val Glu Val Asp Leu Glu Gly Asn Gly Ser Tyr
35 40 45
Lys Gln Leu Val Thr Arg Val Glu Gln Val Asn Ile Asn Ile Glu Trp
50 55 60
Ser Ala Trp Ser Gly Asp Gly Gly Asp Ser Tyr Lys Val Tyr Phe Asp
65 70 75 80
Asp Met Leu Val Asn Glu Gly Thr Leu Ala Ala Gly Ser Lys Ser Gly
85 90 95
Thr Ile Thr Phe Pro Tyr Asp Lys Ala Gly Arg His Thr Met Tyr Val
100 105 110
Glu Leu Cys Glu Gly Gly Thr Thr Cys Ala Arg Ser Ala Gly Lys Pro
115 120 125
Ile Val Ile Ala Asp Thr Asp Gly Gly His Leu Ala Pro Leu Pro Met
130 135 140
Asp Val Asp Pro Asn Asn Arg Asp Ile Gly Ile Lys Gln Gly Leu Val
145 150 155 160
Thr Gly Ala Tyr Phe Val Glu Trp Gly Ile Tyr Gly Arg Asp Tyr Asp
165 170 175
Val Thr Asn Met Pro Ala Gln Asn Leu Ser His Ile Leu Tyr Gly Phe
180 185 190
Ile Pro Ile Cys Gly Glu Asn Ala Ser Leu Ser Gly Gly Pro Lys Arg
195 200 205
Ala Leu Asp Thr Ala Cys Ala Gly Ser Ala Asp Tyr Glu Val Val Ile
210 215 220
His Asp Pro Trp Ala Ala Val Gln Lys Ala Leu Pro Gly Val Asp Ala
225 230 235 240
Lys Asp Pro Ile Arg Gly Thr Tyr Ser Gln Leu Met Ala Leu Lys Gln
245 250 255
Arg Tyr Pro Asp Ile Lys Ile Leu Pro Ser Val Gly Gly Trp Thr Leu
260 265 270
Ser Asp Pro Phe Gly Gly Phe Thr Asn Lys Ala Asn Arg Asp Thr Phe
275 280 285
Val Ala Ser Met Glu Glu Phe Leu Arg Thr Trp Lys Phe Tyr Asp Gly
290 295 300
Val Asp Ile Asp Trp Glu Phe Pro Gly Gly Asp Gly Pro Asn Pro Asp
305 310 315 320
Ile Gly Asp Pro Ile Asn Asp Gly Pro Ala Tyr Val Ala Leu Met Gln
325 330 335
Glu Leu Arg Ala Met Leu Asp Lys Leu Glu Ala Glu Thr Gly Arg Thr
340 345 350
Tyr Glu Leu Thr Ser Ala Ile Gly Ala Gly Tyr Asp Lys Ile Glu Asp
355 360 365
Val Asp Tyr Gln Ala Ala Ser Gln Tyr Met Asp Tyr Ile Phe Ala Met
370 375 380
Thr Tyr Asp Phe Tyr Gly Ala Trp Ser Asn Val Thr Gly His Gln Thr
385 390 395 400
Ala Leu Tyr Cys Gly Glu His Met Ser Val Gly Gln Cys Asn Gly Thr
405 410 415
Gly Leu Asp Glu Asn Gly Glu Pro Arg Lys Gly Pro Ala Tyr Thr Thr
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Asp Asn Ala Val Gln Leu Leu Leu Ala Gln Asn Val Pro Ser Lys Lys
435 440 445
Ile Val Val Gly Thr Ala Met Tyr Gly Arg Gly Trp Glu Gly Val Tyr
450 455 460
Pro Gln Asn Ala Ala Ile Asp Gly Asn Pro Met Thr Ala Pro Gly Asn
465 470 475 480
Gly Pro Leu Lys Gly Ser Thr Ala Gln Gly Val Trp Glu Asp Gly Val
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Ile Asp Tyr Lys Gly Val Lys Ala Asn Met Ile Gly Ala Ala Gly Thr
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Gly Ile Asn Gly Phe Glu Val Gly Tyr Asp Glu Gln Ala Gln Ala Ala
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Tyr Val Trp Asn Arg Ala Thr Gly Lys Leu Ile Thr Tyr Asp Ser Arg
530 535 540
Lys Ser Val Leu Ala Lys Gly Ala Tyr Val Asn Gln Tyr Asn Leu Gly
545 550 555 560
Gly Leu Phe Ala Trp Glu Ile Asp Ala Asp Asn Gly Asp Ile Leu Asn
565 570 575
Ala Met His Asp Gly Leu Gly Gly Val Val Ala Pro Pro Thr Asn Lys
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Lys Pro Val Val Ser Val Ser Ser Ser Val Ser Val Asn Ser Gly Glu
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Ser Ile Thr Val Thr Ala Ser Ala Thr Asp Ala Asp Asn Asp Pro Leu
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Ser Phe Ser Trp Ser Ala Asp Asn Ala Leu Val Val Ser Gly Gln Asn
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Ser Ala Ser Leu Val Ile Thr Ala Pro Thr Val Thr Ala Asp Thr Gln
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Tyr Val Ala Thr Val Ala Val Ser Asp Gly Glu Ala Thr Val Asn Arg
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Asp Val Val Val Asn Val Ile Ala Pro Thr Ser Gly Gly Glu Asn Thr
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Ala Pro Ser Val Asp Ala Ile Ala Asn Ile Ser Val Glu Glu Gly Ala
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Ser Thr Ser Val Ala Val Val Ala Ser Asp Ala Gln Asn Asp Ala Val
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Thr Tyr Thr Trp Thr Val Pro Ala Gly Leu Thr Leu Val Gly Ser Gly
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Ser Asn Val Thr Ile Glu Ala Gly Ala Val Asp Ala Asp Thr Asp Phe
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Thr Val Ser Val Ala Val Ser Asp Gly Ala Leu Thr Thr Thr Gln Ser
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Phe Ser Val Thr Val Thr Asn Val Asp Thr Thr Asn Pro Pro Thr Gly
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Ser Trp Asp Ala Ser Val Ala Tyr Val Gly Gly Asp Val Val Thr Tyr
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Asn Gly Val Glu Tyr Lys Ala Lys Trp Trp Thr Gln Gly Glu Arg Pro
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Asp Leu Gly Gly Ala Trp Glu Ala Thr Thr Gln Pro Thr Asp Gly Thr
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Gly Val Ala Val Trp Gln Pro Thr Ala Ile Tyr Asn Ser Gly Asp Glu
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Val Ser Tyr Gln Gly Asn Lys Tyr Gln Ala Lys Trp Trp Thr Gln Gly
850 855 860
Asn Glu Pro Gly Ser Thr Asp Val Trp Leu Ala Leu
865 870 875
Claims (10)
1.一种几丁质酶基因chiC,为SEQ ID No.1所示的核苷酸序列。
2.一种如权利要求1所述的几丁质酶基因chiC的扩增方法,其特征在于,以假交替单胞菌株(Pseudoalteromonas sp.DL-6)基因组DNA为模板,以引物对chiC-F和chiC-R进行PCR扩增;上游引物chiC-F:5’-CCCGGATCCGGCGCCTTCTACACCAAGCATTAATTGG-3’,下游引物chiC-R:5’-CCGCTCGAGAAGTGCTAACCATACATCGG-3’。
3.一种如权利要求1所述的几丁质酶基因chiC表达的几丁质酶。
4.根据权利要求3所述的几丁质酶,其特征在于,具有如SEQ ID No.4所示的氨基酸序列。
5.一种几丁质酶chiC的表达与纯化方法,其特征在于,步骤如下:
(1)以PCR方法扩增如SEQ ID No.1所示的核苷酸序列;
(2)构建大肠杆菌克隆载体pMD19-T-chiC:将PCR扩增的DNA序列和pMD19-T simple载体用同样的限制性内切酶进行双酶切,酶切产物经回收纯化,用T4 DNA连接酶连接纯化的酶切产物,得到克隆载体pMD19-T-chiC;
(3)构建大肠杆菌重组表达载体pET28a-chiC:将克隆载体pMD19-T-chiC质粒转化至E.coli DH5α,得到阳性转化子,用限制性内切酶BamHI和XhoI将chiC基因片段从pMDl9-T-chiC质粒上切下,连接到pET-28a表达载体上,转化至E.coli BL21(DE3),通过菌落PCR、酶切筛选鉴定阳性转化子,得到重组表达载体pET28a-chiC;
(4)构建大肠杆菌重组表达菌株BL21(DE3)-pET28a-chiC:将重组表达载体pET28a-chiC质粒转化至E.coli BL21(DE3),挑选阳性转化子克隆,得到重组表达菌株BL21(DE3)-pET28a-chiC;
(5)体外诱导表达:将重组表达菌株BL21(DE3)-pET28a-chiC接入含卡那霉素的LB培养基中,于37℃过夜培养14h后,按1%的接种量接种到含卡那霉素的LB培养基中,当OD600nm=0.6-0.8,加入终浓度为0.2mM的IPTG,于30℃,100rpm低温诱导6h;
(6)几丁质酶chiC的纯化:体外诱导表达结束后于4℃,5,000×g离心5min收集菌体,用PBS缓冲液洗涤菌体三次,再用PBS缓冲液重悬菌体,之后置于冰上超声破碎三次,每次30s,每次间隔1min,然后于4℃,12,000×g离心20min收集上清液,即为粗蛋白,粗蛋白用Ni2+-NTA柱进行亲和层析纯化,得到几丁质酶chiC。
6.一种如权利要求3所述的几丁质酶在降解含几丁质底物中的应用。
7.根据权利要求6所述的几丁质酶chiC的应用,其特征在于,适宜的酶解条件为:酶解温度30℃,pH值为9.0。
8.根据权利要求6所述的几丁质酶chiC的应用,其特征在于,所述的几丁质酶chiC对胶体几丁质具有高降解活性可达1.569±0.017U/ml。
9.根据权利要求6所述的几丁质酶chiC的应用,其特征在于,所述的几丁质酶chiC降解α几丁质和β几丁质生成几丁二糖。
10.一种具有几丁质酶的功能的产品,具有如SEQ ID No.4所示的氨基酸序列。
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