CN114807190B - 一种南极地衣链霉菌壳聚糖酶基因及其应用 - Google Patents
一种南极地衣链霉菌壳聚糖酶基因及其应用 Download PDFInfo
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- CN114807190B CN114807190B CN202110110583.3A CN202110110583A CN114807190B CN 114807190 B CN114807190 B CN 114807190B CN 202110110583 A CN202110110583 A CN 202110110583A CN 114807190 B CN114807190 B CN 114807190B
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- chitosanase
- chitosan
- streptomyces
- antarcticus
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Abstract
本发明提供了一种南极地衣链霉菌壳聚糖酶基因及其应用。本发明根据大肠杆菌密码子的偏好性,对链霉菌的壳聚糖酶编码基因进行了优化,优化后的核酸序列与原始核酸序列的同源性为73.39%,该壳聚糖酶SPAMC‑CSN在大肠杆菌BL21(DE3)中成功表达,获得的壳聚糖酶SPAMC‑CSN能高效降解壳聚糖,获得聚合度为2‑4的壳寡糖,其中壳三糖为主要降解产物;并且对不同脱乙酰度的壳聚糖底物有较高的水解活性,获得不同脱乙酰度的复杂壳寡糖。本发明提供的壳聚糖酶可用于壳寡糖及几丁寡糖等的规模制备,具有良好的工业应用前景。
Description
技术领域
本发明属于微生物基因工程领域,具体的,本发明涉及一种南极地衣链霉菌壳聚糖酶基因及其应用。本发明的壳聚糖酶可以用于催化壳聚糖制备壳寡糖。
背景技术
壳寡糖是一类聚合度一般为2-10、可溶于水的氨基糖类化合物,也是目前发现的自然界中唯一带正电荷的寡糖。研究证明壳寡糖具有杀菌、抗肿瘤、调节人体免疫、降血糖血脂、改善骨质疏松症以及神经保护等功效,极具开发潜力,已广泛应用于生物医药、保健食品、精细化工、农林畜牧等领域。
研究发现,壳寡糖的功能与聚合度有密切关系,如聚合度为2-3的壳寡糖具有爽口甜味,由于其降血糖的生理功能,且在人体内很少被消化道吸收,是糖尿病人和肥胖病人理想的功能性甜味剂;聚合度4-7的壳寡糖具有抑制癌细胞生长和转移的效果。
特定聚合度壳寡糖的主要制备方法包括化学法和酶解法。化学法虽然能够降解壳聚糖生成壳寡糖,但存在环境污染、过程繁琐、重复性差、产物聚合度不可控等弊端。酶法制备具有反应条件温和、催化效率高、重复性好、产物聚合度可控、绿色环保等优点。壳聚糖酶(Chitosanases,EC.3.2.1.132)是一种糖苷水解酶,主要来源于古菌、细菌、真菌以及植物等,以内切方式催化水解壳聚糖中的β-1,4-氨基葡萄糖苷键,生成壳寡糖。按照糖活性酶数据库(Carbohydrate-Active enZYmes,CAZY),壳聚糖酶在糖苷水解酶(GlycosideHydrolases,GH)家族5、7、8、46、75及80中均有分布,其中,家族46、75及80中仅包含壳聚糖酶。
在工业上,由于缺乏经济高效的特异性壳聚糖酶,往往使用蛋白酶及纤维素酶等非特异性商品酶类水解壳聚糖以制备壳寡糖。由于具有壳聚糖酶水解活性的酶类在这些商品酶中所占比例极低,用酶量较大,壳寡糖的生产成本也相应增加。因此,迫切需要研发出一系列经济高效的壳聚糖酶以满足工业壳寡糖生产的需求。
另外,目前的壳聚糖酶大多存在专一性差的问题,导致产物聚合度范围不可控。因此,获得能够专一性降解壳聚糖生成特定聚合度壳寡糖的壳聚糖酶,具有重要的理论意义和广阔的应用前景。
发明内容
本发明目的在于提供一种南极地衣链霉菌壳聚糖酶的基因及该基因表达的壳聚糖酶。
本发明的另一目的是提供包含一种南极地衣链霉菌壳聚糖酶基因的重组载体以及高效表达壳聚糖酶的重组菌株。
为实现上述目的,本发明采用的技术方案为:
本发明提供了一种南极地衣链霉菌壳聚糖酶基因,所述南极地衣链霉菌壳聚糖酶基因的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种南极地衣链霉菌壳聚糖酶SPAMC-CSN,所述南极地衣链霉菌壳聚糖酶SPAMC-CSN由上述的基因表达而成。
根据本发明所述的南极地衣链霉菌壳聚糖酶SPAMC-CSN,其中,该酶的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了包含上述南极地衣链霉菌壳聚糖酶基因的重组表达载体,优选重组表达载体所用载体为质粒pET22b。
本发明还提供了包含上述重组表达载体的重组菌株。进一步优选地,所述重组菌株为大肠杆菌BL21(DE3)。
本发明的上述南极地衣链霉菌壳聚糖酶SPAMC-CSN的制备方法,包括以下步骤:
1)构建表达编码南极地衣链霉菌壳聚糖酶的基因序列,获得上述链霉菌壳聚糖酶基因,然后构建上述重组载体;
2)用步骤1)的重组载体转化宿主细胞,得到重组菌株;
3)培养重组菌株使其发酵,诱导南极地衣链霉菌壳聚糖酶表达;
4)回收并纯化所表达的南极地衣链霉菌壳聚糖酶。
本发明还提供了上述南极地衣链霉菌壳聚糖酶基因在降解壳聚糖或几丁质中的应用。同时,提供了上述南极地衣链霉菌壳聚糖酶在降解壳聚糖或几丁质中的应用。
优选地,本发明所述的表达载体为质粒pET22b-SPAMC-CSN,由壳聚糖酶基因和表达载体pET-22b载体组成。将所述表达壳聚糖酶基因的质粒pET22b-SPAMC-CSN导入到大肠杆菌BL21(DE3)菌株中进行高效表达,目的蛋白质的产量达到了~1300mg/L,并通过体外实验证实了所述的壳聚糖酶SPAMC-CSN的酶活。
本发明的壳聚糖酶SPAMC-CSN可以用于高效催化降解壳聚糖或几丁质制备聚合度2-4为主的壳二糖,壳三糖和壳四糖。具体方法:壳聚糖水解酶SPAMC-CSN水解高脱乙酰度(脱乙酰度>94%)壳聚糖或几丁质制备壳寡糖时,可以得到聚合度2-4为主的壳寡糖。
本发明所具有的优点:本发明的壳聚糖酶能够专一降解不同脱乙酰度壳聚糖或几丁质的酶,为不同脱乙酰度壳聚糖或几丁质的降解提供了一种工具酶。
本发明所述的壳聚糖酶可单独或者与其他壳聚糖酶或几丁质酶类组合用于壳聚糖及几丁质等的降解,从而制备获得壳寡糖或几丁寡糖。
附图说明
图1为本发明壳聚糖酶基因的表达质粒结构示意图。
图2为本发明表达壳聚糖酶SPAMC-CSN镍柱纯化的SDS-PAGE电泳图(框内的条带为纯化的目的蛋白质SPAMC-CSN)。
图3为壳寡糖标准品的HPLC图(D:氨基葡萄糖);
图4为本发明表达壳聚糖酶SPAMC-CSN催化降解壳聚糖反应产物的HPLC图(D:氨基葡萄糖)。
图5为本发明表达壳聚糖酶SPAMC-CSN催化水解壳聚糖的LC-ESI质谱(D:氨基葡萄糖)
图6为本发明表达壳聚糖酶SPAMC-CSN催化水解脱乙酰度30%壳聚糖的MALDI-TOF质谱图(A:N-乙酰氨基葡萄糖;D:氨基葡萄糖)。
具体实施方式
下面结合实施例对本发明的技术方案进行详细说明。以下采用的试剂和生物材料如未特别说明,均为商业化产品。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。
本发明中质粒提取采用OMEGA公司Plasmid Mini Kit I试剂盒(D6943-01);BL21(DE3)感受细胞来自中国科学院过程工程研究所糖生物工程课题组。
实施例1壳聚糖水解酶SPAMC-CSN表达菌株的构建
本发明参照编码壳聚糖水解酶SPAMC-CSN的基因(GenBank No:AGJ58226.1),通过密码子优化,委托华大基因有限公司合成了编码壳聚糖水解酶(不包括信号肽)的基因,共有867个碱基,核甘酸序列如SEQ ID NO.1所示,克隆载体为pET22b,克隆位点BamHI和XhoI,载体抗性为氨苄青霉素(Amp),优化物种E.coli(图1)。对携带表达质粒pET22b-SPAMC-CSN的大肠杆菌DH5α进行培养,使用Plasmid Mini Kit I试剂盒提取质粒,然后将表达质粒导入感受态大肠杆菌BL21(DE3)中,获得重组菌株。氨基酸序列如SEQ ID NO.2所示,含有288个氨基酸,预测蛋白分子量为29.37kDa。
SEQ ID NO.1:
(1)序列特征
长度:867bp
类型:碱基序列
链型:双链
拓扑结构:线性
(2)分子类型:DNA
(3)假设:否
(4)反义:否
(5)最初来源:AGJ58226.1
(6)特异性名称:南极地衣链霉菌壳聚糖水解酶SPAMC-CSN基因。
SEQ ID NO.2:
MHHPHTSTSR RTTPLTRTAG LAVLALGLTF TAIPATAQPG VPTSSAAHLEAAATGLDDPA 60
KKDIAMQLVS SAENSTLDWK AQYGYIEDIG DGRGYTAGII GFCSGTGDMLDLVELYTERE 120
PGNALASYLP ALREVDGTDS HEGLDPGFTD AWAEAASDPA FEQAQNDERDRVYFDPAVRQ 180
GKADGLGTLG QFAYYDAIVM HGGGTDATSF GSIRQRALAV ASPPSQGGDETAYLDAFLDA 240
RVWAMKQEEA HSDTSRVDTA QRVFLRNGNL DLDPPLDWKV YGDSFHIG 288
(1)序列特征
长度:288
类型:氨基酸序列
(2)分子类型:蛋白质
(3)假设:否
(4)反义:否
(5)最初来源:AGJ58226.1
(6)特异性名称:南极地衣链霉菌壳聚糖水解酶SPAMC-CSN。
实施例2壳聚糖水解酶SPAMC-CSN的表达与检测
(1)配制含有Amp抗性的LB培养基:5g/L酵母提取物,10g/L胰蛋白胨,10g/L氯化钠,120℃灭菌20min,冷却到室温加入Amp使其终浓度100μg/mL。
(2)将实施例1得到的重组菌株,接种到含有Amp抗性的固体LB培养基上,37℃过夜培养,挑取单菌落接种到10mL含有Amp抗性的液体LB培养基中,37℃,200rmp摇床培养24小时后,将上述菌液接种到600mL含有Amp抗性的液体LB培养基中,37℃、200rmp摇床培养至OD600nm=0.6时,加入0.25mM IPTG,16℃、200rmp诱导表达12小时,4000rmp离心,收集菌体。
(3)取少量菌体进行SDS-PAGE分析,上清溶液中检测到目的蛋白质的表达。
实施例3壳聚糖水解酶SPAMC-CSN的表达、纯化与检测
(1)将实施例2收集到菌体,悬浮到缓冲溶液A(50mM Tri-HCl,pH 7.9,500mMNaCl)中进行超声破碎,12,000rmp离心收集上清,进行SDS-PAGE检测(图2)预测蛋白分子量29.37kDa。
(2)使用镍柱对上述蛋白进行纯化:
1.缓冲溶液A(50mM Tris/HCl,pH 7.9,0.5M NaCl)平衡柱子,流速1mL/min;
2.上样,流速1mL/min,收集穿透;
3.缓冲溶液A洗涤柱子,流速1mL/min,洗涤40mL;
4.缓冲溶液A+20mM咪唑洗脱,流速1mL/min,洗涤40mL,每4min收集一管;
5.G250检测收集样品,看最后一管是否还有蛋白被洗脱下来,若没有蛋白,则进行下一浓度咪唑洗脱;
6.缓冲溶液A+200mM咪唑洗脱,流速1mL/min,洗涤40mL,每4min收集一管;
7.G250检测收集样品,看最后一管是否还有蛋白被洗脱下来,若没有蛋白则进行下一浓度咪唑洗脱;
8.缓冲溶液A+500mM咪唑洗脱,流速1mL/min,洗涤40mL,每5min收集一管;
9.G250检测收集样品,看最后一管是否还有蛋白被洗脱下来,若没有蛋白,则进行下一浓度咪唑洗脱;
10.选取每个咪唑浓度洗脱蛋白含量高的组、原液、上样穿透、缓冲溶液A洗脱,进行SDS-PAGE分析(图3)。
合并比较纯的壳聚糖水解酶SPAMC-CSN酶组分,用14,000Da的透析袋进行透析。
在本实施例中,壳聚糖水解酶SPAMC-CSN在大肠杆菌中获得高效表达,纯化的蛋白质产量达到了~1300mg/1L培养基,为壳聚糖水解酶的应用开发奠定了基础。
实施例4壳聚糖水解酶SPAMC-CSN水解高脱乙酰度壳聚糖制备聚合度2-4的壳寡糖
称取0.05g壳聚糖(DDA,Degree of Deacetylation,脱乙酰度>94%),加至10mL1.5%乙酸水溶液中,(pH 5-6)。充分溶解后,加入4mL纯化的壳聚糖水解酶SPAMC-CSN溶液,37℃下震荡反应24小时。反应结束后,加入等体积的乙腈,离心去除不容物,配置成浓度为2.5mg/mL的溶液,过滤后用于高效液相色谱及质谱分析。高效液相色谱仪连接蒸发光散射检测器用于壳寡糖的信号检测,使用XAmide色谱柱(华谱新创科技有限公司)对壳寡糖进行分离,乙腈浓度递减(70%-50%)方式洗脱,柱温为30℃,检测器气压23psi,流速:1mL/min。流动相为0.1M甲酸胺(pH 3.2)、乙腈及水溶液。洗脱时间:40min。结果如图4所示:产物为聚合度2-4的壳寡糖。并对降解产物进行LC-ESI质谱分析(图5)。
实施例5壳聚糖酶水解低脱乙酰度壳聚糖制备壳寡糖
称取0.05g壳聚糖(DDA:~30%),加至10mL 1.5%乙酸水溶液中,(pH 5-6)。充分溶解后,加入4mL纯化的壳聚糖水解酶SPAMC-CSN溶液,37℃下震荡反应24小时。反应结束后,加入等体积的乙腈,离心去除不容物,配置成浓度为2.5mg/mL的溶液。由于该产物组分较为复杂,难以通过液相对组分进行有效分离,因此采用MALDI-TOF质谱方法对其组分进行分析。具体方法为:称取一定量制备的壳寡糖,配制成浓度为2mg/mL的水溶液,过滤后吸取1μL点样至样品板上,待其自然干燥后,加入1μL基质2,5-二羟基苯甲酸(DHB)溶液,待其干燥后使用autoflexⅢsmartbeam型MALDI-TOF质谱仪(Bruker公司)进行检测(正离子反射模式)。质谱检测结果如图6所示:为便于区分,以A代表N-乙酰氨基葡萄糖,D代表氨基葡萄糖,随后的数字代表含有该单糖的个数,二者加和为寡糖的聚合度。质谱方法可检测到聚合度3-15的寡糖。
当然,本发明还可以有多种实施例,在不背离本发明精神及其实质的情况下,熟悉本领域的技术人员可根据本发明的公开做出各种相应的改变和变形,但这些相应的改变和变形都应属于本发明的权利要求的保护范围。
序列表
<110> 中国科学院过程工程研究所
<120> 一种南极地衣链霉菌壳聚糖酶基因及其应用
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cacgaagggc tggaccccgg attcaccgac gcctgggccg aggcggcctc ggaccccgcg 480
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Claims (10)
1.一种南极地衣链霉菌壳聚糖酶基因,其特征在于:所述基因的核苷酸序列如SEQ IDNO.1所示。
2.一种南极地衣链霉菌壳聚糖酶,其特征在于:由权利要求1所述南极地衣链霉菌壳聚糖酶基因表达而成。
3.根据权利要求2所述的南极地衣链霉菌壳聚糖酶,其特征在于,所述酶的氨基酸序列如SEQ ID NO.2所示。
4.包含权利要求1所述南极地衣链霉菌壳聚糖酶基因的重组表达载体。
5.根据权利要求4所述的重组表达载体,其特征在于:所述重组表达载体所用载体为质粒pET22b。
6.包含权利要求4或5所述重组表达载体的重组菌株。
7.根据权利要求6所述的重组菌株,其特征在于,所述重组菌株为大肠杆菌BL21(DE3)。
8.一种南极地衣链霉菌壳聚糖酶的制备方法,包括以下步骤:
1)构建表达编码南极地衣链霉菌壳聚糖酶的基因序列,获得权利要求1所述的南极地衣链霉菌壳聚糖酶基因,然后构建权利要求4或5所述的重组表达载体;
2)用权利要求4或5所述的重组表达载体转化宿主细胞,得到重组菌株;
3)培养重组菌株使其发酵,诱导南极地衣链霉菌壳聚糖酶表达;
4)回收并纯化所表达的南极地衣链霉菌壳聚糖酶。
9.权利要求1所述的南极地衣链霉菌壳聚糖酶基因在降解壳聚糖或几丁质中的应用。
10.权利要求2或3所述的南极地衣链霉菌壳聚糖酶在降解壳聚糖或几丁质中的应用。
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