CN114507615A - 一种解淀粉芽孢杆菌及其生产纤溶酶的应用 - Google Patents
一种解淀粉芽孢杆菌及其生产纤溶酶的应用 Download PDFInfo
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Abstract
本发明名称为一株解淀粉芽孢杆菌GXU‑1及其生产纤溶酶的应用,属微生物应用领域,该菌株分离自海洋方格星虫肠道,命名为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU‑1,菌种于广东省微生物菌种保藏中心,保藏号为GDMCC No.61785,可应用于纤溶酶的生产,所产纤溶酶在20~45℃、pH 5~11具有良好的水解纤维蛋白的活性,酶活性受到Fe2+、Mn2+、Fe3+、EDTA、PMSF等物质的抑制,属于丝氨酸金属蛋白酶,具有直接纤溶活性。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一株解淀粉芽孢杆菌GXU-1及其生产纤溶酶的应用。
背景技术
解淀粉芽孢杆菌(Bacillus amyloliquefaciens)是一种非致病性需氧革兰氏阳性菌,属芽孢杆菌,与枯草芽孢杆菌具有很高的亲缘性,广泛分布于土壤、空气、海洋、污泥中。解淀粉芽孢杆菌可产生大量具有抗菌活性、免疫活性、抗氧化活性的胞外代谢物,如大环内酯类、蛋白质类、酯肽类抗生素等;同时,可以产生大量的淀粉酶和蛋白酶。该类菌属表现出较强的抗逆性和适应性,并且在促进植物生长方面发挥重要作用,被广泛应用于水产、农作物的病害防治,环境保护、动物饲料等多个领域。
纤溶酶全称为纤维蛋白溶解酶,是一类作用于肽键的水解酶,具有溶解血栓的功能。血栓的形成是引发心肌感染、深静脉血栓形成以及心脑血管疾病的主要原因,世界卫生组织数据显示,2016年全球便有1790万人死于血栓性疾病,血栓性疾病已成为造成人类死亡的第一大元凶。目前临床上用于治疗血栓性疾病的手段可分为三种,分别是外科手术、抗凝治疗以及药物溶栓。血栓性疾病的患者大都是65岁以上的老年人,外科手术治疗风险大,愈后效果难以保证;抗凝治疗所用的华法林、双嘧达莫、阿司匹林等药物,生产成本较高,存在引发不良出血、胃肠道不适、肝损伤、过敏反应、白细胞碎屑血管炎和脱发等副作用;临床上所用的溶栓药物,如尿激酶和链激酶,也有很大概率导致出血等致命的并发症,尤其是对于患有脑出血的患者。
寻找更为安全有效的纤溶酶来源是目前治疗血栓性疾病的研究热点,已有报道表明微生物源纤溶酶具有较高的底物特异性,且生产成本低、可大量集中生产。公开号为CN113234627 A的中国专利公开了一株高产纳豆激酶的枯草芽孢杆菌及其应用,该发明从麦秆自然发酵物中分离得到枯草芽孢杆菌(Bacillus subtilis)JNFE0127,发酵生产纤溶酶的条件为豆类浸泡6~24 h后,于100℃蒸0.5~1.5 h后接种菌种,于37~41℃发酵12~96 h,发酵产物纳豆激酶酶活24673 IU/g。该发明专利采用固体发酵,发酵步骤较繁琐,发酵时间较长,所得纤溶酶需要采用特定的分离技术,不易实现工业化量产。公开文献《Characterization of an Intracellular Alkaline Serine Protease from Bacillusvelezensis SW5 with Fibrinolytic Activity》报道了分离自鱼露的一株芽孢杆菌,可生产一种胞内碱性丝氨酸蛋白酶ISP-SW5,其最适pH和温度分别为8.0、40℃,纤溶酶活性为1261 U/mg,能够被PMFS(苯甲基磺酰氟)抑制其酶活,而Ca2+和Zn2+能够增强其酶活性,但是由于胞内酶的分离纯化难度较大,其最适温度高于人体温度,该菌株生产的酶不适于溶栓药物的开发应用。
海洋是药物资源开发的重要宝库,由于其高渗、高压、低温等特殊环境,海洋源的微生物代谢途径可以产生具有独特结构的代谢产物。已有报道的海洋微生物及其在纤溶酶生产应用的研究较少,且微生物的来源、产酶发酵工艺,以及生产的纤溶酶性质均不同。公开文献《一株来自可口革囊星虫产纤溶酶菌株的筛选与鉴定》报道了来自可口革囊星虫肠道的菌株XC5,经鉴定为稻壳芽孢杆菌(Bacillus oryzaecorticis),该菌株在30℃培养18h后检测到最高纤溶酶活力为601.4 U/mL;公开文献《海洋青霉菌代谢纤溶活性化合物的研究》报道了来自南极磷虾的海洋青霉菌A-10,最优发酵温度25℃、pH 6.0、接种量为2%、转速180 rpm、发酵5天,得到纤溶酶的活性为736.44 U/mg;公开文献《产纤溶酶海洋枯草芽抱菌的液体发酵条件优化》报道了分离自海底淤泥的海洋枯草芽孢杆菌(marine Bacillus subtilis )Y-6-A,最佳发酵条件为:可溶性淀粉46.1 g/L,黄豆粕粉23.3 g/L.,温度31.2℃,转速184 rpm,在此发酵条件下得到的酶活为3606.23 IU/mL。
本发明从方格星虫肠道中分离到一株产纤溶酶菌株,经鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),经液体发酵可产生高活性的纤溶酶。
发明内容
本发明的目的是提供了一株分离自方格星虫肠道的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1,菌种保藏号为GDMCC No. 61785。
本发明的另一目的在于提供了解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1在生产纤溶酶的应用。
本发明的另一个目的是提供了解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1生产纤溶酶的方法。
本发明的另一个目的是提供了解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1发酵所产生纤溶酶的酶学性质。
具体的,本发明涉及以下技术方案:
本发明从方格星虫肠道中分离到一株产纤溶酶的解淀粉芽孢杆菌,该菌株命名为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1,于2021年8月7日保藏于广东省微生物菌种保藏中心,菌种保藏号为GDMCC No. 61785,所述GXU-1菌株全基因组序列在GeneBank数据库的登录号为CP065159。
进一步的,所述解淀粉芽孢杆菌在生产纤溶酶中的应用包括利用本发明的解淀粉芽孢杆菌GXU-1或其发酵液生产纤溶酶。
进一步的,所述利用解淀粉芽孢杆菌GXU-1生产纤溶酶的方法为将菌种接种于LB液体培养基进行活化,活化后的菌液按照2%(v/v)的接种量接种于液体发酵培养基,于30℃、200 rpm培养36 h后离心取上清液,获得具有纤溶活性的酶液。
进一步的,向上述酶液中添加硫酸铵至溶液中硫酸铵饱和度(w/v)达到70%,静置过夜后于4℃、10000 rpm离心20 min,收集沉淀后,使用透析袋(MW7000Da)透析,使用含1.9mol/L (NH4)2SO4的0.05 mmol/L的磷酸盐缓冲液(pH 7.0)将透析后的沉淀溶解,于4℃、10000 rpm离心10 min,使用0.22 μm微孔滤膜对上清液进行过滤,而后使用Methy HICSupport疏水层析柱(1.6 cm×10 cm)进行分离,使用1 mL/min流速的0.05 mmol/L PBS缓冲液(pH 7.0)进行梯度洗脱,收集第100 min ~108 min时样品,而后使用Sephacryl S-200凝胶柱(1cm×45cm)进行纯化,使用1mL/min流速的0.05mmol/L PBS缓冲液(pH为7.0)进行梯度洗脱,收集第66 min时洗脱液。将收集到的洗脱液透析后冻干保存,得到纤溶酶,酶活为693.9 U/mL。
进一步的,所述纤溶酶在20~45℃、pH5~11具有良好的水解纤维蛋白的活性, 37℃具有最大酶活性,最适pH为8,酶活性受到K+,Na+,Mg2+,Ca2+,Zn2+、Cu2+、Fe2+、 Mn2+、Fe3+、EDTA、PMSF等物质的抑制,属于丝氨酸金属蛋白酶,具有直接纤溶活性。
与现有技术相比,本发明具有以下有益效果:
本发明提供了一种来源于方格星虫肠道的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1,可发酵产生纤溶酶,采用液体发酵获得高活性的胞外纤溶酶,发酵方法简单易行。
此外, GXU-1菌株近经液体发酵后,得到纤溶酶活693.9 U/mL的纤溶酶,该酶在pH8,37℃具有最大酶活性,具有较强的纤维蛋白直接纤溶活性,该酶最适酶活温度接近人体温度,表现出优良的新型溶栓药物开发潜力。
附图说明
图1是解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1 的全基因组序列与GenBank核酸数据库比对结果。
图2是SDS-PAGE检测目的酶分子量图(注:Marker为已知分子量标准蛋白质,分子量范围为11 KDa ~180KDa,1为分子筛过滤后的纤溶酶)。
图3是纤溶酶的最适温度(A)与温度稳定性(B)曲线。
图4是纤溶酶的pH(A)与pH稳定性(B)曲线。
图5是金属离子以及抑制剂对纤溶酶活力影响。
图6是纤溶酶体外溶栓作用方式示意图。
图7是纤溶酶体外溶栓能力示意图。
具体实施方式
以下结合具体实施例对本发明进行描述,本发明所述的技术方法,如未做特殊说明,均为本领域的常规方法;所用试剂及材料,均来源于商业渠道。本领域技术人员可以在不偏离本发明精神和范围的情况下,对本发明做出各种改变和修改,以使其适用各种用途和条件。
L B液体培养基配方:蛋白胨10 g/L,酵母膏5 g/L,葡萄糖5 g/L,氯化钠5 g/L,调pH至7.2。
液体发酵培养基配方:蛋白胨12 g/L,酵母提取物0.8 g/L,蔗糖17.5 g/L,调pH至7.5。
L B固体培养基配方:蛋白胨10 g/L,酵母膏5 g/L,葡萄糖5 g/L,氯化钠5 g/L,琼脂15 g/L,调pH至7.2。
脱脂乳平板的制作:脱脂乳20 g/L,琼脂15 g/L,分别配置50 mL的脱脂乳溶液与琼脂溶液,分别于121℃灭菌20 min后,于超净台上趁热混匀,倒平板。
纤维蛋白平板的制作:配置10 mL的0.002 g/mL的牛纤维蛋白原生理盐水溶液于37℃水浴保温,
0.2 g琼脂糖溶于10 mL超纯水于121℃高压灭菌20 min,取10 U/mL的凝血酶5 mL于37℃水浴待用。无菌琼脂糖溶液冷却至45℃左右时加入凝血酶,充分混匀后加入牛纤维蛋白原生理盐水溶液,充分混匀后快速倒板,室温放置3 h后使用玻璃棒打孔(直径3 mm)。
纤溶酶活性的测定:配置浓度为100 U/mL、200 U/mL、300 U/mL、400 U/mL、500 U/mL、的尿激酶溶液,以生理盐水为空白对照,每个浓度分别取10μL加入纤维蛋白板孔中,室温静置10 min,37℃温育18h后测定溶解圈垂直直径。以尿激酶纤溶活性的对数(lgC)为横坐标,纤溶圈垂直直径乘积D对数(lgA)为纵坐标,绘制尿激酶酶活力标准曲线线性方程,取lO μL待测液加入到纤维蛋白平板孔中,37℃温育18 h后测定溶解圈垂直直径,根据标准曲线计算酶的活力。
实施例1
解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1的获得,包括如下步骤:
对新鲜方格星虫样本进行清洗后,于无菌条件下取出肠体,加入1:2(w/v)无菌0.85%NaCl溶液充分研磨,进行梯度稀释后选择适宜梯度的稀释液涂布于LB固体培养基上,25℃倒置培养7 d后,挑取单菌落进行分离纯化,使用脱脂乳平板,筛选出具有较强产酶能力的菌株。利用菌株进行液体发酵培养后,获得具有纤溶活性的酶液,使用纤维蛋白平板法进行酶液的纤溶活性测定。对具有较高产纤溶酶的菌株进行分离纯化,将纯化的菌株送至北京百迈客生物科技有限公司进行菌株全基因组测序,与GenBank核酸数据库(www.ncbi.nlm.nih.gov/BLAST)进行比对(图1),确定该菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),命名为GXU-1。将该菌株全基因组序列提交到Gen Bank,登录号为CP065159。该菌株于2021年月8日保藏于广东省微生物菌种保藏中心,菌种保藏号为GDMCC No. 61785。
实施例2
利用解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1生产纤溶酶的应用,包括如下步骤:。
1、菌种活化:将GXU-1菌种接种于LB液体培养基,于30℃、200 rpm培养12 h,得到活化的GXU-1菌液。
2、发酵:将步骤1制得的活化GXU-1菌液按照2%(v/v)的接种量接种于液体发酵培养基,于30℃、200 rpm培养36h后离心取上清液,获得具有纤溶活性的发酵液。
3、纤溶酶样品的获得:向步骤2制得的发酵液中添加硫酸铵至硫酸铵饱和度(w/v)达到70%,静置过夜后于4℃、10000 rpm离心20 min,收集沉淀后,使用透析袋(MW7000Da)透析,将截留液冻干,得到含有纤溶酶的蛋白样品,于-80℃保存。
4、纤溶酶的疏水层析:使用含1.9 mol/L(NH4)2SO4的0.05 mmol/L的PBS缓冲液(pH7.0)将含有纤溶酶的蛋白样品溶解,于4℃、10000 rpm离心10 min,使用0.22 μm微孔滤膜对上清液进行过滤,而后使用Methy HIC Support疏水层析柱(1.6 cm×10 cm)进行分离,使用1 mL/min流速的0.05 mmol/L PBS缓冲液(pH 7.0)进行梯度洗脱,共洗脱得到5处峰,收集第100 min~108 min时的洗脱样品,冻干后,得到纤溶酶粗酶,于-80℃保存。
5、纤溶酶的纯化:使用0.05 mmol/L的PBS缓冲液(pH 7.0)将纤溶酶粗酶溶解,于4℃、10000 rpm离心10 min,收集得到上清液经Sephacryl S-200凝胶柱(1 cm×45 cm)进行纯化,使用1 mL/min流速的0.05 mmol/L PBS缓冲液(pH 7.0)进行梯度洗脱,在第66 min收集该处的洗脱组分,透析后冻干,得到纤溶酶,于-80℃保存。
使用SDS-PAGE对纯化所得样品的分子量进行测定,分离胶浓度为12%,堆积胶浓度为5%,纯化后样品分子量约为30.7 kDa(图2),酶活为693.9 U/mL。使用高效液相凝胶色谱对纯化所得样品的纯度进行检测,仅有一个单峰出现,说明该样品具有较高的纯度。
实施例3
纤溶酶的性质分析,其内容及方法如下:。
1、纤溶酶的最适温度以及温度稳定性
将纤溶酶溶于生理盐水溶液中形成1 mg/mL的样液,取10 μL点样于纤维蛋白平板,而后将平板分别置于20℃、37℃、45℃、50℃、55℃、65℃恒温放置18 h,对纤溶酶最适温度进行测定。
将纤溶酶溶于生理盐水溶液中形成1mg/mL的样液,分别于20℃、37℃、45℃、50℃、55℃、65℃温育1 h后用纤维蛋白平板法测定其剩余酶活,对纤溶酶的温度稳定性进行测定。
由图3所示,该纤溶酶在37℃具有最大酶活性,在20~45℃之间均能保持较好的稳定性。
2、纤溶酶的最适pH值与pH稳定性
将纤溶酶溶于生理盐水溶液中形成1 mg/mL的样液,分别于pH为5、6、7、8、9的纤维蛋白平板上测定酶活,对纤溶酶的最适pH值进行测定。
将纤溶酶溶于生理盐水溶液中形成1 mg/mL的样液,将pH为5、6、7、8、9的缓冲液与酶液进行1:1混合,在pH为7的纤维蛋白平板上测定其剩余酶活,对纤溶酶的pH稳定性进行测定。
由图4所示,该纤溶酶最适pH值为8,在pH为7~9的范围内具有较强的纤溶活性。
3、金属离子以及抑制剂对纤溶活性的影响
分别用10 mM Tris-HCL缓冲液(pH为7)分别配置0.2 mmol/L、0.6 mmol/L、1mmol/L、2 mmol/L、10 mmol/L、20 mmol/L 的KCl、NaCl、MgCl2、MnCl2、ZnCl2、CuCl2、CaCl2、FeCl2、FeCl3、EDTA、PMSF溶液,纤溶酶溶于pH 8的0.1 mol/L的Tris-HCL缓冲液形成1 mg/mL的样液,将与上述各浓度的溶液与样液进行1:1混合,以未做处理的样液为对照,37℃孵育1h后,使用纤维平板法测定其剩余酶活力。由图5所示,各金属离子均表现出抑制作用,最为显著的是10 mmol/L的Fe2+,最弱的是0.3 mmol/L的K+,不同浓度下各金属离子对酶活的抑制效果大都呈先减小后增大的趋势,Fe3+的抑制效果则在0.5 mmol/L达到最小,而后表现出较强的抑制效果体而言,单价离子抑制效果较弱,二价离子抑制效果较强,EDTA的抑制效果极强,可能该酶活性中心需要金属的参与,高浓度下PMSF表现出强烈的抑制效果,说明该酶属于丝氨酸蛋白酶。
4、纤溶酶体外溶栓作用方式的测定
制备纤维蛋白平板,将此平板在85℃恒温箱中加热30 min,使平板中的纤维蛋白原失活,定义为对照平板,未经热处理平板为标准平板,分别将尿激酶与纤溶酶样品加入对照平板和标准平板中,于37℃培养18 h,测量溶解圈直径。在标准平板中,尿激酶与纤溶酶样品形成的溶解圈直径分别为17 mm与17.9 mm,无显著差异,而在对照平板中,尿激酶未出现溶解圈,纤溶酶样品则形成了明显的溶解圈(图6,说明纤溶酶具有较强的直接纤溶活性。
5、纤溶酶体外溶栓能力的测定
采取新鲜人血,将血液与抗凝剂(柠檬酸钠)按照9:1的比例混合,在离心管(W1)中加入0.3 mL血液,而后加入0.2 mL2%(w/v)的CaCl2水溶液,混合后于37℃水浴1.5 h,获得血块。再次称重离心管(W2)后加入0.4 mL的纤溶酶溶液,37℃孵化3 h后倾倒出管内液体,对剩余血块进行称量(W3),使用0.4 mL两种不同浓度的酶液(214U与27U),阴性对照为生理盐水,阳性对照为不同浓度的尿激酶0.5 mL(525U和88U)
由图7所示,高浓度纤溶酶溶液(214U)与尿激酶(525U)对血块的溶解率分别为50.43%与52.83%,二者酶活差别较大,但是对血块的溶解率无显著差别,低浓度纤溶酶溶液(27U)与尿激酶(88U)对血块的溶解率分别为43.50%与23.56%,纤溶酶溶液对血块的溶解率高于尿激酶。结果表明纤溶酶具有良好的纤溶活性。
Claims (3)
1.一株解淀粉芽孢杆菌GXU-1及其生产纤溶酶的应用,其特征在于,所述解淀粉芽孢杆菌GXU-分离自海洋方格星虫肠道的解淀粉芽孢杆菌(Bacillus amyloliquefaciens),该菌株命名为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)GXU-1,菌种保藏号为GDMCC No.61785。
2.权利要求1所述的解淀粉芽孢杆菌GXU-1生产纤溶酶的应用,其特征在于,将菌种GXU-1接种于LB液体培养基活化后,按照2~5%(v/v)的接种量接种于液体发酵培养基,于24~37℃、200~220 rpm培养30~40 h,经硫酸铵沉淀、疏水层析、纯化后,得到纤溶酶,酶活为693.9 U/mL。
3.权利要求1所述的纤溶酶,其特征在于,纤溶酶在20~45℃、pH 5~11具有良好的水解纤维蛋白的活性,酶活性受到K+,Na+,Mg2+,Ca2+,Zn2+、Cu2+、Fe2+、 Mn2+、Fe3+、EDTA、PMSF等物质的抑制,属于丝氨酸金属蛋白酶,具有直接纤溶活性。
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