CN101412982B - 解淀粉类芽孢杆菌p17菌株,由其所得的低温果胶酶及其分离纯化方法 - Google Patents
解淀粉类芽孢杆菌p17菌株,由其所得的低温果胶酶及其分离纯化方法 Download PDFInfo
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Abstract
本发明提供解淀粉类芽孢杆菌(Paenibacillus amylolyticus)P17菌株,以及以该菌株为原料分离纯化所得的低温果胶酶及其分离纯化方法。该方法包括粗酶液的制备、硫酸铵盐析、阴离子交换层析、阳离子交换层析后,收集有活性的组分保存,即得纯酶。本发明的低温果胶酶由于在低温条件下的活性好、对金属离子耐受性较好、对有机溶剂耐受性较好和对高酯化度的果胶底物具有很高的专一性,可广泛应用于冷洗涤行业、重金属污染废水处理、水果加工业、纺织、制浆造纸等工业领域。
Description
技术领域
本发明属于微生物技术领域,具体地,涉及解淀粉类芽孢杆菌(Paenibacillusamylolyticus)P17菌株,由其产生的低温果胶酶及其分离纯化方法。
背景技术:
果胶酶(Pectinases,EC.3.2.1.15)是指能够分解果胶质(由D-半乳糖醛酸以α-1,4糖苷键连接形成的直链聚合物,部分被甲酯化)的,由多种酶组成的一类复合酶,它广泛存在于高等植物、昆虫和微生物中。果胶酶主要有两种分类方法:1)根据底物优先法、水解类型和降解方式可以分为原果胶酶、果胶酯酶、果胶解聚酶2)根据最适作用pH分为酸性果胶酶和碱性果胶酶。
果胶酶主要应用于食品业,尤其是水果加工业,它可以有效地提高水果的出汁率,改善果汁的过滤效率,减少化学澄清剂的用量,改善果汁质量,简化果汁加工工艺,缩短加工时间,提高生产效率、果汁产量和产品质量的稳定性。此外,果胶酶还可以应用于纺织、制浆造纸、麻类脱胶、木材防腐,甚至有报道用于治疗胃结石等领域。
低温果胶酶的发现不仅为果胶物质的低温降解提供了可能,而且扩展了果胶酶的工业应用范围。低温果胶酶多由低温微生物产生。Lund等人在研究果蔬腐坏病过程中发现了微生物果胶酶,在以后的几十年里,国内外研究者对微生物果胶酶进行了大量的研究,但对微生物低温果胶酶的研究则起步较晚,主要研究都集中在最近十多年。国内对低温果胶酶的研究工作刚刚开展,有关低温果胶酶的研究鲜有报道。国外对低温果胶酶的研究主要集中在菌种筛选、鉴定、理化性质分析方面。Takasawa等从小麦幼苗中分离得到的嗜冷性核盘菌(Sclerotinia borealis)产生低温聚半乳糖醛酸酶,最适酶活温度40~50℃,最适pH4.5,在5℃仍有最大活性的30%,室温过夜或50℃保温30min活性损失明显,60℃活性弱。Laurent等从储存于冷库的根芹菜腐烂部位中分离出产生三种果胶酸裂解酶同工酶(PL I、PL II和PLIII)的浅黄金色单胞菌(Chryseomonas luteola)。Truong等从南极冰水中首次分离出一株产低温果胶酶的海洋微生物Pseudoalteromonas haloplanktis,该菌株能产生两种具有低温果胶酸裂解酶活力的酶,最适酶活温度30℃,最适pH分别为9和10。Miura,et al.从深海沉积物中分离的新型隐球酵母菌(Cryptococcus N6)产生的两种低温聚半乳糖醛酸酶(P36和P40),最适作用温度为50℃,在0℃分别保持有最高活性的25%和30%。P36和P40对压力有很好的耐受性,所受的压力缓慢升到100Mpa时,酶活力没有发生变化。Birgisson,et al.从冻土、被冻结的树叶或树枝中分离得到8株分泌胞外冷活性聚半乳糖醛酸酶的酵母,并对其中四株酵母进行了产酶条件的研究。最近关于微生物低温果胶酶的报道,来自于Margesin,et al,关于两株木克拉酵母(Mrakia frigida A15和AG25)的研究。A15和AG25分别分离自高山冰川的冰尘和西伯利亚北部地区的沉积物中,它们的最适产酶温度分别为5℃和1℃,其产酶温度均低于它们的最适生长温度。Margesin等对Mrakia frigida A15和AG25产生的低温果胶酸裂解酶研究表明,最适酶活温度30℃,最适pH分别为9.0和8.5,在0℃分别保持有最高活性的21%和16%,Ca2+是两种酶活性的必须金属离子。由于低温果胶酶在低温下仍能保持较高的活力和对热敏感等特点已引起众多学者的广泛关注,已成为近年酶学研究的一个热点。因此,对低温果胶酶的研究不仅在理论上有着重要的意义,而且在工业应用上也具有巨大的潜在价值。
发明内容:
本发明的目的是提供一株产生低温果胶酶的菌株P17,经16S rRNA鉴定,将此菌株初步鉴定为解淀粉类芽孢杆菌(Paenibacillus amylolyticus)。
本发明的另一目的是提供一种在低温条件下具有较高活性的低温果胶酶及其分离纯化方法。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
解淀粉类芽孢杆菌(Paenibacillus amylolyticus)P17菌株,菌种保藏号为CGMCC No.2640。
低温果胶酶,从解淀粉类芽孢杆菌(Paenibacillus amylolyticus)P17菌株产生,其酶学性质为:最适反应温度为40℃,并在0-10℃保持稳定;最适pH为8.0,在pH6.0-10.0保持稳定;Mg2+、Na+、Zn2+和Cd2+对其有激活作用,Li+、Cu2+、Mn2+、Ca2+、Fe2+、Ba2+、Ni2+、Pb2+、Co2+、Al3+和Fe3+对酶有轻微的抑制作用,Hg2+对酶有显著的抑制作用;鳌合剂EDTA和变性剂SDS对其有抑制作用;丝氨酸蛋白酶专一抑制剂苯甲基磺酰氟化物对其没有抑制作用;其酶活力随着有机溶剂如甲醇、乙醇、丙酮和乙腈浓度的升高而提高,当浓度达到一定值50%时,随着其浓度的升高低温果胶酶活力反而下降;10%浓度的二甲基亚砜对酶有激活作用,但随着其浓度的升高,对低温果胶酶有抑制作用;10%浓度的甲醛对酶表现出较强的抑制作用;随着果胶酯化程度的增加,酶活力表现出相应的提高,其最适底物为90%酯化度的果胶;但该酶不能降解多聚半乳糖醛酸;低温果胶酶在4℃时的Km值为0.233mg/mL,远远低于其最适温度40℃时的Km值1.362mg/mL。
低温果胶酶,从解淀粉类芽孢杆菌(Paenibacillus amylolyticus)P17经下述纯化方法获得:粗酶液的制备:将解淀粉类芽孢杆菌菌株P17接种于液体发酵培养基中,培养至稳定期初期,离心,过滤,收集上清液;硫酸铵盐析:往粗酶液中加入硫酸铵,离心取上清,再加入硫酸铵,离心取沉淀,在上样缓冲液中透析;阴离子交换层析:酶液上样,收集有活性的透过液;阳离子交换层析:酶液上样,洗脱,收集有活性的组分-20℃甘油保存,即纯酶。
低温果胶酶,从解淀粉类芽孢杆菌(Paenibacillus amylolyticus)P17经下述纯化方法获得:将解淀粉类芽孢杆菌菌株P17接入液体LB培养基中,在13~25℃的条件下振荡培养至指数生长期,将处于指数生长期的菌液转接于液体发酵培养基中,13~25℃振荡培养至稳定期初期即为产酶高峰期,将发酵液离心,取上清,滤纸过滤,得粗酶液;用截留分子量为10KDa的膜包将粗酶液的体积浓缩至原来的1/10;浓缩后的酶液加硫酸铵至60%~70%饱和度,离心取上清,继续加硫酸铵至90%~100%,离心取沉淀,在pH8.0的KPB缓冲液透析过夜;将透析后的酶液离心,取上清液,上样于已用pH8.0的KPB缓冲液平衡好的DEAE-Sepharose预装柱,收集有活性的透过峰酶液,并用截留分子量为10KDa的浓缩管进行浓缩;用pH6.0的KPB缓冲液将浓缩酶液透析过夜,离心取上清,上样于已用pH6.0的KPB缓冲液平衡好的CM-Sepharose预装柱,洗脱,收集有活性的部分,即为纯酶。
解淀粉类芽孢杆菌属P17低温果胶酶的纯化方法包括下列步骤:
(1)粗酶液的制备:将菌株P17接入液体LB培养基中,在13~25℃的条件下振荡培养至指数生长期,将处于指数生长期的菌液转接于液体发酵培养基中,13~25℃振荡培养至稳定期初期即为产酶高峰期,将发酵液离心,取上清,滤纸过滤,得粗酶液;
(2)用截留分子量为10KDa的膜包将粗酶液的体积浓缩至原来的1/10;
(3)浓缩后的酶液加硫酸铵至60%~70%饱和度,离心取上清,继续加硫酸铵至90%~100%,离心取沉淀,在pH8.0的KPB缓冲液透析过夜;
(4)将透析后的酶液离心,取上清液,上样于已用pH8.0的KPB缓冲液平衡好的DEAE-Sepharose预装柱,收集有活性的透过峰酶液,并用截留分子量为10KDa的浓缩管进行浓缩;
(5)用pH6.0的KPB缓冲液将浓缩酶液透析过夜,离心取上清,上样于已用pH6.0的KPB缓冲液平衡好的CM-Sepharose预装柱,洗脱,收集有活性的部分,即为纯酶。
发酵培养基的组成为:酵母粉0.2g/L,葡萄糖1g/L,硫酸铵2g/L,磷酸氢二钾1g/L,氯化钾0.5g/L,硫酸亚铁0.01g/L,硫酸镁0.5g/L,pH9.0。
本发明所说的菌株解淀粉类枯草芽孢杆菌P17于2008年8月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为:CGMCC No.2640。样品的来源为禄劝彝族苗族自治县轿子雪山的土壤。菌株的筛选分离和培养方法为取少量土壤样品于富集培养基中13℃振荡培养3~5天,稀释涂布于固体LB培养基,于13℃倒置培养。待完全长出菌落后,用划线法将单菌落划线至新鲜的固体LB培养基平板上,于13℃恒温培养箱中倒置培养。如此重复直至获得纯培养物。将纯培养物接种到液体LB培养基中,于13℃恒温培养箱中振荡培养。经酶活检测,筛选出产生低温果胶酶的菌株。所说的培养基如下:
富集培养基:果胶2g/L,硫酸镁0.5g/L,氯化钾0.5g/L,硫酸铁0.01g/L,磷酸氢二钾1g/L,硝酸钠3g/L,PH7.0。
固、液体LB培养基:NaCl10g/L,酵母粉5g/L,蛋白胨10g/L,[琼脂15g/L],pH7.0。
本文所说的菌株解淀粉类芽孢杆菌属P17低温果胶酶具有以下酶学性质:
1、菌株解淀粉类芽孢杆菌属P17低温果胶酶最适酶活温度40℃,在0℃和4℃还分别具有最高活性的8%和10%,表现出明显的低温果胶酶特性。
2、菌株解淀粉类芽孢杆菌属P17低温果胶酶在0~10℃之间活力较为稳定,保温1小时,还保持80%以上的酶活力。温度一旦超过50℃,酶活力下降很快。在50℃保温10min也会导致酶活力下降近80%。在70℃保温10min酶活完全丧失,表明菌株解淀粉类芽孢杆菌属P17低温果胶酶属于典型的低温果胶酶。
3、菌株解淀粉类芽孢杆菌属P17低温果胶酶最适pH为8.0,pH小于5或大于11检测不到酶活力。
4、菌株解淀粉类芽孢杆菌属P17低温果胶酶pH6.0~10.0之间酶活力稳定,酶活力均在80%以上。
5、Mg2+、Na+、Zn2+和Cd2+对菌株解淀粉类芽孢杆菌属P17低温果胶酶有激活作用,Li+、Cu2+、Mn2+、Ca2+、Fe2+、Ba2+、Ni2+、Pb2+、Co2+、Al3+和Fe3+对酶有轻微的抑制作用,Hg2+对酶有显著的抑制作用。
6、鳌合剂EDTA和变性剂SDS对菌株解淀粉类芽孢杆菌属P17低温果胶酶有较为明显的抑制作用。丝氨酸蛋白酶专一抑制剂苯甲基磺酰氟化物(PMSF)对菌株解淀粉类芽孢杆菌属P17低温果胶酶没有抑制作用,表明了菌株解淀粉类芽孢杆菌属P17低温果胶酶的活性中心不含有丝氨酸。
7、菌株解淀粉类芽孢杆菌属P17低温果胶酶活力随着有机溶剂如甲醇、乙醇、丙酮和乙腈浓度的升高而提高,当有机溶剂的浓度达到一定值(50%),随着有机溶剂浓度的升高菌株解淀粉类芽孢杆菌属P17低温果胶酶活力反而下降。低浓度的二甲基亚砜(10%)对菌株解淀粉类芽孢杆菌属P17低温果胶酶有激活作用,但随着有机溶剂浓度的升高,有机溶剂对菌株解淀粉类芽孢杆菌属P17低温果胶酶有抑制作用。低浓度的甲醛(10%)对菌株解淀粉类芽孢杆菌属P17低温果胶酶表现出较强的抑制作用。
7、随着果胶(Sigma)酯化程度的增加,菌株解淀粉类芽孢杆菌属P17低温果胶酶活力表现出相应的增加,其最适底物为90%酯化度的果胶。但该酶不能降解多聚半乳糖醛酸(Sigma)。
8、菌株解淀粉类芽孢杆菌属P17低温果胶酶在4℃时的Km(0.233mg/mL)值远远低于其最适温度40℃时的Km值(1.362mg/mL)。低Km值可以使低温果胶酶在低温条件下与果胶底物结合能力加强,弥补了低温导致化学反应速率降低而带来的不利影响和保证了低温果胶酶在低温下保持较高的催化能力。
本发明低温果胶酶的优点是:
1、菌株P17所产低温果胶酶在低温下仍具有较高的酶活力,在冷洗涤行业和生物精练行业有一定的工业应用价值。
2、菌株P17所产低温果胶酶对金属离子耐受性较好,除了重金属离子Hg+2能明显抑制酶活力外,一些重金属离子如Ba2+、Ni2+、Pb2+、Co2+只部分抑制酶活力(残留酶活力均在83%以上)和重金属离子Cd2+反而对酶有激活作用。这在重金属污染废水处理中有很大的发展空间。
3、菌株P17所产低温果胶酶对有机溶剂耐受性较好,即使浓度为50%的甲醇、乙醇和丙酮不但没有抑制酶活力,反而对酶有很好的激活作用,在工业上具有良好的应用价值。
4、天然果胶一般以高酯化度的形式存在。菌株解淀粉类芽孢杆菌属P17低温果胶酶对高酯化度的果胶具有很高的专一性。这在水果加工业、纺织、制浆造纸、麻类脱胶和治疗胃结石等领域有着很大的发展空间。
附图说明:
图1为菌株P17所产低温果胶酶的反应温度和相对活性的关系曲线;
图2为菌株P17所产低温果胶酶的反应pH和相对活性的关系曲线;
图3为菌株P17所产低温果胶酶的热稳定性曲线;
图4为菌株P17所产低温果胶酶的pH稳定性曲线。
具体实施方式:
下面结合附图,用本发明的实施例来进一步说明本发明的实质性内容,但不以此来限定本发明。
实施例1:
菌株解淀粉类芽孢杆菌属P17的分离提取:
样品的来源为轿子雪山的土壤。菌株的筛选分离和培养方法为取少量土壤样品于富集培养基中13℃振荡培养3~5天,稀释涂布于固体LB培养基,于13℃倒置培养。待完全长出菌落后,用划线法将单菌落划线至新鲜的固体LB培养基平板上,于13℃恒温培养箱中倒置培养。如此重复直至获得纯培养物。所说的培养基如下:
富集培养基:果胶2g/L,硫酸镁0.5g/L,氯化钾0.5g/L,硫酸铁0.01g/L,磷酸氢二钾1g/L,硝酸钠3g/L,PH7.0。
固、液体LB培养基:NaCl 10g/L,酵母粉5g/L,蛋白胨10g/L,[琼脂15g/L],pH7.0。
由该方法得到的菌株解淀粉类芽孢杆菌属P17于2008年8月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为:CGMCC No.2640。
实施例2:
菌株解淀粉类芽孢杆菌属P17低温果胶酶的分离纯化:
(1)粗酶液的制备:将菌株P17接入液体LB培养基中,在13~25℃的条件下振荡培养至指数生长期,将处于指数生长期的菌液转接于液体发酵培养基(其组成为:酵母粉0.2g/L,葡萄糖1g/L,硫酸铵2g/L,磷酸氢二钾1g/L,氯化钾0.5g/L,硫酸亚铁0.01g/L,硫酸镁0.5g/L,pH9.0)中,13~25℃振荡培养至稳定期初期即为产酶高峰期,将发酵液离心,取上清,滤纸过滤,得粗酶液。
(2)用截留分子量为10KDa的膜包将粗酶液的体积浓缩至原来的1/10。
(3)浓缩后的酶液加硫酸铵至60%~70%饱和度,离心取上清,继续加硫酸铵至90%~100%,离心取沉淀,在pH8.0的KPB缓冲液透析过夜。
(4)将透析后的酶液离心,取上清液,上样于已用pH8.0的KPB缓冲液平衡好的DEAE-Sepharose预装柱,收集有活性的透过峰酶液,并用截留分子量为10KDa的浓缩管进行浓缩。
(5)用pH6.0的KPB缓冲液将浓缩酶液透析过夜,离心取上清,上样于已用pH6.0的KPB缓冲液平衡好的CM-Sepharose预装柱,洗脱,收集有活性的部分,即为纯酶。
实施例3:
低温果胶酶的最适反应温度:
1准确称取0.020g的果胶底物溶解于10mL的50mM pH8.0磷酸盐缓冲液中。
2、取上述底物溶液分装于16个Eppendorf管中,每管190μl。其中8管为测定管,另外8管为对照管,分别置于4℃、10℃、20℃、30℃、40℃、50℃、60℃和70℃等8个不同的温度下平衡1h。
3、加入10μl合适的稀释酶液,以灭活的酶液为对照。
4、反应30min。反应结束后,置于冰水混合液中冷却。
5、在波长为230nm处测其吸光度。
6、以酶活最高的那个温度下所测得的值为参照,其余温度下所测得的酶活与其相对比,以相对酶活绘制酶催化温度曲线。
实施例4:
低温果胶酶的最适pH的试验:
1、准确称取0.020g的果胶底物溶解于10mL不同pH值的9种缓冲液中,所用到的缓冲液有:50mM的pH3、4、5的柠檬酸-柠檬酸钠缓冲,pH6、7、8的磷酸氢二钾-磷酸二氢钾缓冲液,pH9、10的甘氨酸-氢氧化钠缓冲液,pHl1的碳酸钠-碳酸氢钠缓冲液。
2、取上述底物溶液分装于18个Eppendorf管中(每种pH值2管),每管190μl。其中9管为测定管,另外9管为对照管,置于40℃恒温干燥器平衡20min。
3、加入10μl合适的稀释酶液,以灭活的酶液为对照。
4、反应30min。反应结束后,置于冰水混合液中冷却。
5、在波长为230nm处测其吸光度。
6、以酶活最高的那个pH下所测得的值为参照,其余pH下所测得的酶活与其相对比,以相对酶活绘制酶催化pH曲线。
Claims (2)
1.解淀粉类芽孢杆菌(Paenibacillus amylolyticus)P17菌株,菌种保藏号为CGMCC No.2640。
2.由权利要求1所述的低解淀粉类芽孢杆菌P17菌株产生低温果胶酶的纯化方法,将权利要求1所述的解淀粉类芽孢杆菌菌株P17接入液体LB培养基中,在13~25℃的条件下振荡培养至指数生长期,将处于指数生长期的菌液转接于液体发酵培养基中,13~25℃振荡培养至稳定期初期即为产酶高峰期,将发酵液离心,取上清,滤纸过滤,得粗酶液;用截留分子量为10KDa的膜包将粗酶液的体积浓缩至原来的1/10;浓缩后的酶液加硫酸铵至60%~70%饱和度,离心取上清,继续加硫酸铵至90%~100%,离心取沉淀,在pH8.0的KPB缓冲液透析过夜;将透析后的酶液离心,取上清液,上样于已用pH8.0的KPB缓冲液平衡好的DEAE-Sepharose预装柱,收集有活性的透过峰酶液,并用截留分子量为10KDa的浓缩管进行浓缩;用pH6.0的KPB缓冲液将浓缩酶液透析过夜,离心取上清,上样于已用pH6.0的KPB缓冲液平衡好的CM-Sepharose预装柱,洗脱,收集有活性的部分,即为纯酶,
所述的发酵培养基的组成为:酵母粉0.2g/L,葡萄糖1g/L,硫酸铵2g/L,磷酸氢二钾1g/L,氯化钾0.5g/L,硫酸亚铁0.01g/L,硫酸镁0.5g/L,pH9.0。
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CN101608169B (zh) * | 2009-06-25 | 2011-06-29 | 昆明理工大学 | 嗜热短小芽孢杆菌株Tamy12及其产高温淀粉酶 |
BR112017006629B1 (pt) * | 2014-09-30 | 2021-09-21 | Auburn University | Inoculante para uma planta, sementes de plantas e método para aumento do crescimento ou da saúde de uma planta |
CN110055197B (zh) * | 2019-05-07 | 2021-02-02 | 农业部沼气科学研究所 | 一种解淀粉类芽孢杆菌brec-10及其菌剂和应用 |
CN111004788B (zh) * | 2019-12-17 | 2020-12-04 | 中国农业科学院麻类研究所 | 一种果胶酯酶及其制备方法和应用 |
CN114317364B (zh) * | 2021-12-30 | 2023-06-13 | 中国科学院青岛生物能源与过程研究所 | 一株高地芽孢杆菌及其在产高稳定性碱性果胶酶中的应用 |
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