CN103525717A - 一株厌氧海藻酸分解菌及其应用 - Google Patents
一株厌氧海藻酸分解菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株厌氧海藻酸分解菌Marinicatena alginatilytica SH-52,该菌株保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M2013073,本发明菌株为能有效降解海藻酸的厌氧微生物,可用于海藻酸裂解酶生产,且时间周期短,酶产量高,收集简单,操作方便,易于工业产业化实施。
Description
技术领域
本发明涉及一株厌氧以海藻酸分解菌及其应用,属于海藻化工领域。
背景技术
随着对海洋资源的逐步开发,海藻中的多糖类物质尤其是海藻酸被广泛应用于各种工业生产中。由海藻酸降解得到的海藻酸寡糖,由于分子量不同、M/G不同、结构不同,其生物学活性也不同,使其在医药、食品、农业等诸多领域都有广泛应用。在医药领域,硫酸寡糖SPMG(sulfated polymannuroguluronate)能够抑制HIV-1进入细胞【Geng M, Li F, Xin X, et al(2003). The potential molecular targets of marine sulfated polymannuroguluronate interfering with HIV-1 entry. Interaction between SPMG and HIV-1 rgp120 and CD4 molecule. Antiviral Res, 59(2):127-135】。海藻酸寡糖还可以通过提高生物机体对肿瘤细胞的防御能力和增强宿主免疫***来对抗肿瘤细胞【Yamamoto Y, Kurachi M, Yamaguchi K, et al (2007). Stimulation of multiple cytokine production in mice by alginate oligosaccharides following intraperitoneal administration. Carbohydr Res, 342(8):1133-1137.】。在食品领域,海藻酸寡糖可以在体外促进双歧杆菌和乳酸菌的生长,同时抑制有害菌的生长,是一种新的益生元,用于保健食品的制作【Wang Y, Han F, Hu B, et al (2006). In vivo prebiotic properties of alginate oligosaccharides prepared through enzymatic hydrolysis of alginate. Nutr.Res., 26(11):597-603】。在农业领域,海藻酸寡糖可以促进种子的萌发和根的生长【Yonemoto Y, Tanaka H, Yamashita T, et al(1993). Promotion of germination and shoot elongation of some plants by alginate oligomers prepared with bacterial alginate lyase. J. Ferment. Bioeng., 75(1):68-70】。
目前,海藻酸降解为寡糖主要有酸水解法和酶解法【Haug A, Larsen B, Smidsr?d O (1967). Studies on the sequence of uronic acid residues in alginic acid. Acta Chemica Scandinavica, 21: 691-04. Park HH, Kam N Lee EY, et al (2011). Saccharification of alginate by using exolytic oligoalginate lyase from marine bacterium Sphingomonas sp. MJ-3. Journal of Industrial and Engineering Chemistry, 17(5–6): 853–58】。酸水解法需要消耗大量盐酸等高浓度酸,水解废渣对环境污染严重,酸水解没有特异性,不能有效控制寡糖的大小和其中甘露糖醛酸和古洛糖醛酸的组成,这些缺点都限制了海藻酸寡糖的大规模应用。而酶解法以其反应条件温和,水解效率高,对环境无污染等优点,可以逐步取代酸水解法。同时,现在以发现的海藻酸裂解酶有polyM 、polyG和polyMG底物特异性【Rahman MM, Wang L, Inoue A, et al(2012). cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula. Carbohydr Res. 360: 69-77】,可以有效的剪切不同G/M组成的海藻酸寡糖,并且海藻酸裂解酶有内切型和外切型【In Lee S, Choi SH, Lee EY, et al. Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2. Appl Microbiol Biotechnol, 2012, 95(6): 1643-53 Rahman MM, Inoue A, Tanaka H, et al (2011). cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme. Biochimie. 93(10): 1720-30】,这两种酶联合利用可轻易的获得目的大小的海藻酸寡糖。所以,开发多种类型的高活性的海藻酸裂解酶前景广阔,海藻酸裂解酶多来源于海洋细菌,筛选产高活性海藻酸裂解酶的海洋细菌行之有效。
发明内容
本发明针对现有技术的不足,提供一株厌氧海藻酸分解菌Marinicatena alginatilytica SH-52,其保藏于中国典型培养物保藏中心(地址:中国武汉,武汉大学),保存日期为2013年3月10日,保藏编号为CCTCC NO:M2013073。
本发明另一目的是将厌氧海藻酸分解菌应用在生产海藻酸裂解酶中,且是利用海藻酸为碳源生产海藻酸裂解酶。
本发明中所述的厌氧海藻酸分解菌SH-52具有以下微生物学特征:
本菌株分离及培养的基础培养基采用改良的海洋培养基,培养基组分如下:NH4SO4 5g,NaCl 25g,KH2PO4 0.75g, K2HPO4 1.5g,MgCl2.H2O 0.4g,CaCl2 0.13g,蛋白胨 0.5g,酵母膏 0.5g,海藻酸 10g,半胱氨酸盐酸盐 0.75g,刃天青 0.5mg,微量元素溶液SL-10 1ml。各组分分别溶解后定容至1000ml,培养基加热从蓝色变为无色后,采用铜柱除氧***除氧,分装到充满氮气的亨特试管中,每管装5ml,121℃灭菌后备用。
微量元素溶液SL-10的组成成分为:HCl (22%;7.7M)10ml,FeCl2.4 H2O 0.200 g,MnCl2.4 H2O 0.100 g,CoCl2.6 H2O 0.170 g,CaCl2.2 H2O 0.100 g,ZnCl2 0.100 g,CuCl2 0.020 g, H3BO3 0.010 g Na2MoO4.2 H2O 0.010 g,NiCl2.6 H2O 0.026 g,NaCl 1.000 g,Na2SeO3 .5 H2O 0.020 g,首先将FeCl2.4 H2O溶解在HCl中,然后加入蒸馏水,再溶解其他盐,定容到1000ml备用。
除温度、pH实验外的其他实验均在30℃,pH值7.5的条件下培养接种量均为2%,所有实验都设三组平行试验。
1、形态学特征
本发明菌株SH-52是从广东省汕头市沿海沉积物中分离得到的,用改良的海洋培养基培养,菌落为白色,相差显微镜观察,菌体为长杆状。
2、生理生化特征
菌株SH-52为革兰氏阴性专性厌氧菌,生长温度范围为20℃-45℃,最适生长温度为30℃;生长的pH范围为6-8.5,最适pH为7.5,生长的NaCl浓度范围为0-9%(w/v),最适的生长盐浓度为2%(w/v)。菌株SH-52可将硫和硫代硫酸钠中的硫元素还原为H2S。
本发明中菌株SH-52碳源利用情况如下(见表1):
表1:本发明菌株的碳源利用结果
注:“+”表示生长;“-”表示不生长。
与Sunxiuqinia elliptica sp. NCCB 100301相比,不同点见表2。
表2:本发明菌株SH-52与Sunxiuqinia elliptica sp. NCCB 100301的不同点
注:(“+”代表以生长,“-”代表不能生长)
本发明涉及上述厌氧海藻酸分解菌的应用,将该菌用于生产海藻酸裂解酶,即将该菌株以2%的接种量在含1%海藻酸钠的改良海洋培养基中培养,30℃厌氧环境下静置培养36h,利用海藻酸为碳源生产海藻酸裂解酶。
接种的菌株是将处于菌种保藏环境下的厌氧海藻酸分解菌SH-52依次进行菌种活化处理和细菌培养处理。
本发明所述菌株保藏环境是在-80℃的低温冰箱中添加甘油做保护剂进行冷冻保藏,将微生物处于冷冻状态,使其代谢作用停止以达到保藏菌种的目的。
所述的细菌培养处理是指:含海藻酸钠或葡萄糖的厌氧改良海洋培养基,将培养基在121℃灭菌,将菌种活化处理后的SH-52厌氧海藻酸分解菌接种到液体培养基中并在30℃厌氧条件下培养12h。
本发明菌株(G+C)mol%为44%。
3、***进化分析
本发明的菌种SH-52的16SrRNA基因序列比对结果显示,和Sunxiuqinia elliptica sp. NCCB 100301的序列相似性为93%,根据上述结果说明本发明菌株SH-52属于新种。
本发明具有以下优点及效果:
1、采用本发明菌种生产海藻酸裂解酶时,是采用厌氧发酵,培养基盐浓度高,可有效避免杂菌污染;
2、利用本发明菌种生产海藻酸裂解酶,时间周期短,酶产量高,收集简单,操作方便,易于工业产业化实施;
3、本发明菌株生产的海藻酸裂解酶,活性高,粗酶液比酶活力可达到134U/mg。
附图说明
图1为本发明厌氧海藻酸分解菌SH-52的菌体形态照片示意图;
图2为本发明厌氧海藻酸分解菌SH-52菌株在30℃,pH7.5条件下培养时的生长曲线示意图;
图3为本发明厌氧海藻酸分解菌SH-52菌株不同温度下的生长情况示意图;
图4为本发明厌氧海藻酸分解菌SH-52菌株在不同pH下的生长情况示意图;
图5为本发明厌氧海藻酸分解菌SH-52菌株在不同NaCl浓度下的生长情况示意图;
图6为本发明厌氧海藻酸分解菌SH-52菌株的产酶情况示意图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中方法如无特殊说明的采用常规方法,使用的实际如无特殊说明的使用常规市售试剂或按常规方法配置的试剂。
实施例1:厌氧菌株SH-52的筛选分离
将从广东省汕头市沿海采集的海底沉积物按1:10(W/V)稀释,接种到装有100ml改良海洋培养基的250ml厌氧试剂瓶中(碳源为10g/L的海藻酸钠),30℃培养,培养两天后转接到新鲜的厌氧改良海洋培养基中,培养到吸光度OD=0.8,转接到新鲜的试管培养基中,反复接种三次(接种量为2%)。将培养物梯度稀释(10-1、10-2、10-3、10-4、10-5、10-6),采用亨特滚管(培养基为改良的海洋培养基,碳源为10g/L的海藻酸钠,加1.5%的琼脂)分离,挑单菌落接种于碳源为海藻酸钠的改良海洋培养基中,筛选出能利用海藻酸钠的菌株,编号为SH-52,通过分子***发育学分析,命名为:Marinicatena alginatilytica SH-52,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2013073,保存日期为2013年3月18日。
实施例2:生理生化检测
1、将实施例1中得到的菌株以2%的接种量接入到海藻酸钠浓度为10g/L的改良海洋培养基中,分别在20℃、25℃、30℃、35℃、40℃、45℃、50℃培养,测得细菌的温度生长范围为20℃到45℃,最适温度为30℃(见图3)。
2、配置pH梯度的改良海洋培养基,pH梯度为5、5.5、6、6.5、7、7.5、8、8.5、9。测得细菌pH生长范围为6到8.5,最适生长pH为7.5(见图4)。
3、配置NaCl浓度梯度改良海洋培养基,NaCl浓度梯度为0%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%(w/v),测得细菌在NaCl浓度梯度培养基上的生长范围为0%到9%,最适生长浓度为2%(见图5)。
4、将2g/L(w/v)的硫和7g/L(w/v)的硫代硫酸铵分别加入到改良海洋培养基中,接种本发明菌株,培养48h,吸取上层气体,打入到GuSO4溶液中,检到有GuS黑色沉淀产生,即说明菌株SH-52产生了H2S气体。
5、利用苯酚-氯仿法提取菌株基因组DNA,利用高氯酸水解DNA,用高效液相色谱分析核酸碱基;色谱条件:柱温为35℃,流速0.5ml/min,流动相为20mmol/L KH2PO4:甲醇(90:10),色谱柱为inertsil ODS-SP,进样量为20ul,结果菌株(G+C)mol%为44%。
6、将葡萄糖、半乳糖、山梨糖、甘露糖、***糖、鼠李糖、木糖、麦芽糖、乳糖、蔗糖、纤维二糖、山梨醇、木糖醇、甘露醇、葡萄糖酸钠、淀粉、明胶、燕麦木聚糖、Avicel、CMC为唯一碳源配制厌氧改良海洋培养基,底物浓度为1%(w/v),检测细菌能否生长利用,结果见表1。
实施例3:海藻酸裂解酶粗酶液的制备
将实施例1中得到的菌株以2%的接种量接入到海藻酸钠浓度为10g/L的厌氧海洋培养基中,于30℃下静置培养36h,10000g离心10min收集菌体,以pH7.5的50mM Tris-HCl缓冲液重悬菌体,用超声波破碎仪破碎菌体,功率为200W,超声处理3s,暂停3s,运行时间8min,之后12000g离心15min,收集上清液,得到粗酶液。
实施例4:菌种SH-52最佳产酶时间及酶活检测实验
将实施例1中得到的菌株以2%的接种量接入到海藻酸钠浓度为10g/L的厌氧海洋培养基中,于30℃下静置培养,分别在0h、8h、12h、24h、36h、48h、60h、72h测定粗酶液(粗酶液的制备方法参照实施例3)酶活和细菌生长的OD值,酶活测定方法为:在235nm处测定底物吸光度变化,具体内容为:取0.9ml 3g/L海藻酸钠溶液(0.3g海藻酸铵溶于100ml pH7.5,0.05M的Tri-HCl冲液中),加入0.1ml酶液,30℃保温30min,以0min处底物溶液为空白,迅速测定反应体系在235nm的吸光值。酶活定义为每分钟,使上述反应体系的OD值变化0.01所需的酶量。结果最佳产酶时间为培养36h(图6)。最高酶产量达到1升培养基中含酶33880U,粗酶液比酶活力为134U/mg,而已报道的Vibrio sp.QY101的酶活只有3370U/L培养基,比酶活力只有5.03U/mg。菌株SH-52的酶产量比Vibrio sp.QY101的酶产量高出10倍,比酶活力高出26倍。
Claims (2)
1.一株厌氧海藻酸分解菌Marinicatena alginatilytica SH-52,其在中国典型培养物保藏中心的保藏编号为CCTCC NO:M 2013073。
2.权利要求1所述厌氧海藻酸分解菌在生产海藻酸裂解酶中的应用。
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CN105255923A (zh) * | 2015-10-28 | 2016-01-20 | 昆明理工大学 | 一种海藻酸裂解酶sha-4基因及其原核表达载体 |
CN105255922B (zh) * | 2015-10-28 | 2018-08-10 | 昆明理工大学 | 一种海藻酸裂解酶sha-5基因及其原核表达载体 |
CN105255923B (zh) * | 2015-10-28 | 2018-08-31 | 昆明理工大学 | 一种海藻酸裂解酶sha-4基因及其原核表达载体 |
CN108148852A (zh) * | 2018-01-26 | 2018-06-12 | 昆明理工大学 | 一种海藻酸裂解酶sha-6基因及应用 |
CN108148852B (zh) * | 2018-01-26 | 2020-12-15 | 昆明理工大学 | 一种海藻酸裂解酶sha-6基因及应用 |
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