CN116286474A - 一种低温降解半纤维素的微生物菌株 - Google Patents
一种低温降解半纤维素的微生物菌株 Download PDFInfo
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Abstract
一种低温降解半纤维素的微生物菌株，所述菌株为沙雷氏菌（Serratia sp.）SL‑1，保藏于中国普通微生物菌种保藏管理中心，保藏地址为中国北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：25984，保藏日期为2022年10月28日。本发明中的沙雷氏菌SL‑1在较低温度下可以快速生长并高效产出木聚糖酶和几丁质酶，产酶最优温度低至15℃，15℃低温下生产的木聚糖酶的酶活达到37℃下产的木聚糖酶活的1.4倍，所产的木聚糖酶的酶活可达到46.502IU/mL，同时其在低温下也能产几丁质酶，酶活为30.427 IU/mL，所产的木聚糖酶和几丁质酶在较低催化温度下依然具有高酶活力，且具有优异的pH耐受性，适用于在低温环境下对半纤维素的降解。
Description
技术领域
本发明涉及微生物工程技术领域，具体涉及一种低温降解半纤维素的微生物菌株。
背景技术
木聚糖(xylan)是一种广泛存在于植物细胞壁中的异质多糖，约占植物细胞干重的15％～35％，也是植物半纤维素的主要成分，是自然界除纤维素之外另一丰富的再生多糖。大多数木聚糖是一种结构复杂的、具有高度分枝的异质多糖，含有许多不同的取代基。木聚糖的生物降解也因此需要一个复杂的酶系统，木聚糖酶(xylanase)属于水解酶类，是通过其中各种组分的相互协同作用来降解木聚糖，将木聚糖转化为低聚木糖或木糖的复合酶系，因此，木聚糖酶是一组酶，而非一种酶，在农业、能源、饲料、造纸、食品等领域具有广阔的应用前景。该酶在自然界的来源广泛，其中利用微生物生产的木聚糖酶是主要的来源之一。
沙雷氏菌(Serratia sp.)属于肠杆菌科，是一类兼性厌氧菌，广泛分布于植物、土壤和水体中。沙雷氏菌可以在10～37℃、pH 5～9、含有0％～7％(w/v)的NaCl条件下生长，具有强大的生存能力，且有产气、产酸、产脂肪酶、产抑制物的能力，可应用于土壤改良、污水处理和秸秆降解等方面，且在其他方面也具有潜在的应用价值。
自然环境中存在很多产木聚糖酶的微生物，但是大多菌株在环境温度较低时菌体生长速度会明显受到限制，产酶能力剧降或者产酶的酶活效果大打折扣，在北方长期在寒冷环境中，导致在如降解秸秆还田等的应用时受到限制。鉴于此，对于低温环境中高效生长并产酶的微生物的研究至关重要。沙雷氏菌通常是常温下高产木聚糖酶，较低温度下产木聚糖酶研究报道较少。
发明内容
基于上述问题，本发明目的在于提供一种低温下高产木聚糖酶和几丁质酶的沙雷氏菌。该菌在较低温度下高效生长，同时在低温环境下可高产木聚糖酶和几丁质酶。
本发明目的通过如下技术方案实现：
一种低温降解半纤维素的微生物菌株，其特征在于：所述菌株为沙雷氏菌(Serratia sp.)SL-1，保藏于中国普通微生物菌种保藏管理中心，保藏地址为中国北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：25984，保藏日期为2022年10月28日。
菌株SL-1为一种杆状细菌，体积较大、菌落呈白色、表面光滑且中央突起。
进一步，上述沙雷氏菌SL-1的发酵温度为4～37℃，优选15℃。
进一步，所述沙雷氏菌SL-1在15℃下同时生产几丁质酶和木聚糖酶，生产的几丁质酶和木聚糖酶的酶解温度为30～70℃，优选50℃。
进一步，所述沙雷氏菌SL-1生产的木聚糖酶的酶解pH为3～11，pH优选为7。
本发明具有如下技术效果：
本发明中的沙雷氏菌SL-1在较低温度下可以快速生长并高效产出木聚糖酶和几丁质酶，产酶最优温度低至15℃，15℃低温下生产的木聚糖酶的酶活达到37℃下产的木聚糖酶活的1.4倍，所产的木聚糖酶的酶活可达到46.502IU/mL，同时其在低温下也能产几丁质酶，酶活为30.427IU/mL，所产的木聚糖酶和几丁质酶在较低催化温度下依然具有高酶活力，且具有优异的pH耐受性，因而为该菌株在低温环境下降解半纤维素提供了较好的条件。
附图说明
图1：菌株SL-1菌落形态及刚果红透明圈情况。
图2：菌株SL-1的系统进化发育树。
图3：菌株SL-1的生长曲线。
图4：发酵温度对菌株SL-1产木聚糖酶活力的影响。
图5：反应温度对菌株SL-1木聚糖酶活力的影响。
图6：反应pH对菌株SL-1木聚糖酶活力的影响。
具体实施方式
下面通过实施例对本发明进行具体的描述，有必要在此指出的是，以下实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，该领域的技术人员可以根据上述本发明内容对本发明作出一些非本质的改进和调整。
实施例1：原始菌株SL-1的筛选及鉴定
(一)菌株的分离
(1)将采集于长白山常年低温苔原带土壤样品，至于土壤富集盐溶液(0.006mMFeSO4·7H2O；0.01mM CaCO3·7H2O；0.08mM MgSO4·7H2O；0.07mM MnSO4·7H2O；0.006mMZnSO4·7H2O)制备土壤菌悬液。
(2)配制固体培养基：0.5g酵母浸出粉、0.5g蛋白胨、0.5g酪蛋白水解物、0.5g可溶性淀粉、0.5g葡萄糖、0.024g无水硫酸镁、0.3g磷酸二氢钾、15.0g琼脂、0.3g丙酮酸钠、1000蒸馏水，pH为7.2±0.2；将菌悬液按照梯度(10-3～10-8)稀释涂布于固体培养基中，在10℃下倒置培养3-10天。
(3)根据菌落形态、颜色、大小及光泽度，进行不同的单菌落的挑取。
(二)菌株SL-1的筛选
(1)将挑取的单菌落用质量浓度为0.6％的氯化钠溶液溶解形成菌液，用接种环沾取适量菌液在LB固体培养基进行划线培养，经多次传代，挑取菌落形态单一且状态一致的单菌落置于LB液体培养基中，在180rpm和10℃的条件下摇瓶培养3～4d。
(2)取摇瓶培养的菌液200ul，测定其OD600值，利用灭菌牙签沾取OD600在0.6-0.8之间的菌液，用灭菌牙签沾取菌液点样至木聚糖-刚果红固体培养基中，将接菌的固体平板转移至10℃恒温培养箱中，静置培养，所述木聚糖-刚果红固体培养基是由1.5％商品木聚糖、4g/L硫酸铵、0.5g/L磷酸二氢钾、2g/L磷酸氢二钾、0.1g/L MgSO4·7H2O、6g/L氯化钠、0.1g/L氯化钙、20g/L琼脂、0.5g/l酵母提取物和0.2g/L刚果红组成。
(3)对培养7～10d的木聚糖-刚果红固体培养基进行肉眼观察，主要对菌落生长状态及会否产生透明圈进行考察。结果显示菌落附近有明显的透明圈出现，说明该沙雷氏菌具有产木聚糖酶的能力。
此外，将SL-1采用上述方法进行产几丁质酶检测，使用的培养基为含有几丁质筛选培养基，其成分为：胶体几丁质10g/L，(NH4)2SO4 10g/L，K2HPO40.7g/L，KH2PO4 0.3g/L，NaCl 0.5g/L，Mg SO4·7H2O 0.5g/L，FeSO4·7H2O 0.01g/L，Zn SO4 0.01g/L，琼脂粉20g/L，pH＝7.0。对培养7～10d的筛选培养基进行肉眼观察，对菌落生长状态及会否产生透明圈进行考察，结果显示菌落附近有明显的透明圈出现，说明沙雷氏菌SL-1同样具有产几丁质酶的能力。
通过检测各酶活可知，本发明的菌株SL-1具有在低温环境下同时产木聚糖酶和几丁质酶的能力。
(三)鉴定
菌株SL-1用LB平板培养出来适量菌体，通过革兰氏染色，观察菌株的形态、颜色等，菌株SL-1为一种杆状细菌，体积较大、菌落呈白色、表面光滑且中央突起。并对菌株进行生理生化实验鉴定，测定的生理生化指标如表2所示，“+”代表阳性，“-”代表阴性。
表2：菌株SL-1生理化指标的测定
| 试验 | Serratia sp.SL-1 | 试验 | Serratia sp.SL-1 |
| 革兰氏染色 | - | 7％NaCl | + |
| 葡萄糖酸生产 | + | 蔗糖酸生产 | + |
| 甘露醇酸生产 | + | 淀粉水解 | - |
| 麦芽糖水解 | + | V-p测定 | + |
| 接触角 | + | 甲基红 | + |
对SL-1菌落进行PCR扩增16S rRNA后，将PCR产物送生工测序。使用blast与NCBI数据库中所有可用的16S rRNA序列进行比较，分析对比显示SL-1菌和沙雷氏菌属其它种的菌株的一致性较高，有同源性，结合菌落形态、颜色及革兰氏染色结果确定菌属，该菌为沙雷氏菌属。
实施例2：沙雷氏菌SL-1的生长及产酶分析
(一)沙雷氏菌SL-1的生长特性分析
(1)对通过分离纯化的菌株SL-1进行生长特性的评估。取菌株SL-1活化对数期菌液以1％接种量接种至LB培养基中培养，置于不同温度(37℃、30℃、25℃、20℃、15℃、10℃、4℃)，转速180rpm的振荡培养箱中进行摇瓶培养，以SL-l的菌浓度(OD600)来评价菌株SL-1的生长特性。
(二)沙雷氏菌SL-1的产酶特性分析
(1)将对数期菌液接种于发酵产酶培养基中在不同温度(4、10、15、25、30、37℃)下，接菌量为200uL，转速为180rpm，进行振荡培养48h，取样保存于-20℃冰箱中。所用的产酶培养基由硫酸铵4g/l，磷酸氢二钾2g/L，七水硫酸镁0.1g/L，酵母提取物10g/L，蛋白胨1g/L，木聚糖/CMC-Na 1.5％组成；
(2)将保存的发酵液室温下冻融后，8000rpm，离心5min，取发酵上清液进行木聚糖酶活力测定，计算相对酶活力。
将菌株SL-1在发酵温度4℃、10℃、15℃、20℃、25℃、30℃及37℃培养下，观察其生长状况，通过结果分析，如图3所示，10～15℃是菌株SL-1最佳的生长温度。如图4所示，菌株SL-1的酶活力在发酵温度为15～20℃之间最佳(于酶催化温度为50℃下检测)，与生长特性结果相比较后，综合确定菌株SL-1的最佳生长及产酶温度为15℃，此时产生的木聚糖酶酶活是37℃下产生的酶活的1.4倍。
实施例3：沙雷氏菌SL-1的酶学性质表征
(1)保持其它测酶活力步骤不变，改变酶催化温度(30、40、50、60、70℃)，分别检测其对应催化温度下沙雷氏菌SL-1产酶的酶活力。并计算相对酶活力。
结果如图5所示，催化温度在40℃-60℃之间，菌株SL-1产生的木聚糖酶的酶活力较高，这为后续低温酶解提供参考。
(2)保持其它测酶活力步骤不变，改变酶催化pH值(3、4、5、6、7、8、9、10)，分别检测其对应pH值下的酶活力。
结果如图6所示，当pH在3～11之间时，木聚糖酶均具有较优异的酶活性质，木聚糖酶最佳反应pH为中性左右，高于或低于7，酶活力有一定程度损失，但整体损失较少，因此说明菌株SL-1产生的酶具有较好的pH耐受性。
定量测定产酶酶活：
(1)将沙雷氏菌SL-1接种至LB液体培养基中，180rpm，10℃下振荡培养，将对数期菌液进行甘油保菌(700uL菌液中加入300uL质量分数为50％甘油)。
(2)将沙雷氏菌SL-1(10℃、180rpm)进行发酵培养，并取样分别进行木聚糖酶和几丁质酶的测定。木聚糖酶活力和几丁质酶均采用DNS法，分别取25ul酶液加入125ul含有0.2g/mL的木聚糖底物和含有0.2g/mL的几丁质底物中，50℃水浴30min，加入DNS试剂100ul，沸水煮沸10min，对照氨基葡萄糖和木糖标准曲线，得生成的还原糖浓度。
酶活的单位定义是：国际上酶分钟产生1μmol氨基葡萄糖的酶量定义为一个酶活单位，其计算公式如下：
A：酶活，单位是IU/mL；
C：反应中产生的氨基葡萄糖或木糖浓度，单位是mg/mL；
V1：反应体系的体积，单位是mL；
D：酶液的吸湿稀释倍数；
1000：将氨基葡萄糖或木糖的mg数换算成μg数；
M：生成的单糖的摩尔质量，单位是g/mol，氨基葡萄糖是180，木糖是150；
V2：加入酶液的体积，单位是mL；
通过计算，菌株SL-1产酶活力如表1所示。
表1：
| 菌株 | 酶活最佳温度(℃) | 木聚糖酶(IU/mL) | 几丁质酶(IU/mL) |
| 菌株SL-1 | 50 | 46.502 | 30.427 |
综上所述，本发明提供的在较低温度环境下高产木聚糖酶和几丁质酶的菌株，经过菌株鉴定为沙雷氏菌SL-1，将该菌株通过发酵培养后获得的木聚糖酶，最优产酶温度为15℃，最终发酵产酶水平是木聚糖酶为46.502IU/mL、几丁质酶为30.427IU/mL。木聚糖酶具有最适pH为7.0、最适温度50℃的特性，可见，本发明的沙雷氏菌SL-1在低温15℃时具有高效产木聚糖酶和几丁质酶的能力，所产的木聚糖酶具有优异的pH耐受性和温度适应性，用于低温降解半纤维具有极大潜能。
Claims (2)
1.一种低温降解半纤维素的微生物菌株，其特征在于：所述菌株为沙雷氏菌，沙雷氏菌（Serratia sp.）SL-1，保藏于中国普通微生物菌种保藏管理中心，保藏地址为中国北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO：25984，保藏日期为2022年10月28日。
2.如权利要求1所述的一种低温降解半纤维素的微生物菌株，其特征在于：所述沙雷氏菌SL-1的发酵温度为15℃。
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Effective date of registration: 20240327 Address after: Room 202-25, Building H1, Phase III, Changchun Beihu Science and Technology Park, No. 3333 Shengbei Street, Beihu Science and Technology Development Zone, Changchun City, Jilin Province, 130000 Patentee after: Jilin Gerunjia Biotechnology Co.,Ltd. Country or region after: China Address before: Room 605, Unit 3, Building 19, Block B, Keyuan Community, Changdongbei Science City, South of Bingba Road, High tech Development Zone, Changchun City, Jilin Province, 130000 Patentee before: Changchun Deyou Agricultural Technology Co.,Ltd. Country or region before: China |
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