CN1914326B - 维生素c的微生物生产方法 - Google Patents
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Abstract
本发明提供了一种从底物，例如L-山梨糖酮，来生产维生素C的方法，其中使用属于Ketogulonicigenium属的微生物。
Description
本发明涉及对L-抗坏血酸(维生素C)进行的微生物生产。
维生素C对人来说是非常重要且不可缺少的营养因子之一，通过所谓的“Reichstein方法”已能对其进行商业生产，该方法作为技术上成熟的方法已被人们所熟知。但是该方法包含大量复杂的步骤，并且难于获得总产量的任何提高。因此，已有大量建议，企图用于减少步骤数量和/或提高总产量。
本发明提供了一种生产维生素C的方法，所述方法包括：在培养基中，使用属于Ketogulonicigenium属的微生物将底物转化为维生素C。
底物到维生素C的转化指，导致产生维生素C的底物转化是通过属于Ketogulonicigenium属的微生物来进行的，即，底物可被直接转化为维生素C。在允许上文定义的此类底物转化进行的条件下，对所述微生物进行培养，例如，将微生物与底物直接接触。还可以使用例如静止细胞、丙酮处理过的细胞、被冻干的细胞、被固定的细胞等形式的微生物，以在底物上直接发挥作用。通过使用通气，可使用本身已知能作为用于微生物的培养技术的方法的任何手段，优选地，搅拌深浸式发酵罐。进行反应的优选细胞浓度范围为每ml大约10mg至大约700mg湿细胞，更优选地，每ml大约30mg至大约500mg湿细胞。
本文中使用的培养基可以是用于生产维生素C的任何合适的培养基。典型地，该培养基是水性培养基，其中包含，例如，盐、底物并且具有一定的pH。
碳源可以作为底物使用，例如D-山梨糖醇、L-山梨糖、L-山梨糖酮、L-葡萄糖或L-古洛糖-γ-内酯。优选地，底物选自D-山梨糖醇、L-山梨糖或L-山梨糖酮，更优选地，L-山梨糖酮。
属于Ketogulonicigenium属的合适的微生物可以例如，选自Ketogulonicigenium robustum、Ketogulonicigenium vulgare或其能够进行本发明的底物到维生素C转化的突变体。在本发明的一个方面，属于Ketogulonicigenium属的微生物选自Ketogulonicigenium robustum、Ketogulonicigenium vulgare或其突变体，但排除Ketogulonicigenium vulgareDSM 4025或其突变体。
在一种实施方式中，本发明提供了从L-山梨糖酮生产维生素C的方法，所述方法包括：将选自Ketogulonicigenium robustum、Ketogulonicigenium vulgare或其突变体的微生物与L-山梨糖酮在反应混合物中接触，以及从反应混合物中对维生素C进行分离和纯化。
应当理解，“Ketogulonicigenium robustum”和“Ketogulonicigeniumvulgare”还包括上述物种的具有同样的生理属性的异名(synonym)或基源异名(basonym)，如International Code of Nomenclature of Prokaryotes所定义的。
在本文中，上文提到的微生物的“突变体”指其基因组序列发生了改变的、但仍能进行本发明的方法所提供的从底物(例如，L-山梨糖酮)到维生素C的转化的微生物。可以通过任何传统方法来获得突变体，包括，例如，化学和UV诱变接着针对目标表型进行筛选或选择，体外构建功能缺陷基因，这可以通过下述方法来进行，所述方法包括：用于对微生物基因组中完整基因段进行替换的重组技术，单双交叉重组以及其它已知技术。见，Sambrook，et al.，Molecular Cloning，A Laboratory Manual，2nd Ed.，Cold Spring Harbor Laboratory Press(1989)和Harwood and Cutting，Molecular Biology Methods For Bacillus，John Wiley and Sons(1990)，pp.27-74。合适的诱变剂包括但不限于，紫外射线、X-射线、γ-射线和化学诱变剂，例如，氮芥或N-甲基-N’-硝基-N-亚硝基胍。此外，可以通过本身为本领域技术人员所熟知的用于此目的的任何方法，对通过自发突变产生的克隆进行分离来获得突变体。
在一种优选的实施方式中，用于本发明方法的微生物选自由K.robustum NRRL B-21627、K.vulgare NRRL B-30035、K.vulgare NRRL B-30036、K.vulgare NRRL B-30037N及其各自的突变体所构成的组。
K.robustum NRRL B-21627被描述于US 5,834,231中。菌株K.vulgareNRRL B-30035、K.vulgare NRRL B-30036和K.vulgare NRRL B-30037N被描述于US 6,316,231B1中。公众可从Agricultural Research CultureCollection(NRRL)，1815N.University Street，Peoria，Illinois 61604，USA获得这些菌株。
在一种实施方式中，本发明提供了从L-山梨糖酮生产维生素C的方法，所述方法包括将属于Ketogulonicigenium属的微生物与L-山梨糖酮在反应混合物中接触，以及从反应混合物中对维生素C进行分离和纯化。
本文中使用的“将微生物与L-山梨糖酮在反应混合物中接触”包括：在含有L-山梨糖酮的培养基中培养微生物。还可以使用例如静止细胞、丙酮处理过的细胞、被冻干的细胞、被固定的细胞等形式的微生物，以在底物(即L-山梨糖酮)上直接发挥作用。通过使用通气，可使用本身已知能作为与用于微生物的培养技术相关的方法的任何手段，优选地，搅拌深浸式发酵罐。开展反应的优选细胞浓度范围为每ml大约10mg至大约700mg湿细胞，更优选地，每ml大约30mg至大约500mg湿细胞。
合适的“反应混合物”可以是水或本领域内已知用于培养微生物的任何营养培养基。此类营养培养基包括碳源、氮源和其它无机盐，其可被微生物所利用。通常用于使微生物更好地生长的多种营养物质可能被适当地包括进培养基。
此类营养物的例子，例如可吸收的碳源包括但不限于，甘油、D-甘露醇、赤藻糖醇、核糖醇、木糖醇、阿糖醇、肌醇、卫矛醇、D-核糖、D-果糖、D-葡萄糖和蔗糖。可消化的氮源，例如有机物质，包括但不限于，蛋白胨、酵母提取物、烘焙酵母、尿素、氨基酸和玉米浸出液。多种无机物质也可作为氮源使用，例如硝酸盐和铵盐。此外，培养基通常含有无机盐，例如，硫酸镁、磷酸钾和碳酸钙。
对本发明微生物的培养可在大约4.0至大约9.0的pH下进行，其中，可以优选保持为大约5.0至大约8.0的pH。培养期随pH、温度和所用的营养培养基而变化，其优选为大约1至5天，最优选为大约1至3天。优选用于开展本发明方法的温度是大约13至大约36℃之间的温度，更优选的是大约18至大约33℃之间的温度。
虽然底物例如L-山梨糖酮的浓度可以随反应条件变化，但是反应优选在底物浓度为大约2至大约120mg/ml的条件下进行，更优选地，浓度为大约4至大约100mg/ml。在一种实施方式中，本发明的方法在L-山梨糖酮浓度为大约2至大约120mg/ml的条件下进行，更优选地，浓度为大约4至大约100mg/ml。
可通过可适于利用产物性质的任何已知的传统方法，来分离和纯化由此产生并积聚在反应混合物中的维生素C，维生素C可被分离为游离的酸或钠、钾、钙、铵等盐。
特别地，分离可以通过下述步骤的任何合适的组合或重复来进行：通过形成盐，通过利用产物和周围杂质间属性(例如，溶解度、可吸收性和溶剂间的分配系数)的差异，通过吸收(例如在离子交换树脂上)进行。上述任何过程单独或组合使用，就构成了用于分离产物的传统方法。由此获得的产物可用传统手段进行进一步分离，例如，通过重结晶或色谱。
根据本发明，在减少步骤数量方面的改进是明显的，因为其获得了用于从底物(例如，L-山梨糖酮)生产维生素C的单步骤途径。
在下述实施例中，将对本发明的方法进行更为详细的阐述。
实施例1用静止细胞系统从L-山梨糖酮来生产维生素C
于30℃在Triptic Soy Agar(Difco，Becton，Dickinson and Company，Sparks，MD，USA)上对K.robustum NRRL B-21627和K.vulgare菌株NRRL B-30035、NRRL B-30036和NRRL B-30037N进行3天的培养。从平板收获细胞，将其悬浮到1ml 50mM磷酸钾缓冲液(pH7.0)中，用同样的缓冲液洗两次。对菌株NRRL B-21627、NRRL B-30035、NRRL B-30036和NRRL B-30037N而言，在600nm处测得的细胞悬浮液光学密度分别为12.2、12.5、16.2和11.2。这些数字分别对应于每ml 31.7、32.5、42.1和29.1mg的湿细胞重量。
反应混合物(试管中，5ml)含有0.9ml的细胞悬浮液和0.1ml的50mg/ml L-山梨糖酮，其处于50mM磷酸钾缓冲液(pH7.0)。通过加入细胞悬浮液来开始反应。在30℃，于颠倒式振荡器上180rpm下对反应混合物进行3小时的培养。反应之后，在8000×g下对反应混合物进行10分钟的离心，以获得上清液。用HPLC来测量上清液中的维生素C含量。
柱：YMC-Pack Polyamine II(150×4.6mm i.d.)，YMC Co.Ltd，Kyoto，Japan
洗脱剂：50mM NH4H2PO4/乙腈＝30/70
流速：1ml/分钟
检测：在250nm处的UV吸收
在上述HPLC条件下，维生素C的驻留时间为7.7分钟。用该HPLC分析，所有被测试菌株的反应产物都被证实为维生素C。
表1显示了菌株NRRL B-21627、NRRL B-30035、NRRL B-30036和NRRL B-30037N产生的维生素C的量。
表1：从L-山梨糖酮进行的维生素C生产
| 菌株 | 产生的维生素C(mg/ml) |
| K.robustum NRRL B-21627 | 0.21 |
| K.vulgare NRRL B-30035 | 0.44 |
| K.vulgare NRRL B-30036 | 0.36 |
| K.vulgare NRRL B-30037N | 0.46 |
Claims (7)
1.一种生产维生素C的方法，所述方法包括：在培养基中，用属于Ketogulonicigenium属的微生物将L-山梨糖酮转化为维生素C，其中，所述微生物选自由Ketogulonicigenium robustum NRRL B-21627、Ketogulonicigenium vulgare NRRL B-30035、Ketogulonicigenium vulgareNRRL B-30036、Ketogulonicigenium vulgare NRRL B-30037所构成的组。
2.如权利要求1所述的生产维生素C的方法，所述方法包括：将所述微生物与L-山梨糖酮在反应混合物中接触，以及从所述反应混合物中对维生素C进行分离和纯化。
3.如前述权利要求中任意一项所述的方法，其中，所述方法在4.0至9.0的pH下和13至36℃的温度下进行。
4.如前述权利要求中任意一项所述的方法，其中，所述方法在5.0至8.0的pH下和18至33℃的温度下进行。
5.如前述权利要求中任意一项所述的方法，其中，所述方法在L-山梨糖酮浓度为2至120mg/ml的条件下进行。
6.如权利要求5所述的方法，其中，所述方法在L-山梨糖酮浓度为4至100mg/ml的条件下进行。
7.属于Ketogulonicigenium属的微生物用于在培养基中将L-山梨糖酮转化为维生素C的用途，其中，所述微生物选自由Ketogulonicigeniumrobustum NRRL B-21627、Ketogulonicigenium vulgare NRRL B-30035、Ketogulonicigenium vulgare NRRL B-30036、Ketogulonicigenium vulgareNRRL B-30037所构成的组。
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| DE602005021619D1 (de) | 2010-07-15 |
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