CN109337938A - 利用食烷菌发酵降解石蜡生产2-氧代己二酸的方法 - Google Patents
利用食烷菌发酵降解石蜡生产2-氧代己二酸的方法 Download PDFInfo
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- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
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Abstract
本发明公开了一种利用食烷菌发酵降解石蜡生产2‑氧代己二酸的方法，属于生物工程技术领域。本发明的技术方案要点为：利用食烷菌发酵降解石蜡生产2‑氧代己二酸的方法，所述食烷菌Alcanivoraxsp.Y5为经离子束照射诱变的变异菌株，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2018461。该方法以石蜡为唯一碳源来生产2‑氧代己二酸，不仅克服了常规生物合成方法产量低的障碍，而且成本低廉，也提高了石油工业副产品的利用率，减轻传统化学合成法对环境造成的污染。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种利用食烷菌发酵降解石蜡生产2-氧代己 二酸的方法。
背景技术
2-氧代己二酸(2-Ketoadipic acid；2-Oxoadipic acid)是己二酸(Adipic acid)的前体 物质。己二酸又称肥酸，能够发生成盐反应、酯化反应、酰胺化反应等，并能与二元胺或 二元醇缩聚成高分子聚合物等。己二酸是工业上具有重要意义的二元羧酸，在化工生产、 有机合成工业、医药、润滑剂制造、工程塑料等领域应用广泛。预计2020年，世界己二酸产能将达510万吨，居所有二元羧酸中的第二位(姬亚非.国内外己二酸市场分析[J].化 工管理,2017(5):136)。目前己二酸的主要生产方式是化学合成，主要以环己酮混合物与环己醇为原料，通过化学反应合成。生产原料及中间产物毒性强，且生产过程中产生大量有害气体，对大气及河流环境污染严重。因此，己二酸的生物合成法越来越受到全世界的关注，近年来关于微生物发酵法生产己二酸的报道也越来越多。
微生物发酵法生产己二酸的研究主要集中在以葡萄糖为底物，通过微生物如大肠杆菌 (Escherichia coli)发酵合成己二酸前体顺，顺-粘康酸(cis,cis-muconic acid)，然后再利 用金属催化剂催化合成己二酸(Zhang HR,Pereira B,Li ZJ,,etal.Engineering Escherichia coli coculture systems for the production ofbiochemical products.PNAS 2015,112 (27):8266–8271)。邓禹等通过大肠杆菌配合底物乙酰CoA和琥珀酰CoA全生物合成己二 酸(邓禹,张晓娟,毛银.一种己二酸的全生物合成方法:CN105112436A[P].2015.)。此外， 酿酒酵母(Saccharomyces cerevisiae)发酵油脂(Coconut oil)以及不动杆菌(Acinetobacter) 氧化环己醇也可以产生己二酸(BeardsleeT,Picataggio S.Bio-based adipic acid from renewable oils.Lipid Technol,2012,24(10):223–225；Cheng Q,Thomas SM,Kostichka K,et al. Genetic analysis of agene cluster for cyclohexanol oxidation in Acinetobacter sp.strain SE19by invitro transposition.J Bacteriol,2000,182(17):4744–4751)。虽然目前理论上可以实现己二 酸的生物合成，但由于产率低，成本高等原因，生物法生产己二酸达不到工业化的要求。
截至目前为止，利用Alcanivorax sp.液体发酵降解长链烷烃获得2-氧代己二酸的工艺 尚属首例。作为己二酸的前体物质，2-氧代己二酸可以与氢气加成反应，然后再利用醇的 消去反应成双键，然后再与氢气加成反应生成己二酸。也可利用黄鸣龙还原反应或者 CLEMMENSEN还原反应生成己二酸，步骤简单(Li JJ.Name reactions:a collection ofdetailed reaction mechanisms[J].Springer,2014)。不同于目前的常规方法以葡萄糖等底物为 原料生物合成己二酸前体物及己二酸，尝试以微生物降解长链烷烃的方式获得己二酸前体 物2-氧代己二酸是一种新的思路。
发明内容
本发明解决的技术问题是提供了一种利用食烷菌发酵降解石蜡生产2-氧代己二酸的方 法，该方法以石蜡为唯一碳源来生产2-氧代己二酸，不仅克服了常规生物合成方法产量低 的障碍，而且成本低廉，也提高了石油工业副产品的利用率，减轻传统化学合成法对环境 造成的污染。
本发明为解决上述技术问题采用如下技术方案，利用食烷菌发酵降解石蜡生产2-氧代 己二酸的方法，其特征在于：所述食烷菌Alcanivorax sp.Y5为经离子束照射诱变的变异菌 株，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2018461，保藏日期为 2018年7月9日，保藏单位地址为中国武汉武汉大学。
本发明所述的利用食烷菌发酵降解石蜡生产2-氧代己二酸的方法，其特征在于具体过 程为：将含有上述食烷菌Alcanivorax sp.Y5的菌悬液按照体积百分数为5％的接种量接种 于发酵培养基装液量为50mL的250mL三角瓶中，发酵培养基的配方为(NH4)2SO48.0g/L、 NH4NO3 2.0g/L、K2HPO4 1.0g/L、KH2PO4 1.0g/L、MgSO4 0.2g/L、FeCl3 0.03g/L、CaCl2 0.02g/L、NaCl 15.0g/L、石蜡6.0g/L、吐温80体积百分含量为0.1％，发酵培养基的初始 pH为7.0，液体发酵温度控制在28℃，转速为150rpm，发酵时长为5～7d。
优选的，所述菌悬液的浓度为106CFU/mL。
优选的，所述石蜡为53#固体石蜡或液体石蜡中的一种或多种，其中53#固体石蜡需要 切碎后加入发酵培养基灭菌使用。
优选的，以液体石蜡为碳源时，Alcanivorax sp.Y5发酵液中2-氧代己二酸的含量为 3.5g/L，转化率高达58.33％。
本发明利用生物降解途径生产己二酸的前体物2-氧代己二酸，底物成本低，工艺简单， 在尼龙工程塑料等领域具有广阔的应用前景。
附图说明
图1为发酵液中2-氧代己二酸的LC-MS检测图谱。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明，但不应该将此理解为本发明 上述主题的范围仅限于以下的实施例，凡基于本发明上述内容实现的技术均属于本发明的 范围。
1.实验菌株
食烷菌Alcanivorax sp.Y5(CCTCC NO：M 2018461)。
2.菌株的活化
将保藏的食烷菌划线或者涂布在海生菌培养基2216(美国BD公司，琼脂添加量为20g/L)上，28℃培养7d。
3.菌悬液的制备
刮取1～2环固体培养基上的菌苔，转移至装有无菌水的灭菌EP管(离心管)中，旋涡 仪混匀后，置于4℃冰箱待用。
4.发酵条件
将菌悬液(菌悬液浓度＝106CFU/mL)按照体积百分数为5％的接种量接种于发酵培养 基装液量为50mL的250mL三角瓶中，发酵培养基的配方为(NH4)2SO4 8.0g/L、NH4NO32.0g/L、K2HPO4 1.0g/L、KH2PO4 1.0g/L、MgSO4 0.2g/L、FeCl3 0.03g/L、CaCl2 0.02g/L、NaCl 15.0g/L、石蜡6.0g/L、吐温80体积百分含量为0.1％，发酵培养基的初始pH为7.0，液体发酵温度控制在28℃，转速为150rpm，发酵时长为7d。
5.2-氧代己二酸的测定
测定样本的制备过程如下：发酵结束，取1mL菌液于灭菌的EP管中，8000rpm低温离心10min，取上清800μL，用孔径为0.22μm的滤膜过滤除菌，低温保存待用。取100μL微生 物发酵液样本，加入400μL提取液(甲醇、乙腈与水的体积比为2:2:1)，涡旋混匀30s。冰 水浴超声处理10min后将样本4℃、12000rpm离心15min。小心地取出0.35mL上清于2mL甲 烷硅基化的进样瓶中真空干燥。干燥后加入100μL提取液(乙腈与水的体积比为1:1)复溶， 涡旋30s，冰水浴超声10min后将样本4℃、12000rpm离心15min。小心地取出60μL上清于2mL 进样瓶，每个样本各取10μL混合成样本，再取60μL上机检测。
2-氧代己二酸利用LC-MS系统测定，LC-MS系统由安捷伦1290超高效液相串联ABSciex TripleTOF 6600高分辨质谱仪组成。所使用的色谱柱为购自Waters的UPLC BEHAmide色谱柱(1.7μm，2.1*100mm)。进样体积为2μL，流动相为水和乙腈。质谱条件为： AB6600Triple TOF质谱仪能够在控制软件(Analyst TF 1.7，AB Sciex)控制下基于IDA功 能进行一级、二级质谱数据采集。
6.测定结果
LC-MS检测结果如图1所示：图中最高峰出峰时间为11.28min，二级定性结果为2-氧代己二酸。结合质谱定量定性分析结果可知，2-氧代己二酸的产量达2.97g/L。因此，固体石蜡中的烷烃经过一系列的氧化降解如α，β，ω氧化，使长链烃变为短链烃，在此过程中，2-氧代己二酸得到大量积累。
实施例
1.实验菌株
食烷菌Alcanivorax sp.Y5(CCTCC NO：M 2018461)。
2.菌株的活化
将保藏的Alcanivorax sp.Y5划线或者涂布在海生菌培养基2216(美国BD公司，琼脂 添加量为20g/L)上，28℃培养7d。
3.菌悬液的制备
刮取1～2环固体培养基上的菌苔，转移至装有无菌水的灭菌EP管中，旋涡仪混匀后， 置于4℃冰箱待用
4.发酵条件
将菌悬液(菌悬液浓度＝106CFU/mL)按照体积百分数为5％的接种量接种于发酵培养 基装液量为50mL的250mL三角瓶中，发酵培养基的配方为：
配方一：(NH4)2SO4 8.0g/L、NH4NO3 2.0g/L、K2HPO4 1.0g/L、KH2PO4 1.0g/L、MgSO40.2g/L、FeCl3 0.03g/L、CaCl2 0.02g/L、NaCl 15.0g/L、53#固体石蜡6.0g/L、吐温80体积百分含量为0.1％，发酵培养基的初始pH为7.0。
配方二：(NH4)2SO4 8.0g/L、NH4NO3 2.0g/L、K2HPO4 1.0g/L、KH2PO4 1.0g/L、MgSO40.2g/L、FeCl3 0.03g/L、CaCl2 0.02g/L、NaCl 15.0g/L、液体石蜡6.0g/L、吐温80体积百分含 量为0.1％，发酵培养基的初始pH为7.0。
发酵培养基的初始pH为7.0，发酵温度控制在28℃，转速为150rpm，发酵时长均为7d， 每个配方做3个生物学重复。
5.2-氧代己二酸的测定
测定样本的制备过程如下：发酵结束，取1mL菌液于灭菌的EP管中，8000rpm低温离心10min，取上清800μL，用孔径为0.22μm的滤膜过滤除菌，低温保存待用。取100μL微生 物发酵液样本，加入400μL提取液(甲醇、乙腈与水的体积比为2:2:1)，涡旋混匀30s。冰 水浴超声处理10min后将样本4℃、12000rpm离心15min。小心地取出0.35mL上清于2mL甲 烷硅基化的进样瓶中真空干燥。干燥后加入100μL提取液(乙腈与水的体积比为1:1)复溶， 涡旋30s，冰水浴超声10min后将样本4℃、12000rpm离心15min。小心地取出60μL上清于2mL 进样瓶，每个样本各取10μL混合成样本，再取60μL上机检测。2-氧代己二酸利用LC-MS系 统测定，LC-MS系统由安捷伦1290超高效液相串联AB Sciex TripleTOF 6600高分辨质谱仪 组成。所使用的色谱柱为购自Waters的UPLC BEH Amide色谱柱(1.7μm，2.1*100mm)。 进样体积为2μL，流动相为水和乙腈。质谱条件为：AB 6600Triple TOF质谱仪能够在控制 软件(Analyst TF 1.7，AB Sciex)控制下基于IDA功能进行一级、二级质谱数据采集。
6.实验结果
不同石蜡碳源对2-氧代己二酸产量的影响如表1所示：
表1不同石蜡碳源对2-氧代己二酸产量的影响
根据实验结果可知，以液体石蜡为碳源时，Alcanivorax sp.Y5发酵液中2-氧代己二酸 的含量为3.5g/L，转化率高达58.33％。高于以53#固体石蜡作为碳源时2-氧代己二酸的产 量，可能的原因是由于液体石蜡烃类碳链的长度比固体石蜡短，微生物更容易吸收利用， 故转化率较高。
以上实施例描述了本发明的基本原理、主要特征及优点，本行业的技术人员应该了解， 本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在 不脱离本发明原理的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发 明保护的范围内。
Claims (5)
1.利用食烷菌发酵降解石蜡生产2-氧代己二酸的方法，其特征在于：所述食烷菌Alcanivorax sp.Y5为经离子束照射诱变的变异菌株，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2018461。
2.根据权利要求1所述的利用食烷菌发酵降解石蜡生产2-氧代己二酸的方法，其特征在于具体过程为：将含有食烷菌Alcanivorax sp.Y5的菌悬液按照体积百分数为5%的接种量接种于发酵培养基装液量为50mL的250mL三角瓶中，发酵培养基的配方为(NH4)2SO48.0g/L、NH4NO3 2.0g/L、K2HPO4 1.0g/L、KH2PO4 1.0g/L、MgSO4 0.2g/L、FeCl3 0.03g/L、CaCl2 0.02g/L、NaCl 15.0g/L、石蜡6.0g/L、吐温80体积百分含量为0.1%，发酵培养基的初始pH为7.0，液体发酵温度控制在28℃，转速为150rpm，发酵时长为5~7d。
3.根据权利要求2所述的利用食烷菌发酵降解石蜡生产2-氧代己二酸的方法，其特征在于：所述菌悬液的浓度为106CFU/mL。
4.根据权利要求1或2所述的利用食烷菌发酵降解石蜡生产2-氧代己二酸的方法，其特征在于：所述石蜡为53#固体石蜡或液体石蜡中的一种或多种，其中53#固体石蜡需要切碎后加入发酵培养基灭菌使用。
5.根据权利要求2所述的利用食烷菌发酵降解石蜡生产2-氧代己二酸的方法，其特征在于：以液体石蜡为碳源时，Alcanivorax sp.Y5发酵液中2-氧代己二酸的含量为3.5g/L，转化率高达58.33%。
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