CN104404089B - 一种通过添加葡萄糖酸提高乙偶姻产量的方法 - Google Patents
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Abstract
添加葡萄糖酸提高乙偶姻产量的方法，属于发酵工程领域。利用本实验室前期筛选得到的高产乙偶姻野生型枯草芽孢杆菌B.subtilis JNA(保藏号CCTCC M209309；专利申请公布号CN 101864381A)进行发酵，通过调节葡萄糖与葡萄糖酸的比例改变胞内辅酶变化水平从而改变乙偶姻与2,3‑丁二醇比例。最终在较低NADH浓度及NADH/NAD+比例下(50g/L葡萄糖和50g/L葡萄糖酸)，转化约为55.8g/L的乙偶姻，副产物2,3‑丁二醇仅为4.1g/L；与使用葡萄糖作为唯一碳源相比较，乙偶姻生产效率上升到0.78g/(L·h)，乙偶姻产量提高近86％，副产物2,3‑丁二醇积累量下降80％。为国内外首次利用混合还原态底物降低NADH浓度及其NADH/NAD+比例发酵生产乙偶姻，实现了提高生产效率、减少NADH依赖型副产物的目的。
Description
技术领域
通过在发酵培养基中添加葡萄糖酸，降低发酵前期副产物2,3-丁二醇的积累，提高乙偶姻的产量，本发明属于发酵工程领域。
技术背景
乙偶姻在食品调香、生化及药理方面有着广泛的应用价值，目前国内外对其合成和生产都有着比较深入的研究。自然界中的某些细菌具有生产乙偶姻的能力，主要包括克雷伯氏菌属(Klebisella)、肠杆菌属(Enterobacter)、芽孢杆菌属(Bacillus)、沙雷氏菌属(Serratia)以及乳球菌属(Lactococcus)等。但是在大多数菌株代谢过程中，乙偶姻是作为2，3-丁二醇和丁二酮代谢的副产物而存在的，积累浓度较低，从而直接导致了难以利用这些微生物菌种工业化发酵生产乙偶姻。
本实验室保藏有一株以葡萄糖为底物高产乙偶姻的枯草芽孢杆菌(保藏号CCTCCM2093092009.12.21；专利申请公布号CN 101864381 A)，在摇瓶发酵研究中发现了发酵过程中乙偶姻和2,3-丁二醇相互转化的现象：在以150g/l葡萄糖为底物的前提下，发酵前期主要积累2,3-丁二醇；发酵中期(96h左右)2,3-丁二醇含量达到最高值约43g/L，乙偶姻约为15g/L，此时乙偶姻和2,3-丁二醇含量比例大约为1：3；发酵后期伴随着菌体老化裂解大部分形成芽孢等现象，中间代谢产物乙偶姻开始积累，到发酵终止时(144h)乙偶姻和2,3-丁二醇含量比例大约为3：1,此时它们含量分别为39.5g/L和14g/L左右，导致乙偶姻产量无法进一步提高。本实验室研究发现，发酵前期由于受到糖酵解途径影响，乙偶姻需转化成2,3-丁二醇为细胞生长提供所需的NAD+，导致2,3-丁二醇大量积累，NADH浓度上升。因此考虑通过改变底物还原态，从而改变胞内NADH水平和NADH/NAD+比例来调控乙偶姻及2,3-丁二醇的生成。通过利用葡萄糖酸发酵，减少糖酵解途径代谢，降低NAD+需求，实现降低NADH水平和NADH/NAD+比例。
本发明通过调节葡萄糖与葡萄糖酸的比例改变胞内辅酶变化水平从而改变乙偶姻与2,3-丁二醇比例。利用葡萄糖为底物促进细胞生长，同时利用葡萄糖酸为底物降低NADH水平和NADH/NAD+比例，最终达到提高生产效率，减少NADH依赖型副产物2,3-丁二醇的目的，实现高效发酵生产乙偶姻。
发明内容
本发明所使用的菌种为B.subtilis JNA，该菌株前期发酵葡萄糖合成2,3-丁二醇，后期逆向转化为乙偶姻，已保藏于中国典型培养物保藏中心，保藏编号为：CTCCM209309。
本发明的主要研究内容：本发明根据研究B.subtilis JNA发酵生产乙偶姻的过程中的NADH及NAD+变化，通过调节葡萄糖与葡萄糖酸的比例改变辅酶变化水平从而改变乙偶姻与2,3-丁二醇比例。最终在一个较低NADH浓度及NADH/NAD+比例下(50g/L葡萄糖和50g/L葡萄糖酸)发酵72h，转化约为55.8g/L的乙偶姻，副产物2,3-丁二醇仅为4.1g/L较原始菌株下降80％，乙偶姻生产效率上升到0.78g/(L·h)，产量提高近86％。
本发明的优点和积极效果是：
(1)本发明首次报道了通过利用不同还原态底物调节胞内NADH水平和NADH/NAD+比例来减少NADH依赖型副产物的生成，为枯草芽孢杆菌发酵生产乙偶姻减少NADH依赖型副产物提供了一定的理论可能。
(2)本发明通过调节葡萄糖与葡萄糖酸的比例改变辅酶变化水平从而改变乙偶姻与2,3-丁二醇比例。，将50g/L葡萄糖和50g/L葡萄糖酸转化约为55.8g/L的乙偶姻副产物，2,3-丁二醇仅为4.1g/L较原始菌株下降80％，乙偶姻生产效率上升到0.78g/(L·h)，产量提高近86％。
具体实施方式
实施例1：B.subtilis JNA发酵生产乙偶姻胞内辅酶变化检测
(1)种子培养
从活化平板上挑取单菌落接种于种子培养基中，种子培养温度37℃，摇床转速160r/min，培养时间为12h左右，种子培养基组成：酵母提取物5g/L，胰蛋白胨10g/L，NaCl10g/L葡萄糖40g/L。
(2)发酵培养
初始发酵培养体积为2L，采用的发酵培养基成分如下：
发酵培养基成分：牛肉浸膏5g/L，玉米浆20g/L，尿素2g/L，葡萄糖100g/L。将上述发酵培养基用5mol/L的NaOH调节其pH至6.5，在121℃下高温灭菌30min。
发酵条件：将上述培养好的种子液按5％接种量接种于发酵培养基中进行发酵培养，发酵温度37℃，空气流量为120m3/h·m3培养基，搅拌转速为300r/min。定时取样测定细胞浓度、乙偶姻和2,3-丁二醇及其它NADH依赖型副产物的产量。发酵结束后，发酵液中产物2,3-丁二醇和乙偶姻用气相色谱测定(GC-1690J气相色谱仪，杭州科晓化工仪器公司)。色谱条件如下：毛细管柱，30m×0.32mm色谱柱中固定液为AT.SE-30，检测器为FID，柱温150℃，汽化室与检测器的温度均为250℃，载气为N2，流速0.1Mpa，进样量2μL，采用外标法定量。
(3)胞内辅因子水平测定
重组菌株胞内辅因子水平检测利用AAT Bioquest公司试剂检测盒。通过荧光酶标仪，在Ex/Em＝540/590nm下检测NADH、NAD+浓度和NADH/NAD+比例。
结果表明，发酵前期受到糖酵解途径影响，乙偶姻转化为2,3-丁二醇为细胞提供所需的NAD+，导致2,3-丁二醇大量积累，NADH浓度上升。
实施例2：混合葡萄糖与葡萄糖酸发酵生产乙偶姻、2,3-丁二醇及副产物性能检测
(1)种子培养
从活化平板上挑取单菌落接种于种子培养基中，种子培养温度37℃，摇床转速160r/min，培养时间为12h左右，种子培养基组成：酵母提取物5g/L，胰蛋白胨10g/L，NaCl10g/L葡萄糖40g/L。
(2)发酵培养
初始发酵培养体积为2L，采用的发酵培养基成分如下：
发酵培养基成分：牛肉浸膏5g/L，玉米浆20g/L，尿素2g/L，葡萄糖/葡萄糖酸(9:1，3:7，5:5，7:3，9:1)100g/L。将上述发酵培养基用5mol/L的NaOH调节其pH至6.5，在121℃下高温灭菌30min。
发酵条件：将上述培养好的种子液按5％接种量接种于发酵培养基中进行发酵培养，发酵温度37℃，空气流量为120m3/h·m3培养基，搅拌转速为300r/min。定时取样测定细胞浓度、乙偶姻和2,3-丁二醇及其它NADH依赖型副产物的产量。发酵结束后，发酵液中产物2,3-丁二醇和乙偶姻用气相色谱测定(GC-1690J气相色谱仪，杭州科晓化工仪器公司)。色谱条件如下：毛细管柱，30m×0.32mm色谱柱中固定液为AT.SE-30，，检测器为FID，柱温150℃，汽化室与检测器的温度均为250℃，载气为N2，流速0.1Mpa，进样量2μL，采用外标法定量。
最终B.subtilis JNA在一个较低NADH浓度及NADH/NAD+比例下(50g/L葡萄糖和50g/L葡萄糖酸)，转化约为55.8g/L的乙偶姻，副产物2,3-丁二醇仅为4.1g/L较原始菌株下降80％，乙偶姻生产效率上升到0.78g/(L·h)，产量提高近86％。
Claims (1)
1.一种发酵策略在发酵生产乙偶姻中的应用，其特征是：所述策略为利用葡萄糖和葡萄糖酸作为混合碳源发酵生产乙偶姻；
具体的，将保藏编号为CTCCM 209309的B.subtilis JNA接种于10mL LB培养基中，37℃振荡培养7-9h后，取1mL转接于2瓶50mL含40g/L葡萄糖的LB培养基中37℃振荡培养，当培养至OD600＝5.0-6.0时，加入含1.9L发酵培养基的5L发酵反应器中培养，最终发酵72h；所述的发酵培养基的配方为：牛肉浸膏5g/L，玉米浆20g/L，尿素2g/L，50g/L葡萄糖和50g/L葡萄糖酸，去离子水配置，pH 6.5。
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| WO2012104244A1 (de) * | 2011-01-31 | 2012-08-09 | Gpawacker Chemie Ag | Verfahren zur fermentativen herstellung von 2,3-butandiol |
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