CN112795599A - 一种利用玉米芯同步糖化发酵生产d-1,2,4-丁三醇的方法 - Google Patents
一种利用玉米芯同步糖化发酵生产d-1,2,4-丁三醇的方法 Download PDFInfo
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Abstract
本发明提供了一种利用玉米芯同步糖化发酵生产D‑1,2,4‑丁三醇的方法,该方法通过构建克隆表达2‑酮酸脱羧酶和D‑木糖脱氢酶,木糖酸脱水酶和醇脱氢酶的基因,并将构建好的基因转入宿主菌的细胞内得重组大肠杆菌,培养重组大肠杆菌并接种至含有经预处理过的玉米芯的培养基中发酵生产D‑1,2,4‑丁三醇。本发明以玉米芯为原料,同步糖化发酵生产丁三醇,D‑1,2,4‑丁三醇产量为6.44g/L。利用玉米芯同步糖化发酵生产1,2,4‑丁三醇,操作简便,产量较高,适合产业化生产。
Description
技术领域
本发明属于生物技术领域,具体涉及一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法。
背景技术
D-1,2,4-丁三醇是一种无色无味、透明、粘稠的四碳多元醇;在水和醇类物质中溶解度较高,具有吸湿性。在军事上,D-1,2,4-丁三醇可以用来合成作为火箭推进剂的丁三醇三硝酸酯(BTTN),在医药方面,D-1,2,4-丁三醇可以作为缓释剂,制备抗病毒化合物的中间体,D-1,2,4-丁三醇也可以作为高分子材料的交联剂,增加材料的强度和硬度。
目前,D-1,2,4-丁三醇的生产主要集中于化学合成,即利用 NaBH4还原苹果酸及其衍生物。但化学合成D-1,2,4-丁三醇反应需在2900-5000psi的H2压力下和60-160ºC的条件下,反应条件十分苛刻,操作存在安全威胁,且环境污染严重。因此,继续开发能用于商业化生产D-1,2,4-丁三醇的方法。
生物法合成D-1,2,4-丁三醇由于安全可靠,来源易得等优点,目前备受瞩目。生物法合成D-1,2,4-丁三醇主要以木糖为底物,经过D-木糖脱氢酶,木糖酸脱水酶、2-酮酸脱羧酶和醇脱氢酶四步酶反应合成。目前生物法合成D-1,2,4-丁三醇主要涉及两种方法:一种是大量培养表达上述四种酶的微生物,培养结束后收集细胞,利用收集的细胞将木糖转化为D-1,2,4-丁三醇,即全细胞催化法;这种方法具有转化率较高的优势,但过程中步骤繁琐,不利于大规模生产;另一种是在培养表达上述四种酶的微生物的过程中,加入底物木糖,直接生产BT。利用微生物直接发酵生产BT成本低廉,过程中操作简便,有利于大规模生产。
玉米芯是一种常见的农业废弃物,其主要成分有纤维素,半纤维素,木质素等,其中,半纤维素为木聚糖型,经过半纤维素酶处理,即可转化为木糖,玉米芯的应用价值和经济潜力都是十分巨大的。利用玉米芯为原料通过微生物发酵转化为目标产物可以有两种途径:分步糖化发酵和同步糖化发酵。分步糖化发酵先将玉米芯糖化,后使用含有单糖的水解液进行发酵。CN106148429B公开了一种生物转化纤维素水解液生产D-1,2,4-丁三醇的方法。该方法为构建克隆表达2-酮酸脱羧酶和D-木糖脱氢酶,木糖酸脱水酶和醇脱氢酶的基因,并将构建好的基因转入敲除木糖异构酶的宿主菌的细胞内得基因工程菌,培养基因工程菌并接种至纤维素水解液中发酵生产D-1,2,4-丁三醇。该方法采用稀酸预处理玉米芯得到含有木糖的水解液,稀酸预处理过程会产生糠醛等对菌株生长不利的发酵抑制剂,且水解液pH很低,需要预先经过氢氧化钙中和硫酸,活性炭脱毒等步骤才能应用到发酵过程中。步骤较多,不利于大批量发酵。同步糖化发酵将酶解过程与发酵过程耦合,玉米芯酶解与菌株发酵同步进行,构成同步糖化发酵过程,操作简便,生产成本低廉,有较大的商业化生产空间。
发明内容
针对现有技术的不足,本发明的目的在于提供一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,降低了生产成本的同时,提高了1,2,4-丁三醇的产量,本发明使用碱预处理玉米芯,使玉米芯易于酶解,使用处理后的玉米芯残渣作为底物,除水洗残渣外无需中和、脱毒等步骤,节省成本,操作更加简便,且产量有进一步提升。
为解决现有技术问题,本发明采用的技术方案:
一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,通过构建克隆表达2-酮酸脱羧酶和D-木糖脱氢酶,木糖酸脱水酶和醇脱氢酶的基因,并将构建好的基因转入宿主菌的细胞内得重组大肠杆菌,培养重组大肠杆菌并接种至含有经预处理过的玉米芯的培养基中发酵生产D-1,2,4-丁三醇。
作为改进的是,上述利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,具体步骤为:
步骤1,构建克隆表达2-酮酸脱羧酶(mdlC),D-木糖脱氢酶(xylB),木糖酸脱水酶(yjhG)和醇脱氢酶(adhP),并将构建好的基因转入宿主菌的细胞内得重组大肠杆菌;
步骤2,玉米芯预处理
将氢氧化钠溶液和玉米芯混合后的悬浮水解液经121℃处理,过滤得到玉米芯残渣,将残渣用纯水洗至中性;
步骤3,发酵培养基的配制
配制5g/L酵母粉,10g/L蛋白胨,5g/L氯化钠的LB培养基,并封装于500mL锥形瓶中,装液量50mL,向LB培养基中添加2g预处理后的玉米芯残渣制成发酵培养基,灭菌待用;
步骤4,将重组大肠杆菌接种至发酵培养基中,加入半纤维素酶,再加入IPTG诱导发酵得产物D-1,2,4-丁三醇。
优选的是,步骤1中所述宿主菌为大肠杆菌Trans1 T1。
优选的是,步骤2中氢氧化钠的浓度为1mol/L,玉米芯与氢氧化钠溶液的质量体积比为1:10。
优选的是,步骤4中重组大肠杆菌的接种量4%,IPTG终浓度1mmol/L,初始培养温度37℃,加入诱导剂后33℃培养至发酵结束。
优选的是,步骤4中半纤维素酶添加量为400IU。
有益效果:
现有技术通常使用木糖作为D-1,2,4-丁三醇生产的底物,价格较高,应用于大规生产往往成本较高。玉米芯含有大量的半纤维素,经酶解可释放大量的木糖,但结构紧密,酶解效率低。相比于稀酸预处理直接释放大量木糖,碱预处理主要作用是打破玉米芯的紧密结构,使半纤维素易被半纤维素酶酶解,且酶解过程不会产生发酵抑制物,对发酵过程不良影响少。将酶解过程与发酵过程耦合,玉米芯酶解与菌株发酵同步进行,构成同步糖化发酵过程,操作简便,生产成本低廉,有较大的商业化生产空间。
本发明以玉米芯为原料,碱预处理玉米芯,使酶解效率提升,通过使用预处理后的残渣同步糖化发酵方法来生产D-1,2,4-丁三醇,提高了D-1,2,4-丁三醇的产量,极大地节约了成本,操作简便,有大规模生产的潜力。
附图说明
图1为重组大肠杆菌以木糖为底物发酵生产D-1,2,4-丁三醇;
图2 为预处理前玉米芯与预处理后残渣酶解24h后木糖产量;
图3为重组大肠杆菌利用玉米芯作为底物同步糖化发酵生产D-1,2,4-丁三醇的情况。
具体实施方案
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
以下实施例中如无特别说明,所用酶及质粒均为购买所得。
所述2-酮酸脱羧酶(mdlc), GenBank: AY143338.1;D-木糖脱氢酶(xylB),GeneID: 7329904;木糖酸脱水酶(yjhG),Gene ID: 946829;醇脱氢酶(adhP),Gene ID:00946036。
另外,本发明所述的“同步”是指纤维素糖化和单糖发酵同时进行。
实施例1 含YjhG基因菌株的构建
(1)利用YjhG基因5’端和3’端引物引入酶切位点(NcoI和HindⅢ),对YjhG基因和pCWJ质粒进行双酶切,然后将YjhG基因连接到pCWJ载体上;
(2)将上述重组载体转入大肠杆菌Trans 1T1的感受态细胞(全式金生物技术有限公司)里,涂布在带有50mg/L氯霉抗性的LB平板,37℃过夜培养;
(3)挑取平板上生长的单菌落,转接到含有50mg/L氯霉素抗性的LB培养基里,然后提取质粒,再经限制性内切酶Spe I和Kpn I进行酶切验证,最后得到了重组质粒pCWJ-YjhG。
参考上述方法,继续制备重组质粒PCWJ-yjhG-adhP和pTRC99a-xylB-mdlC。
实施例2 重组大肠杆菌利用木糖作为底物发酵生产1,2,4-丁三醇
将含有重组质粒pTRC99a-xylB-mdlC和重组质粒PCWJ-yjhG-adhP的基因工程菌在LB平板(链霉抗性和氨苄抗性)上划线活化,挑取单菌落,接入5 mL LB培养基中,37℃,200rpm培养7-10 h。
取一组500ml三角瓶,分别装入50ml含有10g/L木糖的发酵培养基,将重组大肠杆菌制得的种子液按4%的体积比的接种量接种到发酵培养基中,37℃下培养至OD600=2时加入IPTG至终浓度为1 mmol/L,加入IPTG 后在33℃培养66h至发酵结束,每隔12 h取一次样,测定D-1,2,4-丁三醇产量,结果如图1。由图可见,菌株发酵至64h后生产速率明显降低,产量不再增高。
其中,发酵培养基的配方如下:5g/L酵母粉,10g/L蛋白胨,10g/L氯化钠,5 g/L葡萄糖,10g/L木糖。
实施例3 重组大肠杆菌利用玉米芯作为底物同步糖化发酵生产1,2,4-丁三醇
玉米芯粉碎过40目筛,将1 mol/L氢氧化钠溶液和玉米芯按固液比10%混合,混合后的悬浮水解液经高温处理,过滤得到玉米芯残渣,将残渣用纯水洗至中性。
配制5g/L酵母粉,10g/L蛋白胨,5g/L氯化钠的LB培养基。分装于500mL锥形瓶中,装液量50mL,向LB培养基中添加2g预处理后的玉米芯残渣制成发酵培养基,灭菌待用。
将含有重组质粒pTRC99a-xylB-mdlC和重组质粒PCWJ-yjhG-adhP的基因工程菌在LB平板(链霉抗性和氨苄抗性)上划线活化,挑取单菌落,接入5 mL LB培养基中,37℃,200rpm培养7-10 h。
取一组500ml三角瓶,分别装入50ml含有2g玉米芯残渣的发酵培养基,将重组大肠杆菌制得的种子液按4%的体积比的接种量接种到发酵培养基中,37℃下培养2h后加入IPTG至终浓度为1 mmol/L,加入IPTG 后在33℃培养66h至发酵结束,每隔12 h取一次样,测定D-1,2,4-丁三醇产量。结果如图2和图3所示。由图2所示,2g经预处理后的玉米芯残渣酶解24h后产生木糖总量达0.52g,浓度10.45g/L,是玉米芯直接酶解所产木糖的8.75倍,证明碱预处理效果良好。由图3所示,使用玉米芯为底物同步糖化发酵,不仅使丁三醇产量提高,且64h后持续生产丁三醇,证明玉米芯可以持续为发酵过程提供底物。
使用玉米芯同步糖化发酵生产D-1,2,4-丁三醇,将玉米芯酶解与发酵过程耦合,提高了D-1,2,4-丁三醇的产量,提供了一种新的工艺路线。玉米芯在我国年产量极高,价格低廉,使用玉米芯极大的节约生产成本,且操作工艺简单,有大规模生产的潜力。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (6)
1.一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,其特征在于,通过构建克隆表达2-酮酸脱羧酶和D-木糖脱氢酶,木糖酸脱水酶和醇脱氢酶的基因,并将构建好的基因转入宿主菌的细胞内得重组大肠杆菌,培养重组大肠杆菌并接种至含有经预处理过的玉米芯的培养基中发酵生产D-1,2,4-丁三醇。
2.根据权利要求1所述的一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,其特征在于,具体步骤如下:
步骤1,构建克隆表达2-酮酸脱羧酶,D-木糖脱氢酶,木糖酸脱水酶和醇脱氢酶,得重组大肠杆菌; 步骤2,玉米芯的预处理 将氢氧化钠溶液和玉米芯混合后的悬浮水解液经121℃处理,过滤得到玉米芯残渣,将残渣用纯水洗至中性;
步骤3,发酵培养基的配制
配制5g/L酵母粉,10g/L蛋白胨,5g/L氯化钠的LB培养基,并封装于500mL锥形瓶中,装液量50mL,向LB培养基中添加2g预处理后的玉米芯残渣制成发酵培养基,灭菌待用; 步骤4,将重组大肠杆菌接种至发酵培养基中,加入半纤维素酶,再加入IPTG诱导发酵得产物D-1,2,4-丁三醇。
3.根据权利要求2所述的一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,其特征在于,步骤1中所述宿主菌为大肠杆菌Trans1 T1。
4.根据权利要求2所述的一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,其特征在于,步骤2中氢氧化钠的浓度为1mol/L,玉米芯与氢氧化钠溶液的质量体积比为1:10。
5.根据权利要求2所述的一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,其特征在于,步骤4中重组大肠杆菌的接种量4%,IPTG终浓度1mmol/L,初始培养温度37℃,加入诱导剂后33℃培养至发酵结束。
6.根据权利要求2所述的一种利用玉米芯同步糖化发酵生产D-1,2,4-丁三醇的方法,其特征在于,步骤4中半纤维素酶添加量为400IU。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106148429A (zh) * | 2016-08-25 | 2016-11-23 | 南京工业大学 | 一种生物转化纤维素水解液生产d‑1,2,4‑丁三醇的方法 |
CN107699536A (zh) * | 2017-11-27 | 2018-02-16 | 南京工业大学 | 一种基因工程菌及其在生产d‑1,2,4‑丁三醇中的应用 |
CN107988128A (zh) * | 2017-11-27 | 2018-05-04 | 南京工业大学 | 一种产d-1,2,4-丁三醇的基因工程菌及其应用 |
CN109554386A (zh) * | 2018-12-17 | 2019-04-02 | 山东大学 | 一种利用工程大肠杆菌以玉米芯水解液为底物高产d-木糖酸的方法 |
CN111118077A (zh) * | 2020-01-09 | 2020-05-08 | 南京工业大学 | 一种利用玉米芯水解液中木糖一步发酵生产1,5-戊二胺的方法 |
-
2021
- 2021-02-24 CN CN202110206930.2A patent/CN112795599A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106148429A (zh) * | 2016-08-25 | 2016-11-23 | 南京工业大学 | 一种生物转化纤维素水解液生产d‑1,2,4‑丁三醇的方法 |
CN107699536A (zh) * | 2017-11-27 | 2018-02-16 | 南京工业大学 | 一种基因工程菌及其在生产d‑1,2,4‑丁三醇中的应用 |
CN107988128A (zh) * | 2017-11-27 | 2018-05-04 | 南京工业大学 | 一种产d-1,2,4-丁三醇的基因工程菌及其应用 |
CN109554386A (zh) * | 2018-12-17 | 2019-04-02 | 山东大学 | 一种利用工程大肠杆菌以玉米芯水解液为底物高产d-木糖酸的方法 |
CN111118077A (zh) * | 2020-01-09 | 2020-05-08 | 南京工业大学 | 一种利用玉米芯水解液中木糖一步发酵生产1,5-戊二胺的方法 |
Non-Patent Citations (2)
Title |
---|
LI XH等: "Design and construction of a non-natural malate to 1,2,4-butanetriol pathway creates possibility to produce1,2,4-butanetriol from glucose" * |
孙雷;杨帆;朱泰承;李兴华;孙红兵;李寅;许正宏;张延平;: "大肠杆菌合成1,2,4-丁三醇的途径优化" * |
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