CN113684127A - 一种促进嗜盐古菌Haloferax mediterranei合成甜菜碱的方法 - Google Patents
一种促进嗜盐古菌Haloferax mediterranei合成甜菜碱的方法 Download PDFInfo
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- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 title claims abstract description 80
- 229960003237 betaine Drugs 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 17
- 241000203069 Archaea Species 0.000 title claims abstract description 7
- 230000001737 promoting effect Effects 0.000 title claims abstract description 7
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 title claims abstract 7
- 238000000855 fermentation Methods 0.000 claims abstract description 36
- 230000004151 fermentation Effects 0.000 claims abstract description 36
- 238000005273 aeration Methods 0.000 claims description 15
- 230000003834 intracellular effect Effects 0.000 claims description 13
- 230000003068 static effect Effects 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 239000007789 gas Substances 0.000 claims 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims 2
- 239000001301 oxygen Substances 0.000 claims 2
- 229910052760 oxygen Inorganic materials 0.000 claims 2
- 239000001963 growth medium Substances 0.000 claims 1
- 239000002609 medium Substances 0.000 claims 1
- 241000204988 Haloferax mediterranei Species 0.000 abstract description 28
- 230000003204 osmotic effect Effects 0.000 abstract description 9
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
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- 102100024085 Alpha-aminoadipic semialdehyde dehydrogenase Human genes 0.000 description 2
- 108010049668 Betaine-Aldehyde Dehydrogenase Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- SXKNCCSPZDCRFD-UHFFFAOYSA-N betaine aldehyde Chemical compound C[N+](C)(C)CC=O SXKNCCSPZDCRFD-UHFFFAOYSA-N 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
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- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
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- 229930013930 alkaloid Natural products 0.000 description 1
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- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
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- MPTQRFCYZCXJFQ-UHFFFAOYSA-L copper(II) chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Cu+2] MPTQRFCYZCXJFQ-UHFFFAOYSA-L 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
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- 150000004687 hexahydrates Chemical class 0.000 description 1
- XEEYVTMVFJEEEY-UHFFFAOYSA-L magnesium;dichloride;tetrahydrate Chemical compound O.O.O.O.[Mg+2].[Cl-].[Cl-] XEEYVTMVFJEEEY-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- LAIZPRYFQUWUBN-UHFFFAOYSA-L nickel chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Ni+2] LAIZPRYFQUWUBN-UHFFFAOYSA-L 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- -1 sodium molybdate monohydrate Chemical class 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
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- 230000002195 synergetic effect Effects 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
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Abstract
一种促进嗜盐古菌Haloferax mediterranei合成甜菜碱的方法属于生物发酵领域。其特征在于：提高H.mediterranei发酵液的盐度，并通过在外部施加特定强度的磁场，提高甜菜碱产量。即利用渗透冲击使H.mediterranei合成更多的甜菜碱以平衡胞内外渗透压，并将外源磁场作用于H.mediterranei发酵液，实现H.mediterranei甜菜碱产量的提高。
Description
技术领域
本发明提出一种促进嗜盐古菌Haloferax mediterranei(H.mediterranei)合成甜菜碱(glycine betaine)的方法。本发明属于生物发酵领域，具体涉及甜菜碱的生物合成领域。
背景技术
甜菜碱是一种生物碱，广泛存在于动植物与微生物中。甜菜碱是代谢的次生产物，是非常重要的渗透调节物质，可以增强生物的抗逆性。近几年，甜菜碱多被用作添加剂，应用在食品、饲料、化妆品、医药等方面。目前甜菜碱的生产多用化学合成法，但该方法能耗大、时间长、副产物多。
随着分子生物学及代谢工程等生物科学的快速发展，将微生物作为细胞工厂生产目的产品已经成为一种可行手段。H.mediterranei是一种极端嗜盐古菌，能在极高的盐度下生存，在其盐适应和短期的盐胁迫过程中，甜菜碱发挥着十分重要的作用。在高盐条件下，甜菜碱可被嗜盐菌从外界吸收或自身合成来维持胞内渗透压，同时，它可以稳定细胞结构，保持胞内酶、蛋白质的活性。近年来，电场(EFs)、磁场(MFs)或电磁场(EMF)对真核生物和原核生物的生物学效应引起了生物学家的强烈兴趣，因为这些物理条件会深刻影响细胞的代谢、行为和生存。在H.mediterranei盐胁迫过程中，外源磁场可作为一种提高其细胞增殖的手段。
本专利提供了一种通过高渗突变耦合外源磁场的方式来促进H.mediterranei合成甜菜碱的方法。利用该方法可以提高H.mediterranei胞内甜菜碱的含量，提高甜菜碱的产率。
发明内容
本发明的目的是提高嗜盐古菌H.mediterranei的甜菜碱产量。
本发明调控嗜盐古菌H.mediterranei的甜菜碱产量，其特征在于：提高H.mediterranei发酵液的盐度，并通过在外部施加特定强度的磁场，提高甜菜碱产量。即利用渗透冲击使H.mediterranei合成更多的甜菜碱以平衡胞内外渗透压，并将外源磁场作用于H.mediterranei发酵液，实现H.mediterranei甜菜碱产量的提高。
本发明提出一种利用高渗突变耦合外源磁场强化嗜盐古菌H.mediterranei合成甜菜碱的装置，包括：
(1)发酵罐，(2)控温装置，(3)pH自动控制仪，(4)曝气泵，(5)气体流量计，(6)曝气头，(7)静态磁场发生装置。
装置连接流程为：将控温装置(2)安装在发酵罐(1)上；pH自动控制仪(3)与发酵罐(1)连接；将曝气泵(4)、气体流量计(5)、曝气头(6)通过导管连接进行曝气；将发酵罐(1)放置于静态磁场发生装置(7)内。
基于上述装置，以Haloferax mediterranei(菌种编号ATCC 33500)为接种物进行发酵，主要步骤如下：
(1)将嗜盐古菌Haloferax mediterranei(ATCC 33500)接种在灭菌后的富集培养基中进行富集培养，培养时间为72h。富集培养基特征为盐度为150-200gNaCl/L；
(2)将富集培养物接种在发酵罐(1)中，控制富集培养物:发酵液＝1:20(v/v)。发酵液特征为盐度为250-300gNaCl/L；
(3)调节控温装置(2)使温度控制在35-39℃，调节pH自动控制仪(3)使pH值控制在6.9-7.3，调节气体流量计(5)使曝气量控制在1.0-1.5L/min；
(4)调节静态磁场发生装置(7)使磁场强度控制在10-60mT，最优磁场强度是50mT；
(5)连续发酵18-30h。发酵期间不断测量甜菜碱含量。在甜菜碱细胞含量最大时停止发酵。收集细胞提取胞内甜菜碱。
应当理解，在不偏离本发明的精神和范围的情况下，本领域的普通技术人员可以在形式和细节上对其做出各种改变和改进，而这些均被认为落入了本发明的保护范围。
本发明的技术原理：
相似相容性物质是嗜盐菌抵抗渗透压的关键物质，一定量的相似相容性物质可以帮助嗜盐菌在高盐环境更好地生长。不同嗜盐菌中主要的相容性溶质是不同的，甜菜碱是H.mediterranei抵抗高渗透压的主要相似相容性物质。当外界渗透压升高时，H.mediterranei会合成更多的甜菜碱来维持胞内渗透压。此外，外源磁场会强化H.mediterranei通过胆碱氧化途径合成甜菜碱，即胆碱被胆碱脱氢酶氧化为甜菜碱醛，然后被甜菜碱醛脱氢酶氧化为甜菜碱。为此，本发明通过高渗突变耦合外源磁场，促进H.mediterranei中甜菜碱醛脱氢酶的活性，进而促进了甜菜碱的合成。
本发明的益效果：
本发明通过发酵的方式，将H.mediterranei作为细胞工厂，通过高渗突变耦合外源磁场促进H.mediterranei胞内甜菜碱的合成。高渗条件能有效促进H.mediterranei胞内甜菜碱的合成，低强度磁场能促进H.mediterranei在高渗条件下的增殖，二者协同作用能提高甜菜碱总产量。该方法简便、安全、成本低，磁设备可重复利用，物理性的设施无毒性，不会像其他化学方法一样产生二次污染。
附图说明
图1为施加静态磁场条件下，H.mediterranei发酵合成甜菜碱的装置结构示意图。(1)发酵罐，(2)控温装置，(3)pH自动控制仪，(4)曝气泵，(5)气体流量计，(6)曝气头，(7)静态磁场发生装置。
图2为200g/LNaCl与无磁场条件下H.mediterranei生长情况与胞内甜菜碱含量。
图3为300g/LNaCl耦合50mT外源磁场条件下H.mediterranei生长情况与胞内甜菜碱含量。
具体实施方式：
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明，但本发明并不限于以下实施例。
在实施例中，所用菌种为Haloferax mediterranei，菌种编号ATCC 33500。
该菌株的富集培养基配方为：200.0g/LNaCl，2.0g/LKCl，20g/LMgSO4·7H2O，0.01g/LFeSO4，3.0g/LC6H5Na3O7·2H2O，7.5g/LCasein Hydrolysate，10g/LYEAST EXTRACT。同时，用氢氧化钠溶液调节培养基的pH值为7.2。
该菌株的发酵培养液配方为：4.0g/LKCl，0.6917g/LCaCl2·2H2O，1.0g/LNH4Cl，13.0g/LMgCl2·6H2O，20.0g/LMgSO4·7H2O，0.25g/LNaHCO3，0.5g/LNaBr，0.4g/LKH2PO4，0.01g/LFeSO4，5.0g/LGlucose，SL-6微量元素1.0ml/L(SL-6微量元素溶液按公知方法配备：每100ml含七水合硫酸锌0.1g，四水合氯化镁0.03g，硼酸0.3g，六水合氯化钻0.2g，二水合氯化铜0.01g，六水合氯化镍0.02g，一水合钼酸钠0.03g)，用氢氧化钠溶液调节发酵液的pH值为7.2。
实施例1：
(1)将嗜盐古菌Haloferax mediterranei(ATCC 33500)接种在灭菌后的富集培养基中进行富集培养，培养时间为72h。富集培养基特征为盐度为200gNaCl/L；
(2)将富集培养物接种在发酵罐中，配备发酵用发酵液1L，控制富集培养物:发酵液＝1:20(v/v)。发酵液特征为盐度为200gNaCl/L；
(3)调节控温装置使温度控制在37.0℃，调节pH自动控制仪使pH值控制在7.2，调节气体流量计使曝气量控制在1.5L/min；
(4)发酵开始后计时，依次在发周期的6h、18h、30h、42h、54h、66h进行取样，检测菌液的细胞干重和H.mediterranei的胞内甜菜碱含量。
(5)在18h达到最大胞内甜菜碱产量，最大产量为188.27mg。
实施例2：
(1)将嗜盐古菌Haloferax mediterranei(ATCC 33500)接种在灭菌后的富集培养基中进行富集培养，培养时间为72h。富集培养基特征为盐度为200gNaCl/L；
(2)将富集培养物接种在发酵罐中，配备发酵用发酵液1L，控制富集培养物:发酵液＝1:20(v/v)。发酵液特征为盐度为300gNaCl/L；
(3)调节控温装置使温度控制在37.0℃，调节pH自动控制仪使pH值控制在7.2，调节气体流量计使曝气量控制在1.5L/min；
(4)调节静态磁场发生装置使磁场强度控制在50mT；
(5)发酵开始后计时，依次在发周期的6h、18h、30h、42h、54h、66h进行取样，检测菌液的细胞干重和H.mediterranei的胞内甜菜碱含量。
(6)在30h达到最大胞内甜菜碱产量，最大产量为441.20mg。
Claims (1)
1.一种促进嗜盐古菌Haloferax mediterranei合成甜菜碱的方法，其特征在于，该方法所用装置包括：
(1)发酵罐，(2)控温装置，(3)pH自动控制仪，(4)曝气泵，(5)气体流量计，(6)曝气头，(7)静态磁场发生装置；
装置连接流程为：将控温装置(2)安装在发酵罐(1)上；pH自动控制仪(3)与发酵罐(1)连接；将曝气泵(4)、气体流量计(5)、曝气头(6)通过导管连接进行曝气；将发酵罐(1)放置于静态磁场发生装置(7)内；
以Haloferax mediterranei为接种物进行发酵，步骤如下：
(1)将嗜盐古菌Haloferax mediterranei接种在灭菌后的富集培养基中进行富集培养，培养时间为72h；富集培养基盐度为150-200gNaCl/L；
(2)将富集培养物接种在发酵罐(1)中，控制富集培养物:发酵液体积比＝1:20；发酵液特征为盐度为250-300gNaCl/L；
(3)调节控温装置(2)使温度控制在35-39℃，调节pH自动控制仪(3)使pH值控制在6.9-7.3，调节气体流量计(5)使溶解氧控制在2mg/L以上并保持一致；
(4)调节静态磁场发生装置(7)使磁场强度控制在10-60mT，最优磁场强度是50mT；
(5)连续发酵18-30h；在甜菜碱细胞含量最大时停止发酵；收集细胞提取胞内甜菜碱。
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