WO2022062087A1 - 一种含木质素类生物质转化合成姜黄素的方法 - Google Patents
一种含木质素类生物质转化合成姜黄素的方法 Download PDFInfo
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- the fermentation medium in step (3) is soybean meal nutrient medium (g/L): enzymatically hydrolyzed soybean meal powder 50, corn steep liquor powder 30, glucose 10, FeSO4 0.1, MgSO4 0.5, phosphate buffer 50mM.
- Fig. 1 is curcumin and plant extract standard substance HPLC chromatogram
- the selected Bacillus subtilis in the present invention can be various subspecies and strains of Bacillus subtilis species, such as: subtilis (Bacillus subtilis subsp.subtilis), subspecies natto (Bacillus subtilis subsp.natto), subspecies desert ( Bacillus subtilis subsp.inaquosorum), endophytic subspecies (Bacillus subtilissubsp.endophyticus) spitz subspecies (Bacillus subtilis subsp.spizizenii) and other typical strains and atypical strains.
- subtilis Bacillus subtilis subsp.subtilis
- subspecies natto Bacillus subtilis subsp.natto
- subspecies desert Bacillus subtilis subsp.inaquosorum
- endophytic subspecies Bacillus subtilis subsp.endophyticus
- spitz subspecies Bacillus subtilis subsp.spiziz
- Depolymerization in the present invention means that the lignin structure is capable of producing monomeric aromatic compounds such as ferulic acid and p-coumaric acid.
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Abstract
Description
Claims (9)
- 草芽孢杆菌基因工程菌,建议的分类命名为枯草芽孢杆菌Bacillus subtilis,保藏号为:CGMCC No.20353。
- 一种利用权利要求1所述枯草芽孢杆菌基因工程菌转化含木质素类生物质合成姜黄素的方法,其特征在于按照下述步骤进行:(1)含有木质素生物材料的预处理;木质素生物材料为包括秸秆或者蔗渣木屑等农林废弃物生物质原料;所述预处理包括通过气爆、稀酸、稀碱法或者酶预处理;(2)工业木质素或者预处理后的预处理液进一步处理达到解聚木质素的目的,得到的解聚反应液pH调至中性备用;所述解聚木质素的方法包括热裂解、酶法、微波、金属催化剂、碱催化等方法;(3)将基因工程菌(保藏号:CGMCC No.20353)冻存管接种到含有卡那霉素(50μg.ml -1)的LB培养基中37℃活化预培养24h,然后按照1:10(V/V)的比例接种到发酵培养基,以解聚产物作为底物,配制发酵培养基,将预培养的枯草芽孢杆菌基因工程菌接种后在发酵罐中高密度发酵培养24h,培养温度37℃,通过NaOH和HCl调整pH在6.8-7.2,转速在100-300rpm之间,溶氧通过控制通气量设置不低于20%,培养过程中通过补料方式在葡萄糖低于5g/L的时候补充葡萄糖,在发酵OD值30以上时停止发酵,然后将发酵液置换为以木质素解聚产物为单碳源的MM63培养基,在37℃继续培养24h,pH控制在6.8-7.2,以HPLC检测姜黄素合成量;(4)将发酵液离心,固液分离后收集上清液,上清液加入有机溶剂萃取,然后从分离的有机溶剂相中进行回收脂溶性的姜黄素;(5)回收的姜黄素混合液经过硅胶柱、大孔树脂或者离子交换柱进行纯化,然后通过结晶重结晶得到纯化后的姜黄素。
- 根据权利要求2所述枯草芽孢杆菌基因工程菌转化含木质素类生物质合成姜黄素的方法,其特征在于其中步骤(2)所述的热裂解:将5-20%(v/v)的木质素水溶液在反应釜中以250-450℃的温度热裂解10-60分钟;压力控制在30兆帕以内。
- 根据权利要求2所述枯草芽孢杆菌基因工程菌转化含木质素类生物质合成姜黄素的方法,其特征在于其中步骤(2)所述的酶法:将漆酶或者 染料脱色酶以0.1g/L的浓度溶解有乙酸盐缓冲液(pH 4)中,然后将漆酶溶液以1:1(v/v)比例与5-20%的木质素溶液混合均匀,在30℃下反应6-24小时,采用漆酶时可以加入0.01-0.05mmol的2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS),采用染料脱色酶时加入0.05-0.2mmol的H 2O 2。
- 根据权利要求2所述枯草芽孢杆菌基因工程菌转化含木质素类生物质合成姜黄素的方法,其特征在于其中步骤(2)所述的金属催化剂裂解方法:以金属催化剂(镍、钛、锌等)为催化剂,在10%的木质素溶液体系中100-300℃下处理0.5-2h;将木质素以1-20%(g/L)的浓度溶解于2-10%NaCl溶液当中,然后在反应釜中60-200℃反应1-3小时后收集滤液备用。
- 根据权利要求2所述枯草芽孢杆菌基因工程菌转化含木质素类生物质合成姜黄素的方法,其特征在于其中步骤(2)所述的碱水解方法:将不高于10%(g/v)的氢氧化钠溶液加入木质素(溶液中木质素含量不超过20g/L),在80-120℃下处理1-12h。
- 根据权利要求2所述枯草芽孢杆菌基因工程菌转化含木质素类生物质合成姜黄素的方法,其特征在于其中步骤(3)所述预培养培养基为LB培养基(g/L):胰蛋白胨10g酵母提取物5g NaCl 10g。
- 根据权利要求2所述枯草芽孢杆菌基因工程菌转化含木质素类生物质合成姜黄素的方法,其特征在于其中步骤(3)所述发酵培养基为豆粕营养培养基(g/L):酶解豆粕粉50,玉米浆粉30,葡萄糖10,FeSO 40.1,Mg SO 40.5,磷酸盐缓冲液50mM。
- 根据权利要求2所述枯草芽孢杆菌基因工程菌转化含木质素类生物质合成姜黄素的方法,其特征在于其中步骤(3)所述转化培养基为MM63培养基:100mM KH 2PO 4,75mM KOH,15mM(NH4) 2SO 4,1mM MgSO 4,3.9μM FeSO 4;木质素解聚产物含量1-20g/L。
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CN103321074A (zh) * | 2013-04-03 | 2013-09-25 | 中国热带农业科学院热带生物技术研究所 | 一种用枯草芽孢杆菌工程菌降解植物的木质素的方法 |
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DATABASE Nucleotide 11 May 2015 (2015-05-11), ANONYMOUS: "Synthetic construct curcuminoid synthase (cus) gene, complete cds", XP055916617, retrieved from Genbank Database accession no. KP284565 * |
DATABASE Nucleotide 7 April 2019 (2019-04-07), ANONYMOUS: "Synthetic construct clone 4CL2 4-coumarate CoA ligase gene, complete cds", XP055916615, retrieved from Genbank Database accession no. MK035998 * |
ESPARAN VIDA, KRINGS ULRICH, STRUCH MARLENE, BERGER RALF: "A Three-Enzyme-System to Degrade Curcumin to Natural Vanillin", MOLECULES, vol. 20, no. 4, 1 January 2015 (2015-01-01), pages 6640 - 6653, XP055916619, DOI: 10.3390/molecules20046640 * |
HORINOUCHI SUEHARU: "Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms", THE JOURNAL OF ANTIBIOTICS, vol. 61, no. 12, 1 December 2008 (2008-12-01), London, pages 709 - 728, XP055916611, ISSN: 0021-8820, DOI: 10.1038/ja.2008.85 * |
LIU BIN;ZHAO YING;YU SI-LI;ZHAO GUANG-RONG: "Advances on Curcuminoids Production via Metabolic Engineering", FOOD AND FERMENTATION INDUSTRIES, vol. 42, no. 7, 25 June 2016 (2016-06-25), pages 254 - 260+268, XP055916610, DOI: 10.13995/j.cnki.11-1802/ts.201607042 * |
RODRIGUES J. L., PRATHER K. L. J., KLUSKENS L. D., RODRIGUES L. R.: "Heterologous Production of Curcuminoids", MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, vol. 79, no. 1, 1 March 2015 (2015-03-01), US , pages 39 - 60, XP055916621, ISSN: 1092-2172, DOI: 10.1128/MMBR.00031-14 * |
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