CN117247848B - 一株产腈水解酶的菌株、发酵方法及应用 - Google Patents
一株产腈水解酶的菌株、发酵方法及应用 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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Abstract
本发明属于生物技术领域,特别涉及一株产腈水解酶的菌株、发酵方法及应用,所述菌株分类命名为白地霉Ytz23,已于2023年9月19日保存于中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号:CGMCC No.28486。本发明所提供的白地霉Ytz23遗传稳定性好,培养基成分简单原料成本低,产生的腈水解酶酶活力高,对部分脂肪腈具有较高的水解活性。且白地霉腈水解酶对2‑氨基‑4(羟基甲基磷酰基)‑丁腈无光学选择性,无差别水解合成D/L体草铵膦,避免传统酸水解工艺带来的大量副产物盐,减轻后处理工艺的除盐压力,工业应用具有优势。
Description
技术领域
本发明属于生物技术领域,特别涉及一株生产腈水解酶的菌株、发酵方法及应用。
背景技术
草铵膦,又名2-氨基-4-(羟基甲基膦酰基)丁酸铵,属于有机膦类部分内吸除草剂,通过作用于植物体谷氨酰胺合成酶,抑制L-谷氨酰胺合成。随着转基因农作物技术的高速发展以及世界范围内对转基因抗草铵膦作物的大力推广,市场上草铵膦的使用需求一直保持较高速的增长,同时由于抗草甘膦杂草的不断发生和发展,带来的双草复配技术的推广,给草铵膦带来了更大的市场空间。目前国内企业基本都采用Strecke法生产草铵膦,2-氨基-4(羟基甲基磷酰基)-丁腈是生产过程中的关键中间体,经强酸或强碱水解后获得草铵膦,伴随有大量盐类形成,为后续的分离纯化带来困难,也造成一定的环境污染。腈水解酶( EC 3.5.5.1)直接催化2-氨基-4(羟基甲基磷酰基)-丁腈生成草铵膦,反应条件温和、成本低、有效避免化学酸法水解腈带来的副产物盐,减轻环境压力,是合成草铵膦的有效酶源之一。
发明内容
本发明要解决的技术问题在于提供从土壤中分离得到的一株高效生产腈水解酶的菌株,并应用该菌株产生的腈水解酶制备草铵膦的方法。本发明所提供的产腈水解酶的菌株是从烟台海底淤泥中分离得到,这是一株产腈水解酶活力较高的菌株,培养简单、遗传稳定性强,可以直接用于酶法制备草铵膦。
本发明的技术方案如下:
一株产腈水解酶的菌株,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2023年9月19日,分类命名为白地霉(Geotrichum candidum),保藏编号为:CGMCC No. 28486。
进一步,该菌株的18S rRNA基因序列如SEQ.NO.1。
本发明还提供所述菌株发酵生产的腈水解酶的方法,发酵温度为28~37℃,pH7.0。
进一步,所述的发酵培养基的成分及终浓度为:甘油 15 g/L,麦芽浸粉 5g/L,酵母浸粉 5g/L,KH2PO4 2 g/L,MgSO4 0.2 g/L,NaCl 1 g/L,CoCl2 0.1mg/L,FeSO4 2 mg/L,MnSO4 2 mg/L,泛酸钙2 mg/L,叶酸1mg/L,维生素B11mg/L,腈化合物 0.2g/L,琼脂 20g/L,pH6.0。
本发明还提供所述菌株的应用,所述应用为利用所述菌株发酵产生的全细胞酶以2-氨基-4(羟基甲基磷酰基)-丁腈为底物水解合成D/L体草铵膦,水解反应温度为30-40℃,水解反应底物浓度为5-10g/L,催化液中菌体细胞与2-氨基-4(羟基甲基磷酰基)-丁腈质量比为5:1-10:1。
本发明与现有技术相比的有益效果:
1、本发明所提供的白地霉Ytz23遗传稳定性好,培养基成分简单原料成本低,产生的腈水解酶酶活力高,对部分脂肪腈具有较高的水解活性,工业应用具有优势。
2、白地霉腈水解酶对2-氨基-4(羟基甲基磷酰基)-丁腈无光学选择性,无差别水解合成D/L体草铵膦,转化率在84.8%以上。所述方法避免了传统酸水解工艺带来的大量副产物盐,减轻后处理工艺的除盐压力。
具体实施方式
为阐明对本项发明特征的理解,下面结合一些非限定性的实施实例对本发明做进一步阐述。
下述实施例中对酶活测定:酶反应体系:pH 7.0、50 mmol/L磷酸盐缓冲液2.0 mL、发酵液0.1 mL、2-氨基-4(羟基甲基磷酰基)-丁腈(200 g/L)0.4 mL,反应15min,按体积比1∶1加入10%三氯乙酸溶液,4 ℃放置终止反应, HPLC检测草铵膦含量。样品采用邻苯二甲醛衍生:手性液相色谱条件为C18色谱柱(250 mm×4.6 mm, 10 μm),柱温30℃;检测波长:338nm;流动相:0.376%乙酸钠:乙腈(95:5,v/v)流速:0.85 mL/min。手性分析结果用ee值表示,ee值为([S]-[R]/[S]+[R])*100%。酶活力定义:在pH 7.0、温度37 ℃的条件下,每毫升发酵液每l min转化生成1μmol 草铵膦所需的酶量为1个酶活力单位U。
实施例1菌株的分离与鉴定
(1)菌株分离:样本采集自山东烟台近海岸海底淤泥。称取1g样本加入装有100mL无菌水的摇瓶中摇匀,梯度稀释后涂布于筛选平板上(筛选培养基:甘油 15,KH2PO4 2,MgSO4 0.2,NaCl 1,微量元素母液2mL,维生素母液 2 mL,2-氨基-4(羟基甲基磷酰基)-丁腈0.2 g/L,琼脂 20g/L,pH6.0。微量元素母液(g/L):CoCl2 0.28,ZnSO4 7,MnSO4 1,CuSO40.48,FeSO4 1.6,钼酸钠 0.34。维生素母液(g/L):生物素 0.05,泛酸钙 1,叶酸 5, 肌醇25,核黄素 0.1,VB1 1,对氨基苯甲酸钠 0.2。),于30 ℃培养箱中培养48 h,挑选单菌落,并划线分离纯化,将划线后长出的单菌落接入摇瓶发酵培养基(甘油 15 g/L,麦芽浸粉5g/L,酵母浸粉 5g/L,KH2PO4 2 g/L,MgSO4 0.2 g/L,NaCl 1 g/L,CoCl2 0.1mg/L,FeSO4 2mg/L,MnSO4 2 mg/L,泛酸钙2 mg/L,叶酸1mg/L,维生素B11mg/L,2-氨基-4(羟基甲基磷酰基)-丁腈 0.2g/L, pH6.0,于115℃高压蒸汽灭菌20min)中32 ℃培养48h,8000r/min离心10min收集菌体,按1%加量加入含2-氨基-4(羟基甲基磷酰基)-丁腈的催化体系30℃转化6h,采用Berthelot法测定催化产生的铵离子浓度,筛选到一株高活性腈水解酶产生菌株。
(2)菌株鉴定:该菌株在保藏培养基上菌落呈圆形,较薄,正面颜色为白色,菌丝质地为绒毛状兼粉状,放射线状,边缘色浅;通过显微镜观察,孢子呈圆柱或长筒形、两端多圆形、少数平切。
经DNA提取、PCR扩增和测序获得的18S rRNA 基因序列长度为379 bp,在GenBank上进行BLAST比对,得出该菌株与Geotrichum candidum(KT828737.1)碱基相似性达98.3%,将Ytz23菌株鉴定为白地霉(Geotrichum candidum)。18S rRNA序列信息如下:
ttttttcttcgggttatgatatggttaagtttagcgggtaatcctacttgatctgaggttgaatagtgttgtttttcaaacgaatttgattcgaaattttagaaaagcaatgcaattccaagagagaaacaacgctcaaacaagtatactttgggggataccccaaagtgcaatgtgcgttcaaaaactgatgattcacttctgcaattcacaagaaatatcgcgtttcgctgcgttcttcatcgatacgagaaccaagagatccattgttaaaagttttgattatttttgttttgactataaaattatggtttgctgggtaaatttcacaaatatttataattcttaaggatccttccgcaggttcacctacggaag。
(3)腈水解酶底物特异性:考察了白地霉Ytz23腈水解酶的底物特异性。由表2中可见,能够较好地水解各种脂肪腈化合物,尤其是对丙烯腈水解能力最强,但对芳香腈水解能力较差。
表2 底物特异性
。
( 4)遗传稳定性:采用划线法将白地霉Ytz23在固体培养基上连续传代20次,测定菌体形态、生长速度及转化对2-氨基-4(羟基甲基磷酰基)-丁腈的速度和转化率,与原代菌株无明显差异,遗传稳定性良好。
(5)菌种保藏:本项发明所述的白地霉Ytz23,已于2023年9月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号:CGMCC No. 28486,保藏单位地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
实施例2 腈水解酶发酵
(1)菌株活化:挑取菌种划线接种至活化培养基:葡萄糖 5 g/L,麦芽浸粉 5 g/L,酵母浸粉 5 g/L,琼脂 20 g/L,pH6.0,121℃高压蒸汽灭菌20min;划线接种于恒温培养箱28℃培养24h;也可以为24-48h中间的任一时间。
(2)液体种子制备:挑取1环活化菌株接种于装有50mL种子培养基的500mL三角瓶中,液体种子培养基组分:葡萄糖 5 g/L,麦芽浸粉 5 g/L,酵母浸粉 5 g/L,pH6.0,121℃高压蒸汽灭菌20min;八层纱布封口,于28℃, 200r/min摇床振荡培养18h,得液体种子。
(3)液体发酵:按5%接种量向发酵培养基接入步骤2制备的液体种子,装液量50mL/500 mL三角瓶,转速220 r/min,pH7.0,发酵温度28℃,振荡培养48h后测定酶活为1.6U/mL,8000 r/min离心20 min收集菌体细胞备用。
优选地,所述的发酵培养基的制备方法:甘油 15 g/L,麦芽浸粉 5g/L,酵母浸粉5g/L,KH2PO4 2 g/L,MgSO4 0.2 g/L, NaCl 1 g/L,CoCl2 0.1mg/L,FeSO4 2 mg/L,MnSO4 2mg/L,泛酸钙2 mg/L,叶酸1mg/L,维生素B11mg/L,2-氨基-4(羟基甲基磷酰基)-丁腈 0.2g/L, pH6.0,于115℃高压蒸汽灭菌20min。
实施例3 腈水解酶发酵
(1)菌株活化:挑取菌种划线接种至活化培养基:葡萄糖 5 g/L,麦芽浸粉 5 g/L,酵母浸粉 5 g/L,琼脂 20 g/L,pH6.0,121℃高压蒸汽灭菌20min;划线接种于恒温培养箱28℃培养48h;
(2)液体种子制备:挑取1环活化菌株接种于装有50mL种子培养基的500mL三角瓶中,液体种子培养基组分:葡萄糖 5 g/L,麦芽浸粉 5 g/L,酵母浸粉 5 g/L,pH6.0,121℃高压蒸汽灭菌20min;八层纱布封口,于28℃, 200r/min摇床振荡培养18h,得液体种子;
(3)液体发酵:按5%接种量向组成成分为甘油 15 g/L,麦芽浸粉 5g/L,酵母浸粉5g/L,KH2PO4 2 g/L,MgSO4 0.2 g/L,NaCl 1 g/L,CoCl2 0.1mg/L,FeSO4 2 mg/L,MnSO4 2mg/L,泛酸钙2 mg/L,叶酸1mg/L,维生素B11mg/L,2-氨基-4(羟基甲基磷酰基)-丁腈 0.2g/L,pH6.0,115℃高压蒸汽灭菌20min制备的发酵培养基接入液体种子,装液量 50mL/500 mL三角瓶,转速220 r/min,pH7.0,发酵温度37℃,振荡培养72h后测定酶活为2.1U/mL,8000r/min离心20 min收集菌体细胞备用。
实施例4 催化合成草铵膦
50mL三角瓶中循序加入10mL 50mM磷酸钠缓冲液(pH7.0)、0.1g的2-氨基-4(羟基甲基磷酰基)-丁腈、实施例2收集的菌体细胞0.5g,50mM磷酸钠缓冲液(pH7.0)定容至20mL,转速120 r/min,温度30℃反应2h,草铵膦浓度为5.9g/L,转化率为96.7%,ee%为0。
实施例5 催化合成草铵膦
50mL三角瓶中循序加入10mL50mM磷酸钠缓冲液(pH7.0)、0.16g的2-氨基-4(羟基甲基磷酰基)-丁腈、实施例3收集的菌体细胞1g,50mM磷酸钠缓冲液(pH7.0)定容至20mL,转速120 r/min,温度35℃反应5h,草铵膦浓度为8.3 g/L,转化率为84.8%,ee%为0。
实施例6 催化合成草铵膦
50mL三角瓶中循序加入10mL50mM磷酸钠缓冲液(pH7.0)、0.2g的2-氨基-4(羟基甲基磷酰基)-丁腈、实施例3收集的菌体细胞2g,50mM磷酸钠缓冲液(pH7.5)定容至20mL,转速120 r/min,温度40℃反应4h,草铵膦浓度为11.9g/L,转化率为97.5% ,ee%为0。
Claims (4)
1.一株产腈水解酶的菌株,其特征在于,所述菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2023年9月19日,保藏地址为北京市朝阳区北辰西路1号院3号,分类命名为白地霉(Georichum candidum),保藏编号为:CGMCC No. 28486。
2.利用权利要求1所述菌株发酵生产腈水解酶的方法,其特征在于,发酵温度为28~37℃,pH7.0。
3.根据权利要求2所述的发酵生产腈水解酶的方法,其特征在于,发酵培养基的成分及终浓度为:甘油 15 g/L,麦芽浸粉 5g/L,酵母浸粉 5g/L,KH2PO4 2 g/L,MgSO4 0.2 g/L,NaCl 1 g/L,CoCl2 0.1mg/L,FeSO4 2 mg/L,MnSO4 2 mg/L,泛酸钙2 mg/L,叶酸1mg/L,维生素B11mg/L,腈化合物 0.2g/L,pH6.0;
所述的腈化合物为2-氨基-4(羟基甲基磷酰基)- 丁腈、2-氨基-4-甲硫基丁腈、丙烯腈、3-氰基吡啶、苯乙腈中的一种。
4.权利要求1所述菌株的应用,其特征在于,所述应用为利用权利要求1所述菌株发酵产生的全细胞酶以2-氨基-4(羟基甲基磷酰基)-丁腈为底物水解合成D/L体草铵膦,反应温度为30-40℃,反应底物浓度为5-10g/L,催化液中菌体细胞与2-氨基-4(羟基甲基磷酰基)-丁腈质量比为5:1-10:1。
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