CN117603835A - 产6-羟基烟酸的杀鲑气单胞菌hd531及其应用 - Google Patents
产6-羟基烟酸的杀鲑气单胞菌hd531及其应用 Download PDFInfo
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Abstract
本发明属于涉及一种微生物发酵生产6‑羟基烟酸的杀鲑气单胞菌株HD531,其分类属弧菌属科(Vibrionaceae)气单胞菌属(Aeromomas)杀鲑气单胞菌(Aeromonas salmonicida),该菌株在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为:CGMCC No.7.525。本发明给出了利用该菌株发酵烟酸生产6‑羟基烟酸的方法,利用静息细胞法在初始烟酸浓度为10g/L的条件下,其在好氧条件下能显著地积累6‑羟基烟酸,最高达到70.16g/L,可适用于6‑羟基烟酸的工业化生产。
Description
技术领域
本发明涉及微生物发酵产酶技术领域,涉及到产6-羟基烟酸的杀鲑气单胞菌HD531及其应用。
背景技术
6-羟基烟酸又名6-羟基-吡啶-3-甲酸,是一种高价值的精细化工中间体,英文名为6-hydroxynicotinic acid,缩写为6-HNA。6-羟基烟酸具有醇式与酮式结构,难溶于酸,易溶于碱性溶液,是一种合成农药、医药和其他化学药品的重要中间体,已广泛应用于农药、染料、医疗美容及材料等行业。在农药方面,6-羟基烟酸可以用于生产以吡虫啉、吡虫清为代表的吡啶甲胺类农药,对植物真菌病害具有高效、低毒的抑制作用,也可以和氯反应生成一种医药活性成分5,6-二氯烟酸,其可以促进脂肪酶的降解功能,具有减脂作用,可用于制备减肥药。在化学方面,6-羟基烟酸还可以作为底物用于医药中间体的合成,如从6-羟基烟酸出发,以乙腈为溶剂,利用化学法逐步合成得到2-氯-5-氯甲基吡啶。在材料领域,因为6-羟基烟酸中具有富电子能力的N原子,从而具有电催化化学性质,可以用作电极材料,如利用循环伏安法,将6-羟基烟酸聚合至碳糊电极的表面,制备得到6-羟基烟酸膜修饰电极。
由于6-羟基烟酸广阔的开发前景,生产6-羟基烟酸已经成为国内外研究热点。但是,目前6-羟基烟酸很难从化学方法合成,有机合成生产6-羟基烟酸的方法主要以以富马酸或苹果酸为原料,反应过程中需要添加浓硫酸、浓盐酸等试剂,易引起环境污染并造成生产成本的相应增加。此方法副产物多,成本高,污染重。与传统的化学法相比,利用微生物中含有的烟酸脱氢酶生物催化烟酸生产6-羟基烟酸具有绿色友好,成本低,产量高等优点。
烟酸脱氢酶是一种能不同程度上作用于烟酸和各种烟酸类似物的羟化酶类。烟酸脱氢酶的研究是从烟酸代谢菌株中分离纯化烟酸脱氢酶开始的。已报道的烟酸脱氢酶大多来源于好氧的恶臭假单胞菌,例如Xu等人在2005年得到一株可有效催化烟酸生产6-羟基烟酸的菌株Pseudomonas putida NA-1;Luo等人于2010年从菌株Pseudomonas putida BK-1中分离得到烟酸脱氢酶;2017年Chen等人从腐鱼中分离鉴定出一株产烟酸脱氢酶的Pseudomonas putida H9,可以催化烟酸生成6-羟基烟酸;2021年Shang等人得到一株产烟酸脱氢酶的恶臭假单胞菌Pseudomonas putida S14。然而,目前已报道的多数烟酸脱氢酶产生菌仍存在酶活偏低、烟酸转化效率不高的问题,限制了其大规模工业应用。因此,筛选出一株高活性烟酸脱氢酶产生菌,以及对其进行功能表达及高效催化转化研究,对6-羟基烟酸的高效合成及工业化生产具有重要的现实意义。
发明内容
发明目的在于提供一种新型的可产烟酸脱氢酶的杀鲑气单胞菌HD531,该菌株可用于微生物发酵烟酸生产6-羟基烟酸。本发明还给出了利用该菌株发酵生产6-羟基烟酸的方法,该方法以廉价的烟酸为原料,可以高效率合成生产6-羟基烟酸。
产6-羟基烟酸的杀鲑气单胞菌HD531,其分类命名为Aeromonas salmonicida,已于2023年7月10日保藏于北京市朝阳区北辰西路1号院3号,中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.7.525。
菌株HD531,其分类属弧菌属科(Vibrionaceae)气单胞菌属(Aeromomas)杀鲑气单胞菌(Aeromonas salmonicida)。
利用所述菌株进行生长细胞生产6-羟基烟酸的方法,具体为:将菌株划线于固体培养基,于30℃,200rpm培养24h,挑取单菌至液体种子培养基,于30℃,200rpm培养24h,按1~5%的接种量接入液体发酵培养基,在好氧条件下于30℃发酵培养24h,利用高效液相色谱法(HPLC)检测产物6-羟基烟酸浓度。
利用所述菌株进行静息细胞生产6-羟基烟酸的方法,具体为:将菌株菌液先涂布划线到固体平板培养基,在好氧条件下于30℃培养24~36h;然后将培养好的菌株单菌落挑取至一级种子培养基,于30℃好氧培养12~24h;然后将培养好的一级种子按1~10%的接种量接入二级种子培养基中,于30℃好氧培养8~12h,8~12h后将培养好的二级种子按1~10%的接种量再接入液体发酵培养基,在好氧条件下于30℃发酵培养10~12h;发酵结束后,收集发酵液,离心收集菌体,加入1%烟酸转化液,使菌体浓缩20倍进行生物催化转化60~80h。
所述固体培养基组成为:胰蛋白胨17g/L,大豆蛋白胨3g/L,葡萄糖2.5g/L,磷酸氢二钾2.5g/L,氯化钠10g/L,琼脂粉20g/L,余量为水,pH为7.0;所述液体种子培养基的组成为:胰蛋白胨17g/L,大豆蛋白胨3g/L,葡萄糖2.5g/L,磷酸氢二钾2.5g/L,氯化钠10g/L,烟酸2g/L,余量为水,pH为7.0;所述液体发酵培养基的组成为:烟酸10~20g/L,酵母浸提物8~10g/L,蛋白胨8~10g/L,KH2PO4 0.5~1g/L,K2HPO4·3H2O 2~4g/L,余量为水,pH为7.0。
所述一级种子培养基和二级种子培养基的组成均为:烟酸1~2g/L,牛肉浸提物2~5g/L,蛋白胨8~10g/L,NaCl 0.5~1g/L,余量为水,pH为7.0;所述液体发酵培养基的组成与生长细胞法相同。
具体的,所述烟酸转化液为烟酸与缓冲液混合的不同浓度溶液,如100ml磷酸缓冲液加1g的烟酸就配成的是10g/L的烟酸转化液,100ml磷酸缓冲液添加2g的烟酸就是20g/L的烟酸转化液;所述缓冲液由NaH2PO4与Na2HPO4制成,pH为7.0;所述碳源氮源优选为酵母浸粉,牛肉浸提物,蛋白胨。
可通过本领域的常规方法来调节培养基的pH值,如添加盐酸,氢氧化钠等无机酸碱,所述缓冲液的pH值用其组成试剂进行调节,即NaH2PO4与Na2HPO4。所述培养基灭菌条件均为115~121℃,20min。
本发明所达到的有益效果是:
本发明可产烟酸脱氢酶的杀鲑气单胞菌HD531在好氧条件下可显著积累6-羟基烟酸60~70g/L,该方法绿色友好可持续,成本低,产量高,可实现工业化生产,具有较好的应用前景。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1为杀鲑气单胞菌HD531平板菌落照片(×10);
图2为杀鲑气单胞菌HD531扫描电镜照片(×10000);
图3为杀鲑气单胞菌HD531基于BLAST结果构建的进化树。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例
初筛
从河南省开封市不同地区的土壤中取样,取所得土壤加入100ml PBS溶液,于30℃,200rpm条件下震荡12h,取上清液,稀释成一系列浓度梯度:10-1、10-2、10-3。每个梯度取0.2ml涂布于平板分离培养基,平板分离培养基组成为:烟酸5g/L,酵母浸提物5g/L,琼脂20g/L,NaCl 1.0g/L,余量为水,pH为7.0。在好氧条件下于30℃培养48~72h。
待菌落长成后,将平板置于紫外照射下观察,挑取显亮色菌落接种至种子培养基中富集培养(菌落显色越亮,酶活越高),每个显色菌株接种一个摇瓶,种子培养基组成为蛋白胨10g/L、牛肉浸提物5.0g/L、NaCl 1.0g/L,余量为水,pH为7.0。
2、复筛
种子培养基在好氧条件下于30℃培养24h,转接至发酵培养基,发酵培养基组成为酵母浸提物10g/L、蛋白胨10g/L、烟酸2.7g/L、KH2PO4 1.0g/L、K2HPO4·3H2O 3.93g/L,余量为水,pH为7.0。于30℃培养24h。
取1.5ml发酵液经12000rpm离心5min,取上清液,稀释合适倍数后用高效液相色谱法(HPLC)测定6-羟基烟酸产量及烟酸剩余量。HPLC条件如下:色谱柱为CST Daiso C18(5μm,4.6×250mm),流动相为甲醇:水(pH=3.0)=20:80(v/v),检测器:RI检测器,UV检测器(检测波长260nm),柱温为30℃,流速为0.4ml/min,波长为260nm,进样量20μl。下表列出部分试验显色菌株所产6-羟基烟酸含量,36号菌株为所筛新型菌株杀鲑气单胞菌(Aeromonassalmonicida)。
表1部分试验菌株得产6-羟基烟酸情况
杀鲑气单胞菌(Aeromonas salmonicida)HD531的鉴定:
筛选得到的能有效积累6-羟基烟酸的新型菌株杀鲑气单胞菌HD531:该菌株为革兰氏阴性菌,细胞直,杆状,两端圆的,至球菌状,直径1.0~4.4微米,偶尔形成长达8微米的丝状体;单个、成对或成链;以极生鞭毛运动,一般单鞭毛;有些不运动。未知有休眠期。属有机化能菌,呼吸型和发酵型代谢;分解碳水化合物产酸或产酸和气(CO2和H2)
按《伯杰氏细菌鉴定手册(第八版)》进行生理生化特性鉴定(结果见表2)。根据革兰氏染色、在含有7.5%NaCl营养液中生长、丁二醇脱氢酶、由甘油产气、由葡萄糖产气、赖氨酸脱羧酶反应、分解半乳糖、分解甘露醇、分解阿拉伯糖等Aeromonas属的特征生理生化反应结果鉴定该菌株属于Aeromonas属。
同时委托上海生工生物有限公司对菌株进行16S RNA全序列测序。在NCBI网站上用BLAST检索GenBank中相关菌株的16S RNA基因序列(见表3),用MEGA 11.0软件构建进化树分析(见图3)。基于16S RNA序列分析和构建进化树分析得出HD531与Aeromonassalmonicida strain ODK2、Aeromonas salmonicida strain YTU1、Aeromonassalmonicida strain B52亲缘关系较近,菌株HD531可以确定为Aeromonas属,杀鲑气单胞菌(Aeromonas salmonicida)。
由上述鉴定结果可知,菌株HD531属弧菌属科(Vibrionaceae)气单胞菌属(Aeromomas)杀鲑气单胞菌(Aeromonas salmonicida)。该菌株在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为:7.525。
表2生理生化特性鉴定结果对照表
表3 16S RNA序列同源性(或相似性)比较
利用杀鲑气单胞菌(Aeromonas salmonicida)发酵生产6-羟基烟酸:
利用所述菌株进行生长细胞发酵催化烟酸生产6-羟基烟酸的方法,具体为:将菌株菌液先涂布划线到胰蛋白胨大豆肉汤固体培养基(TSA)(所述胰蛋白胨大豆肉汤固体培养基组成为:胰蛋白胨17g/L,大豆蛋白胨3g/L,葡萄糖2.5g/L,磷酸氢二钾2.5g/L,氯化钠10g/L,琼脂粉20g/L,余量为水,pH为7.0),在好氧条件下于30℃培养24h;然后将培养好的菌株单菌落挑取至胰蛋白胨大豆肉汤液体培养基(TSB)(所述胰蛋白胨大豆肉汤液体培养基组成为:胰蛋白胨17g/L,大豆蛋白胨3g/L,葡萄糖2.5g/L,磷酸氢二钾2.5g/L,氯化钠10g/L,烟酸2g/L,余量为水,pH为7.0),于30℃好氧培养24h;然后将培养好的种子液按2%的接种量接入发酵培养基中(所述发酵培养基组成为:烟酸10g/L,酵母浸提物10g/L,蛋白胨10g/L,KH2PO4 1g/L,K2HPO4·3H2O 3.93g/L,余量为水,pH为7.0),于30℃好氧培养24h。
利用高效液相色谱检测得6-羟基烟酸产量为3.71g/L。
利用所述菌株进行静息细胞发酵催化烟酸生产6-羟基烟酸的方法,具体为:将菌株菌液先涂布划线到TSA平板固体培养基,在好氧条件下于30℃培养24~36h;然后将培养好的菌株单菌落挑取至一级种子培养基,于30℃好氧培养12~24h;然后将培养好的一级种子按1~10%的接种量接入二级种子培养基中,于30℃好氧培养8~12h,8~12h后将培养好的二级种子按1~10%的接种量再接入含有10L液体发酵培养基的20L发酵罐中,在好氧条件下于30℃发酵培养10~12h;发酵结束后,收集发酵液,离心收集菌体,加入1%烟酸转化液,使菌体浓缩20倍进行生物催化转化60~80h。
所述烟酸转化液的组成为:20mM缓冲液100ml,烟酸10g/L,调pH至7.0。转化过程中通过补充烟酸使底物浓度维持在10g/L左右。
分别在转化第0h、、4h、6h、12h、16h、20h、24h、28h、36h、40h、44h、48h、52h、56h、60h、64h、68h、72h、76h测定转化液中6-羟基烟酸含量,实验结果见表4。转化76h时产物产量开始下降,结束转化。6-羟基烟酸含量最高达70.16g/L。
表4菌株HD531静息细胞发酵生产的6-羟基烟酸产量
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.产6-羟基烟酸的杀鲑气单胞菌HD531,其分类命名为Aeromonas salmonicida,已于2023年7月10日保藏于北京市朝阳区北辰西路1号院3号,中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.7.525。
2.权利要求1所述的产6-羟基烟酸的杀鲑气单胞菌HD531在产中6-羟基烟酸的应用。
3.权利要求1所述的产6-羟基烟酸的杀鲑气单胞菌HD531的筛选方法,其特征在于,将适量土样用PBS溶液混合均匀,过滤后取上清液稀释涂布于平板分离培养基,于30℃好氧培养24~36h,取紫外照射下可显色的菌株进行发酵筛选;所述平板分离培养基的组成为:烟酸2~5g/L,酵母浸提物2~5g/L,琼脂15~20g/L,NaCl 0.5~1g/L,余量为水,pH为7.0;
发酵筛选时将菌株划线于固体培养基,于30℃,200rpm培养24h,挑取单菌至液体种子培养基,于30℃,200rpm培养24h,按1~5%的接种量接入液体发酵培养基,在好氧条件下于30℃发酵培养24h,利用高效液相色谱法检测产物6-羟基烟酸浓度。
4.产6-羟基烟酸的杀鲑气单胞菌HD531液态发酵产6-羟基烟酸的方法,包括如下步骤:
将菌株划线于固体培养基,于30℃,200rpm培养24h,挑取单菌至液体种子培养基,于30℃,200rpm培养24h,按1~5%的接种量接入液体发酵培养基,在好氧条件下于30℃发酵培养24h,利用高效液相色谱法(HPLC)检测产物6-羟基烟酸浓度。
5.如权利要求4所述的方法,其特征在于,
所述固体培养基组成为:胰蛋白胨17g/L,大豆蛋白胨3g/L,葡萄糖2.5g/L,磷酸氢二钾2.5g/L,氯化钠10g/L,琼脂粉20g/L,余量为水,pH为7.0;
所述液体种子培养基的组成为:胰蛋白胨17g/L,大豆蛋白胨3g/L,葡萄糖2.5g/L,磷酸氢二钾2.5g/L,氯化钠10g/L,烟酸2g/L,余量为水,pH为7.0;所述液体发酵培养基的组成为:烟酸10~20g/L,酵母浸提物8~10g/L,蛋白胨8~10g/L,KH2PO4 0.5~1g/L,K2HPO4·3H2O 2~4g/L,余量为水,pH为7.0。
6.产6-羟基烟酸的杀鲑气单胞菌HD531进行静息细胞产6-羟基烟酸的方法,包括如下步骤:
S1、将菌株菌液先涂布划线到固体平板培养基,在好氧条件下于30℃培养24~36h;然后将培养好的菌株单菌落挑取至一级种子培养基,于30℃好氧培养12~24h;然后将培养好的一级种子按1~10%的接种量接入二级种子培养基中,于30℃好氧培养8~12h,8~12h后将培养好的二级种子按1~10%的接种量再接入液体发酵培养基;
S2、将产6-羟基烟酸的菌株HD531好氧条件下发酵10~12h,然后将发酵液离心收集菌体;
S3、向收集到的菌体内加入原始发酵液体积1/20的1%烟酸转化液,使菌体浓缩后进行转化,每隔4小时补加底物烟酸,使体系烟酸浓度稳定在10g/L;
S4、转化一定时间后,即得含有6-羟基烟酸的转化液。
7.如权利要求6所述的方法,其特征在于,所述1%烟酸转化液的组成为:20mM缓冲液100ml,烟酸10g/L,调pH至7.0。
8.如权利要求6所述的方法,其特征在于,
所述固体培养基组成为:胰蛋白胨17g/L,大豆蛋白胨3g/L,葡萄糖2.5g/L,磷酸氢二钾2.5g/L,氯化钠10g/L,琼脂粉20g/L,余量为水,pH为7.0;
所述液体种子培养基的组成为:胰蛋白胨17g/L,大豆蛋白胨3g/L,葡萄糖2.5g/L,磷酸氢二钾2.5g/L,氯化钠10g/L,烟酸2g/L,余量为水,pH为7.0;所述液体发酵培养基的组成为:烟酸10~20g/L,酵母浸提物8~10g/L,蛋白胨8~10g/L,KH2PO4 0.5~1g/L,K2HPO4·3H2O 2~4g/L,余量为水,pH为7.0。
9.如权利要求6所述的方法,其特征在于,一级种子培养基和二级种子培养基的组成均为:烟酸1~2g/L,牛肉浸提物2~5g/L,蛋白胨8~10g/L,NaCl 0.5~1g/L,余量为水,pH为7.0。
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