CN109609388A - 一种黑曲霉及其应用 - Google Patents
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Abstract
一种黑曲霉及其应用,属于生物工程技术领域。本发明一方面提供了一种黑曲霉(Aspergillus niger)WH‑2,该菌株于2018年12月03日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为CGMCC No.16799,保藏单位地址为:北京市朝阳区北辰西路1号院3号;本发明另一方面提供了该黑曲霉在制备L(+)‑酒石酸或其盐中的应用,为获得L(+)‑酒石酸或其盐提供了新的方法。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种黑曲霉及其应用。
背景技术
酒石酸,一种α-羧酸,又名2,3-二羟基琥珀酸或2,3-二羟基丁二酸。1769年,瑞典化学家Carl Wilhelm Scheele最早在葡萄酒的下脚料粗酒石中发现了L(+)-酒石酸的存在,故名“酒石酸”。L(+)-酒石酸广泛存在于自然界中,故又被称为“天然酒石酸”,特别是罗望子果和葡萄中含量较高,是重要的食品乳化剂、饮料酸味剂、医药拆分剂、石膏缓凝剂、印染防染剂、照相显影剂、金属抛光剂,广泛应用于食品工业、医药化工及建筑业中。微生物转化法是目前L(+)-酒石酸工业化生产的主流方法,即以顺酐为原料,通过水解和环氧化反应变为顺式环氧琥珀酸或其盐,然后利用含有顺式环氧琥珀酸水解酶的微生物,生物催化顺式环氧琥珀酸或其盐生成L(+)-酒石酸或其盐。
目前已报道的产L(+)-酒石酸的菌种有:诺卡氏菌属(Nocardia)、棒杆菌属(Coryncbacterium)、红球菌属(Rhodococcus)、根瘤菌属(Rhizobium)、假单胞菌属(Pseudomonas)、无色杆菌属(Achromobacter)、醋酸杆菌属(Acetobacter)、土壤杆菌属(Agrobacterium)、产碱杆菌属(Alcaligenes)、不动杆菌属(Acinetobacter)、克雷伯氏菌属(Klebsiella)和双头菌(Labrys)。这些已报道的生产L(+)-酒石酸的菌种均为细菌,未见利用真菌生产L(+)-酒石酸的报道。
发明内容
针对现有技术存在的问题,本发明的目的在于设计提供一种黑曲霉及其应用的技术方案。
所述的一种黑曲霉(Aspergillus niger)WH-2,该菌株于2018年12月03日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为CGMCC No.16799,建议的分类命名为:黑曲霉Aspergillus niger,保藏单位地址为:北京市朝阳区北辰西路1号院3号。
所述的一种黑曲霉WH-2在制备L(+)-酒石酸或其盐中的应用。
所述的应用,其特征在于包括以下步骤:
1)在斜面培养基中培养黑曲霉WH-2,28℃培养3~5天至长出浓密孢子,加入孢子悬浮液,刮取孢子,使孢子充分被洗下,用无菌的三层擦镜纸过滤除去菌丝体;
2)过滤后的上述孢子液按3×106个/mL浓度接种至种子培养基中,28℃振荡培养24~36h,得到黑曲霉菌株种子液;
3)取上述黑曲霉菌株种子液转接至产酶培养基中,28℃振荡培养2~3天,获得相应的黑曲霉细胞和发酵液,然后分别将得到的黑曲霉细胞和发酵液加入到顺式环氧琥珀酸或其盐溶液中,30℃振荡培养,转化3天,得到细胞转化液和发酵液转化液;
4)向两种转化液中分别加入过量的CaCl2,得到酒石酸钙沉淀,过滤并用蒸馏水冲洗酒石酸钙沉淀,再用硫酸酸解酒石酸钙沉淀,然后过滤后得到的酸解液经过阴阳离子交换精制、浓缩、结晶、烘干后得到L(+)-酒石酸或其盐的成品。
所述的应用,其特征在于所述的孢子悬浮液:称取0.2g的Tween80和1.8g的NaCl,再加入200ml蒸馏水,混合均匀,121℃高压灭菌20min,放4℃冰箱备用;所述的斜面培养基为PDA培养基;所述的种子培养基为PDB培养基;所述的产酶培养基为PDB无机盐培养基。
所述的应用,其特征在于所述的顺式环氧琥珀酸盐选用顺式环氧琥珀酸钠或顺式环氧琥珀酸钾。
本发明的发明人经过长期和深入的研究,从田园土中分离获得了一种新的微生物菌株,该新的菌株经鉴定为黑曲霉(Aspergillus niger)。本发明的黑曲霉菌株及其发酵液可通过水解顺式环氧琥珀酸或其盐生成L(+)-酒石酸或其盐。从而为获得L(+)-酒石酸或其盐提供了新的方法。
具体实施方式
本发明结合实施例作进一步的说明。
在以下实施例中,所用的孢子悬浮液为:称取0.2g的Tween80和1.8g的NaCl,再加入200ml蒸馏水,混合均匀,121℃高压灭菌20min,放4℃冰箱备用。
所用的各种培养基的组成如下:
(1)平板筛选培养基的配置方法为:称取3g硝酸钠、1g磷酸氢二钾、0.5g七水硫酸镁、0.5g氯化钾、0.01g硫酸亚铁、30g蔗糖、5g顺式环氧琥珀酸钠,20g琼脂,用蒸馏水补至1000ml,121℃高压灭菌20min。
(2)斜面培养基的组成为自制PDA培养基,配制方法为:称取200g去皮的新鲜土豆,将土豆切成小块放入锅中,加水1000ml,加热至沸腾,保持30min。再用双层纱布趁热在量杯上过滤,留下滤液。加入20g葡萄糖和15~20g的琼脂,并将滤液补充至1000ml,121℃高压灭菌20min。灭菌完成后,在紫外线灭菌过的超净台上倒入已灭菌的茄形瓶,待茄形瓶中斜面培养基冷却凝固后,将其密封并室内倒置放一夜。若斜面培养基上没有长菌,将其放入4℃冰箱备用。
(3)种子培养基的组成为自制PDB培养基,配制方法为:称取200g去皮的新鲜土豆,将土豆切成小块放入锅中,加水1000ml,加热至沸腾,保持30min。再用双层纱布趁热在量杯上过滤,留下滤液。加入10g葡萄糖,并将滤液补充至1000ml,121℃高压灭菌20min。
(4)产酶培养基的组成为自制PDB无机盐培养基,配制方法为:称取200g去皮的新鲜土豆,将土豆切成小块放入锅中,加水1000ml,加热至沸腾,保持30min。再用双层纱布趁热在量杯上过滤,留下滤液。加入10g葡萄糖,1.4g硝酸铵,20g酵母粉,1g三水磷酸氢二钾,pH6.0,并将滤液补充至1000ml,121℃高压灭菌20min。
实施例1:黑曲霉(Aspergillus niger)WH-2的筛选
于浙江省杭州市采集有果蔬腐烂的田园土,称取1g土样,迅速倒入装有玻璃珠的无菌生理盐水三角瓶中,混匀,然后用移液管分别稀释到10-2、10-3、10-4、10-5倍。分别吸取10-2、10-3、10-4、10-5倍稀释液注入到平板筛选培养基上,均匀涂布后将培养皿倒置,于28℃恒温培养箱中,培养2~5天。挑取平板上生长的单菌落接种于装有50ml产酶培养基的250ml三角瓶中,28℃、180rpm振荡培养2~3天,分别收集菌体和发酵液。在收集的细胞和发酵液中分别加入10ml1M pH8.0顺式环氧琥珀酸钠悬浮,30℃振荡反应3天后,用偏钒酸铵显色法(刘叶青,严文康,周文龙,等.酒石酸的比色测定法.工业微生物,1983,13:32-37.)鉴定反应液中有无酒石酸产生。
本发明在进行大量筛选后,得到了一株具有将顺式环氧琥珀酸或其盐水解为L(+)-酒石酸或其盐特性的菌株,将其保存于斜面培养基中,经CGMCC证明成活。该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(CGMCC),保藏登记号为CGMCCNo.16799,命名为黑曲霉(Aspergillus niger)WH-2,保藏日:2018年12月03日,保藏单位地址:北京市朝阳区北辰西路1号院中国科学院微生物研究所(邮编:100101)。
实施例2:黑曲霉(Aspergillus niger)WH-2的鉴定
步骤1:菌的形态学鉴定
对分离得到的菌株,按照《真菌鉴定手册》(魏景超主编,上海科学技术出版社,1979)的方法,采用PDA平板、显微镜及扫描电镜对菌落、菌丝、孢子等结构进行观察。结果显示,菌株在PDA培养基上30℃培养3天,菌落呈圆形,表面结构呈颗粒状,孢子为黑色或黑褐色,分生孢子为球形,菌丝发达。参照《真菌鉴定手册》,筛选菌株初步确定为黑曲霉(Aspergillus niger)。
步骤2:菌的分子生物学鉴定
使用真菌基因组试剂盒(购自Takara,Code No.9765)提取黑曲霉(Aspergillusniger)WH-2CGMCC No.16799的基因组DNA。
使用Fungi Identification PCR Kit(购自Takara,Code No.RR178),利用试剂盒自带的rDNA的保守序列为引物,PCR扩增未知真菌的核糖rDNA内部转录间隔区(ITS区域),测序后与GenBank中已知的序列进行同源性比较,将未知真菌鉴定到属或种。
PCR反应体系为:步骤2所得的模板DNA 50~100ng,PCR Premix 25μl,Forwardprimer(20pmol/μl)0.5μl,Reverse primer(20pmol/μl)0.5μl,dH2O补足总体积50μl。使用1%的琼脂糖凝胶电泳进行PCR产物的验证,一般ITS区长度约为300~1000bp(碱基对)不等。
PCR反应条件如下所示:
使用琼脂糖凝胶DNA回收试剂盒(购于Sangon公司,Code No.518131)切胶回收目的片段,并以Seq Reverse Primer和Seq Forward Primer为引物进行DNA测序(Sangon公司)。测序结果显示,黑曲霉(Aspergillus niger)WH-2CGMCC No.16799的ITS核苷酸序列如SEQ ID NO:1所示,即本发明的黑曲霉具有SEQ ID NO:1所示的ITS序列。
将上述实施例1中得到的具有SEQ ID NO:1所示的ITS核苷酸序列,用BLAST程序在NCBI数据库中进行比对分析,其比对结果如表1所示,属于黑曲霉(Aspergillus niger)。
根据上述形态学和分子生物学的鉴定结果,所测试的菌株属于黑曲霉(Aspergillus niger)。
表1黑曲霉(Aspergillus niger)WH-2CGMCC No.16799的ITS序列的BLAST比对结果
实施例3:利用顺式环氧琥珀酸钾和黑曲霉(Aspergillus niger)WH-2CGMCCNo.16799生产L(+)-酒石酸
先在茄子瓶中用斜面培养基培养黑曲霉(Aspergillus niger)WH-2CGMCCNo.16799,28℃培养3~5天至长出浓密孢子,加入5ml孢子悬浮液,用接种铲刮取孢子,使孢子充分被洗下,用无菌的三层擦镜纸过滤除去菌丝体。过滤后的上述孢子液经稀释后用血球计数板计数,按3x106个mL-1的浓度接种孢子至装有50ml的种子培养基的250ml锥形瓶中,28℃180rpm振荡培养24~36h,得到所述的黑曲霉菌株种子液。取上述20ml黑曲霉的种子液接种装有200ml产酶培养基的1000ml锥形瓶中,28℃180rpm振荡培养2~3天,获得相应的黑曲霉细胞和发酵液,然后分别将得到的黑曲霉细胞加入到200ml的浓度为1M的顺式环氧琥珀钾中,30℃,180rpm振荡培养,转化3天,得到细胞转化液和发酵液转化液。再分别向两种转化液中加入过量的CaCl2,得到酒石酸钙沉淀,过滤并用蒸馏水冲洗分别得到37.6g和37.4g酒石酸钙沉淀,再用硫酸酸解酒石酸钙沉淀,再次过滤后得到的酸解液经过阴阳离子交换精制、浓缩、结晶、烘干后分别得到22.8g和22.7g固体产物。经红外光谱、紫外光谱、核磁共振谱、质谱检测,确定该固体产物为酒石酸。
本实施例中,样品的红外光谱用Nicolet-Nexus670傅立叶转换式红外光谱仪检测;样品的核磁共振氢谱和碳谱用Bruker Avance DMX500核磁共振仪检测;样品的质谱用Bruker Esquire 3000plus质谱仪检测;样品的旋光度用WZZ-2B旋光仪检测。经旋光度检测,该固体产物的比旋光度为证明该固体产物为右旋型酒石酸,即L(+)-酒石酸,且纯度为99.9%。
实施例4:利用顺式环氧琥珀酸钠和黑曲霉(Aspergillus niger)WH-2CGMCCNo.16799生产L(+)-酒石酸
先在茄子瓶中用斜面培养基培养黑曲霉(Aspergillus niger)WH-2CGMCCNo.16799,28℃培养3~5天至长出浓密孢子,加入5ml孢子悬浮液,用接种铲刮取孢子,使孢子充分被洗下,用无菌的三层擦镜纸过滤除去菌丝体。过滤后的上述孢子液经稀释后用血球计数板计数,按3x106个mL-1的浓度接种孢子至装有50ml的种子培养基的250ml锥形瓶中,28℃180rpm振荡培养24~36h,得到所述的黑曲霉菌株种子液。取上述20ml黑曲霉的种子液接种装有200ml产酶培养基的1000ml锥形瓶中,28℃180rpm振荡培养2~3天,分别获得相应的黑曲霉细胞和发酵液,然后分别将得到的黑曲霉细胞和发酵液加入到200ml的浓度为1M的顺式环氧琥珀钠中,30℃,180rpm振荡培养,转化3天,得到细胞转化液和发酵液转化液。再向转化液中加入过量的CaCl2,得到酒石酸钙沉淀,过滤并用蒸馏水冲洗分别得到38.5g和38.3g酒石酸钙沉淀,再用硫酸酸解酒石酸钙沉淀,再次过滤后得到的酸解液经过阴阳离子交换精制、浓缩、结晶、烘干后分别得到23.5g和23.4g固体产物。
经红外光谱、紫外光谱、核磁共振谱、质谱检测,确定该固体产物为酒石酸。本实施例中,样品的红外光谱用Nicolet-Nexus670傅立叶转换式红外光谱仪检测;样品的核磁共振氢谱和碳谱用Bruker Avance DMX500核磁共振仪检测;样品的质谱用Bruker Esquire3000plus质谱仪检测;样品的旋光度用WZZ-2B旋光仪检测。经旋光度检测,该固体产物的比旋光度为证明该固体产物为右旋型酒石酸,即L(+)-酒石酸,且纯度为99.9%。
综上可知,本发明的黑曲霉(Aspergillus niger)WH-2CGMCC No.16799及其发酵液具有将顺式环氧琥珀酸或其盐水解为L(+)-酒石酸或其盐的特性。
需要说明的是,本发明所述的顺式环氧琥珀酸盐可以是上述的顺式环氧琥珀酸钾或顺式环氧琥珀酸钠,还可以是顺式环氧琥珀酸钙等其它顺式环氧琥珀酸的盐。
序列表
<110> 浙江科技学院
<120> 一种黑曲霉及其应用
<160> 1
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 黑曲霉(Aspergillus niger)
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ttgattgaat gcaatcagtt aaaactttca acaatggatc tcttggttcc ggcatcgatg 240
aagaacgcag cgaaatgcga taactaatgt gaattgcaga attcagtgaa tcatcgagtc 300
tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc 360
tgccctcaag cccggcttgt gtgttgggtc gccgtccccc tctccggggg gacgggcccg 420
aaaggcagcg gcggcaccgc gtccgatcct cgagcgtatg gggctttgtc acatgctctg 480
taggattggc cggcgcctgc cgacgttttc caaccatttt ttccaggttg acctcggatc 540
aggtagggat acccgctgaa cttaagcata tcaataaagg cggagg 586
Claims (5)
1.一种黑曲霉(Aspergillus niger) WH-2,保藏号为:CGMCC No.16799。
2.如权利要求1所述的一种黑曲霉WH-2在制备L(+)-酒石酸或其盐中的应用。
3.如权利要求2所述的应用,其特征在于包括以下步骤:
1)在斜面培养基中培养黑曲霉WH-2,28℃培养3~5天至长出浓密孢子,加入孢子悬浮液,刮取孢子,使孢子充分被洗下,用无菌的三层擦镜纸过滤除去菌丝体;
2)过滤后的上述孢子液按3×106个/mL浓度接种至种子培养基中,28℃振荡培养24~36h,得到黑曲霉菌株种子液;
3)取上述黑曲霉菌株种子液转接至产酶培养基中,28℃振荡培养2~3天,获得相应的黑曲霉细胞和发酵液,然后分别将得到的黑曲霉细胞和发酵液加入到顺式环氧琥珀酸或其盐溶液中,30℃振荡培养,转化3天,得到细胞转化液和发酵液转化液;
4)向两种转化液中分别加入过量的CaCl2,得到酒石酸钙沉淀,过滤并用蒸馏水冲洗酒石酸钙沉淀,再用硫酸酸解酒石酸钙沉淀,然后过滤后得到的酸解液经过阴阳离子交换精制、浓缩、结晶、烘干后得到L(+)-酒石酸或其盐的成品。
4.如权利要求3所述的应用,其特征在于所述的孢子悬浮液:称取0.2g的Tween80和1.8g的NaCl,再加入200ml蒸馏水,混合均匀,121℃高压灭菌20min,放4℃冰箱备用;所述的斜面培养基为PDA培养基;所述的种子培养基为PDB培养基;所述的产酶培养基为PDB无机盐培养基。
5.如权利要求3所述的应用,其特征在于所述的顺式环氧琥珀酸盐选用顺式环氧琥珀酸钠或顺式环氧琥珀酸钾。
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CN111778165A (zh) * | 2020-08-11 | 2020-10-16 | 哈尔滨工业大学 | 黑曲霉dfy1及其应用 |
CN111778165B (zh) * | 2020-08-11 | 2022-10-25 | 哈尔滨工业大学 | 黑曲霉dfy1及其应用 |
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