CN104560913B - 一种桔青霉半连续发酵制备核酸酶p1的方法 - Google Patents
一种桔青霉半连续发酵制备核酸酶p1的方法 Download PDFInfo
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Abstract
本发明公开了一种桔青霉半连续发酵制备核酸酶P1的方法,使用包含了维生素B类物质的发酵培养基,进行桔青霉半连续发酵制备核酸酶P1。本发明方法单批发酵时间由60h缩短至20h,酶活由3000U/mL提高至6500U/mL,可连续发酵12批以上。该方法可以显著的缩短发酵工时,减少生产成本。
Description
技术领域
本发明涉及微生物发酵领域,具体涉及一种桔青霉半连续发酵生产核酸酶P1的方法。
背景技术
5’-核苷酸已广泛应用于食品、生化工业以及制药工业。在食品行业中,己经由最初的食品助鲜剂,扩展为具有提高生物体免疫功能的功能性食品添加剂,可以添加在面包、饼干等食品中,尤其在婴幼儿食品中的使用效果非常明显,能够有效增强婴幼儿抵抗细菌性痢疾的能力,减少腹泻的发生;在医药领域,5’-核苷酸不但能够作为药物使用,而且还是许多抗病毒抗肿瘤药物的生产原料;此外,核苷酸制剂还可以作为动植物生长调节剂,有着明显的增产、增重功效。
核苷酸生产主要采取三种方法:化学合成法、微生物发酵法及酶解法。酶解法由于降解RNA可以一次得到四种核苷酸的混合物,且酶反应收率较高,国内外工业化生产核苷酸均采用此法。在酶解法制备核苷酸中所需的酶是核酸酶Pl,它是一种能够水解RNA获得四种5’-核苷酸的磷酸二酯酶,其酶解产物杂质少后续分离工艺简单,国外一般采用该方法进行核苷酸的生产。
桔青霉属于不对称青霉组,绒状青霉亚组,桔青霉系,是核酸酶P1的重要生产菌种。国内外生产核酸酶P1主要通过桔青霉液体深层发酵,属于分批发酵,该方法耗时较长。如何高效的生产核酸酶P1,降低生产成本,已成为核苷酸生产的首要任务。
发明内容
本发明索要解决的技术问题是提供一种桔青霉半连续发酵生产核酸酶P1的方法。
为解决上述技术问题,本发明采用的技术方案如下:
一种桔青霉半连续发酵制备核酸酶P1的方法,使用包含了维生素B类物质的发酵培养基,进行桔青霉半连续发酵制备核酸酶P1。
其中,所述的桔青霉为CGMCC No.2014。该菌株记载在中国专利CN101067116A中。
其中,所述的维生素B类物质为泛酸、烟酸、生物素、叶酸、维生素B1、维生素B2、吡哆醇和氰钴胺中的任意一种或几种,优选泛酸、烟酸、生物素、叶酸、维生素B1和维生素B2中的任意一种或几种,最优选泛酸、烟酸和叶酸三者的组合;发酵培养基中维生素B类物质总浓度为0.001g/L~1g/L,优选0.05-0.8g/L,最优选0.1-0.8g/L
其中,所述的发酵培养基组分如下:葡萄糖0.5~50g/L,蛋白胨0.05~5g/L,磷酸二氢钾0.01~5g/L,磷酸氢二钾0.01~5g/L,硫酸镁0.01~5g/L,氯化钙0.01~5g/L,维生素B类物质0.001~1g/L,溶剂为水,pH5-8。
上述桔青霉半连续发酵制备核酸酶P1的方法,优选包括如下步骤:
a桔青霉在发酵罐中发酵一段时间,待核酸酶P1酶活增长至稳定后放出一定体积的发酵液,发酵罐中留存的发酵液作为下一批发酵种子;
b通过向发酵罐补加无菌发酵培养基继续发酵培养,待酶活增至稳定后再次放出一定体积的发酵液,发酵罐中留存的发酵液作为下一批发酵种子;
c重复步骤b直至桔青霉产酶能力下降至一定程度后停止发酵。
步骤a中,所述的发酵罐体积为20L~200T,优选1T~50T;所述的发酵一段时间,时间为10~100h,优选20~60h。
步骤a和b中,所述的酶活增长至稳定,是指酶活为3000~6500U/mL,优选4000~6000U/mL。
步骤a和b中,所述的放出一定体积的发酵液,是指放出发酵罐中发酵液体积的20%~95%的发酵液,优选60-90%。
步骤c中,重复步骤b的次数为2~20次,优选10~15次。
步骤c中,所述的桔青霉产酶能力下降至一定程度,是指核酸酶P1酶活小于3000U/mL。
本发明中,核酸酶P1酶活定义:将1.9ml的底物溶液(含有浓度为1%左右的RNA;0.2M pH5.2的醋酸缓冲液以0.0005M的ZnSO4)于70℃恒温水浴10min后,加入0.1ml经适当稀释的酶液,70℃保温15min后加入2.0ml核酸沉淀剂(0.25%钼酸铵-2.5过氯酸),冰水浴20min后离心、取上清夜用蒸馏水稀释一定倍数,测定其260nm处的吸光值为A260。以先加沉淀剂者作为对照,其它操作同前。在上述条件下,每分钟所生成的核苷酸量在260nm处的吸光值的差值为1.0时定义为1个酶活力单位。
有益效果:本发明采用半连续发酵的方式,通过添加维生素B类物质进行半连续发酵,单批发酵时间由60h缩短至20h,酶活由3000U/mL提高至6500U/mL,可连续发酵12批以上。该方法可以显著的缩短发酵工时,减少生产成本。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1:
发酵培养基的配制:葡萄糖20g/L,蛋白胨2g/L,磷酸二氢钾0.5g/L,磷酸氢二钾0.5g/L,硫酸镁0.2g/L,氯化钙0.2g/L,泛酸0.01g/L,维生素B1 0.05g/L,pH6,121℃灭菌15min,降温至30℃左右备用。
50L搅拌罐中装入30L培养基,灭菌后接入经麦芽汁斜面培养基培养的桔青霉菌种进行发酵培养。间隔一定时间取样测定核酸酶P1的酶活,培养56h后酶活稳定在3300U/mL左右,放出24L发酵液,罐中留存6L作为下一批发酵的种子液。配制24L发酵培养基打入发酵罐再次培养。22h后酶活稳定至4800U/mL,再次放出24L发酵液,同样方法补入24L新鲜培养基。如此循环补料5次,第2、3次补料后培养酶活均在5000U/mL左右,第4次补料酶活下降至3460U/mL,第5次补料后酶活明显下降,只有2850U/mL,停止半连续发酵。
用同样的培养基和培养条件发酵,但是发酵培养基不加泛酸和维生素B1,58h后酶活稳定至3800U/mL,放出24L发酵液,同样方法补入24L新鲜培养基。发酵44h,酶活稳定在2500U/mL,停止半连续发酵。
实施例2:
发酵培养基的配制:葡萄糖30g/L,蛋白胨3g/L,磷酸二氢钾1g/L,磷酸氢二钾1g/L,硫酸镁0.5g/L,氯化钙0.2g/L,生物素0.1g/L,烟酸0.06g/L,维生素B2 0.2g/L,pH7,121℃灭菌15min,降温至30℃左右备用。
2T搅拌罐中装入1.2T培养基,灭菌后接入经麦芽汁斜面培养基培养的桔青霉菌种进行发酵培养。间隔一定时间取样测定核酸酶P1的酶活,培养58h后酶活稳定在3700U/mL左右,放出1T发酵液,罐中留存0.2T作为下一批发酵的种子液。配制1T发酵培养基打入发酵罐再次培养。20h后酶活稳定至5200U/mL,再次放出1T发酵液,同样方法补入1T新鲜培养基。如此循环补料10次,第2-8次补料后培养酶活均在5400U/mL左右,第9次补料酶活下降至4200U/mL,第10次补料后酶活明显下降,只有2800U/mL,停止半连续发酵。
用同样的培养基和培养条件发酵,但是发酵培养基不加生物素、烟酸和维生素B2,57h后酶活稳定至4600U/mL,放出1T发酵液,同样方法补入1T新鲜培养基。55h后酶活稳定至4200U/mL,第3次补料发酵55h后酶活明显下降,只有2650U/mL,停止半连续发酵。
实施例3:
发酵培养基的配制:葡萄糖40g/L,蛋白胨4g/L,磷酸二氢钾1g/L,磷酸氢二钾1g/L,硫酸镁0.5g/L,氯化钙0.2g/L,泛酸0.1g/L、烟酸0.2g/L、叶酸0.5g/L,pH7,121℃灭菌15min,降温至30℃左右备用。
20T搅拌罐中装入12T培养基,灭菌后接入经麦芽汁斜面培养基培养的桔青霉菌种进行发酵培养。间隔一定时间取样测定核酸酶P1的酶活,培养56h后酶活稳定在4500U/mL左右,放出8T发酵液,罐中留存4T作为下一批发酵的种子液。配制8T发酵培养基打入发酵罐再次培养。20h后酶活稳定至5400U/mL,再次放出8T发酵液,同样方法补入8T新鲜培养基。如此循环补料12次,第2-9次补料后培养酶活均在6500U/mL左右,第10次补料酶活下降至4850U/mL,第11次补料酶活下降至3680U/mL,第12次补料后酶活明显下降,只有2400U/mL,停止半连续发酵。
用同样的培养基和培养条件发酵,但是发酵培养基不加泛酸、烟酸和叶酸,57h后酶活稳定至4500U/mL,放出1T发酵液,同样方法补入1T新鲜培养基。55h后酶活稳定至4000U/mL,第3次补料发酵58h后酶活明显下降,只有2700U/mL,停止半连续发酵。
Claims (1)
1.一种桔青霉半连续发酵制备核酸酶P1的方法,其特征在于,
发酵培养基的配制:葡萄糖40g/L,蛋白胨4g/L,磷酸二氢钾1g/L,磷酸氢二钾1g/L,硫酸镁0.5g/L,氯化钙0.2g/L,泛酸0.1g/L、烟酸0.2g/L、叶酸0.5g/L,pH7,121℃灭菌15min,降温至30℃备用;
20T搅拌罐中装入12T培养基,灭菌后接入经麦芽汁斜面培养基培养的桔青霉菌种CGMCC No.2014进行发酵培养;间隔一定时间取样测定核酸酶P1的酶活,培养56h后酶活稳定在4500U/mL,放出8T发酵液,罐中留存4T作为下一批发酵的种子液;配制8T发酵培养基打入发酵罐再次培养;20h后酶活稳定至5400U/mL,再次放出8T发酵液,同样方法补入8T新鲜培养基;如此循环补料12次,第2-9次补料后培养酶活均在6500U/mL。
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