Cytidine 5'-monophosphate(5'-CMP)is an essential nucleotide for additives.In this study,enhanced production of 5'-CMP was realized by the transformation of cytidine using co-immobilized di-enzymes,uridine-...Cytidine 5'-monophosphate(5'-CMP)is an essential nucleotide for additives.In this study,enhanced production of 5'-CMP was realized by the transformation of cytidine using co-immobilized di-enzymes,uridine-cytidine kinase(UCK)and acetate kinase(AcK).The immobilization yield of the enzyme had a clear correlation with the surface charges as zeta potential(ξ).Among them,ε-polylysinefunctionalized sepharose(SA-EPL,ξ=9.31 m V)showed high immobilization yield(78.8%),which was4.9-fold than that of nitrilotriacetic acid functionalized sepharose(SA-NTA,ξ=-12.6 m V).The residual activity of affinity co-immobilized enzyme(EPL-Ni/EPL@Ac K-UCK)was higher than 70.6%after recycled 10 times.Thus,this study provides an effective approach for the production of 5'-CMP with the advantages of low adenosine 5'-triphosphate(ATP)consumption,reduced side reactions,and improved reusability by co-immobilized UCK and Ac K on the functionalized Sepharose.展开更多
[Objective] This study aimed to establish an efficient process for separation of phycoerythrin by using Q Sepharose Fast Flow resin and verity its feasibility for scale-up. [Method] Elution gradient, sample volume and...[Objective] This study aimed to establish an efficient process for separation of phycoerythrin by using Q Sepharose Fast Flow resin and verity its feasibility for scale-up. [Method] Elution gradient, sample volume and flow rate were optimized to determine the optimal separation condition, under which the scale-up process was verified. [Result] The optimal condition for separation of phycoerythrin by using Q Sepharose FF resin was investigated： 30 ml of laver extract was loaded to the Q Sepharose FF column with a bed volume of 8 ml; subsequently, the column was stepwise eluted with 0-0.10-0.35-1.00 mol/L NaCI solution （pH 6.0） at a constant flow rate of 1 ml/min; the elution peak under 0.35 mol/L NaCI solution was collected, and the recovery rate and purity coefficient （A565/A280） of phycoerythrin were determined as 44.3 and 1.15, respectively. Based on the established process, 75 ml of phycoerythrin extract was loaded to the Q Sepharose FF column with a bed volume of 20 ml for separation, while no significant variation was observed in the separation result. [Conclusion] Phycoerythrin can be well separated from laver extract by using Q Sepharose FF resin and the process is feasible for scale-up.展开更多
[目的]为Pleurotus eryngii—Co60-7木质素降解酶的分离纯化和综合利用提供试验依据。[方法]采用DEAE—Sepharose^TM Fast Flow离子交换介质，分别考察缓冲液pH值、流速和洗脱方式等对刺芹侧耳木质素降解酶分离纯化的影响，确定了最佳...[目的]为Pleurotus eryngii—Co60-7木质素降解酶的分离纯化和综合利用提供试验依据。[方法]采用DEAE—Sepharose^TM Fast Flow离子交换介质，分别考察缓冲液pH值、流速和洗脱方式等对刺芹侧耳木质素降解酶分离纯化的影响，确定了最佳分离纯化层析条件。[结果]DEAE-Sephalose^TM Fast Flow分离纯化Pleurotus eryngii-Co60-7木质素降解酶的最佳层析条件为：选择20mmol／L，pH值为5．0醋酸钠一醋酸缓冲体系，3ml／min的流速，进行分步洗脱（100、200～300和1000mmoL／L NaCl的三步洗脱），可较好地实现刺芹侧耳发酵液木质素降解酶初分，该纯化操作目标蛋白回收率达85％，纯化分离因素为2．71。[结论]该技术在分离纯化刺芹侧耳木质素降解酶上可行，具有潜在的工业应用价值。展开更多
Q Sepharose Fast Flow(Q Sepharose F.F.) was adopted to purify the suspensions of type Ⅰ,Ⅱ,Ⅲ oral poliovaccine (OPV), which is prepared from Vero cells. After clarification and concentration by ultrafiltration,th...Q Sepharose Fast Flow(Q Sepharose F.F.) was adopted to purify the suspensions of type Ⅰ,Ⅱ,Ⅲ oral poliovaccine (OPV), which is prepared from Vero cells. After clarification and concentration by ultrafiltration,the recovery of virus infected titre may attain above 85%.Using column chromatography on Q Sepharose F.F. to purify the concentrated virus suspensions,the purified viruses attain 100% recovery of virus infectivity. Dot membrane hybrization was used to detect DNA with the probe of Vero cell genome DNA which was labeled with α P 32 dATP,the contents of residual substrate DNA was less than 100 Pg/dose. The process of downstream had no significant influence on some biological characters of purified viruses,such as virus morphology ,tumorigenicity, rct/40 character and capsid protein.This downstream reseach indicates that Q Sepharose F.F. is an ideal chromatography material for purifying OPV prepared from Vero cells.展开更多
双特异性抗体(BsAbs),简称双抗,是一种能同时结合两个不同靶点或表位的抗体,BsAbs在表达过程中通常伴随产生多种副产物,在纯化工艺中很难被去除。本研究运用高通量筛选技术,通过对SP Sepharose Fast Flow填料的纯化条件进行探索,建立基...双特异性抗体(BsAbs),简称双抗,是一种能同时结合两个不同靶点或表位的抗体,BsAbs在表达过程中通常伴随产生多种副产物,在纯化工艺中很难被去除。本研究运用高通量筛选技术,通过对SP Sepharose Fast Flow填料的纯化条件进行探索,建立基于Sepharose Fast Flow填料的双特异性抗体纯化方法开发的通用流程,每个筛选条件只需要0.4 mg双抗样品,可同时筛选多达32个条件,筛选过程总耗时2 h,而传统柱层析需耗时64 h。通过高通量迅速筛选方法可以有效去除双抗分子副产物,为解决纯化工艺难题提供了一种新的工艺路线。展开更多
基金supported by grants from the National Key Research and Development Program of China(2021YFC2102805,2019YFD1101204)the National Natural Science Foundation of China(21878142,21776132)+3 种基金Key Research and Development Plan of Jiangsu Province(BE2020712)Key Research and Development Plan of Jiangsu Province(BE2019001)Jiangsu Natural Science Fund for Distinguished Young Scholars(BK20190035)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。
文摘Cytidine 5'-monophosphate(5'-CMP)is an essential nucleotide for additives.In this study,enhanced production of 5'-CMP was realized by the transformation of cytidine using co-immobilized di-enzymes,uridine-cytidine kinase(UCK)and acetate kinase(AcK).The immobilization yield of the enzyme had a clear correlation with the surface charges as zeta potential(ξ).Among them,ε-polylysinefunctionalized sepharose(SA-EPL,ξ=9.31 m V)showed high immobilization yield(78.8%),which was4.9-fold than that of nitrilotriacetic acid functionalized sepharose(SA-NTA,ξ=-12.6 m V).The residual activity of affinity co-immobilized enzyme(EPL-Ni/EPL@Ac K-UCK)was higher than 70.6%after recycled 10 times.Thus,this study provides an effective approach for the production of 5'-CMP with the advantages of low adenosine 5'-triphosphate(ATP)consumption,reduced side reactions,and improved reusability by co-immobilized UCK and Ac K on the functionalized Sepharose.
基金Supported by National Natural Science Foundation of China(51143012)Natural Science Foundation of Shandong Province(ZR2009BM006)~~
文摘[Objective] This study aimed to establish an efficient process for separation of phycoerythrin by using Q Sepharose Fast Flow resin and verity its feasibility for scale-up. [Method] Elution gradient, sample volume and flow rate were optimized to determine the optimal separation condition, under which the scale-up process was verified. [Result] The optimal condition for separation of phycoerythrin by using Q Sepharose FF resin was investigated： 30 ml of laver extract was loaded to the Q Sepharose FF column with a bed volume of 8 ml; subsequently, the column was stepwise eluted with 0-0.10-0.35-1.00 mol/L NaCI solution （pH 6.0） at a constant flow rate of 1 ml/min; the elution peak under 0.35 mol/L NaCI solution was collected, and the recovery rate and purity coefficient （A565/A280） of phycoerythrin were determined as 44.3 and 1.15, respectively. Based on the established process, 75 ml of phycoerythrin extract was loaded to the Q Sepharose FF column with a bed volume of 20 ml for separation, while no significant variation was observed in the separation result. [Conclusion] Phycoerythrin can be well separated from laver extract by using Q Sepharose FF resin and the process is feasible for scale-up.
文摘[目的]为Pleurotus eryngii—Co60-7木质素降解酶的分离纯化和综合利用提供试验依据。[方法]采用DEAE—Sepharose^TM Fast Flow离子交换介质，分别考察缓冲液pH值、流速和洗脱方式等对刺芹侧耳木质素降解酶分离纯化的影响，确定了最佳分离纯化层析条件。[结果]DEAE-Sephalose^TM Fast Flow分离纯化Pleurotus eryngii-Co60-7木质素降解酶的最佳层析条件为：选择20mmol／L，pH值为5．0醋酸钠一醋酸缓冲体系，3ml／min的流速，进行分步洗脱（100、200～300和1000mmoL／L NaCl的三步洗脱），可较好地实现刺芹侧耳发酵液木质素降解酶初分，该纯化操作目标蛋白回收率达85％，纯化分离因素为2．71。[结论]该技术在分离纯化刺芹侧耳木质素降解酶上可行，具有潜在的工业应用价值。
文摘Q Sepharose Fast Flow(Q Sepharose F.F.) was adopted to purify the suspensions of type Ⅰ,Ⅱ,Ⅲ oral poliovaccine (OPV), which is prepared from Vero cells. After clarification and concentration by ultrafiltration,the recovery of virus infected titre may attain above 85%.Using column chromatography on Q Sepharose F.F. to purify the concentrated virus suspensions,the purified viruses attain 100% recovery of virus infectivity. Dot membrane hybrization was used to detect DNA with the probe of Vero cell genome DNA which was labeled with α P 32 dATP,the contents of residual substrate DNA was less than 100 Pg/dose. The process of downstream had no significant influence on some biological characters of purified viruses,such as virus morphology ,tumorigenicity, rct/40 character and capsid protein.This downstream reseach indicates that Q Sepharose F.F. is an ideal chromatography material for purifying OPV prepared from Vero cells.
文摘双特异性抗体(BsAbs),简称双抗,是一种能同时结合两个不同靶点或表位的抗体,BsAbs在表达过程中通常伴随产生多种副产物,在纯化工艺中很难被去除。本研究运用高通量筛选技术,通过对SP Sepharose Fast Flow填料的纯化条件进行探索,建立基于Sepharose Fast Flow填料的双特异性抗体纯化方法开发的通用流程,每个筛选条件只需要0.4 mg双抗样品,可同时筛选多达32个条件,筛选过程总耗时2 h,而传统柱层析需耗时64 h。通过高通量迅速筛选方法可以有效去除双抗分子副产物,为解决纯化工艺难题提供了一种新的工艺路线。
