将质量源于设计(QbD)理念应用于苦参生物碱的纯化工艺,建立稳定可控的操作空间.以苦参总生物碱(TASF)、苦参碱(MT)和氧化苦参碱(OMT)的解吸量为工艺评价指标,静态吸附和解吸试验选取性能最优的大孔树脂作为纯化过程中的吸附剂,单因素和...将质量源于设计(QbD)理念应用于苦参生物碱的纯化工艺,建立稳定可控的操作空间.以苦参总生物碱(TASF)、苦参碱(MT)和氧化苦参碱(OMT)的解吸量为工艺评价指标,静态吸附和解吸试验选取性能最优的大孔树脂作为纯化过程中的吸附剂,单因素和多因素显著性试验(PBD)筛选并确定关键工艺参数(CPPs),中心点复合试验(CCD)进一步优化工艺参数,建立工艺参数和工艺评价指标之间的数学模型,以此获得操作空间,在空间内、外选点,对模型及设计空间进行验证.NK-109型大孔树脂吸附性能良好,为该试验的最优树脂.上样液pH值、洗脱剂乙醇浓度和洗脱流速为该试验的显著性影响因素.关键工艺参数与工艺评价指标之间回归方程的P值均小于0.05,而失拟项均大于0.05,表明回归方程显著.在苦参总生物碱解吸量≥0.87 mg/g,苦参碱解吸量≥32.50μg/g,氧化苦参碱解吸量≥244.10μg/g的工艺指标下,设计空间为:洗脱流速≤25.0 m L/h,洗脱剂乙醇体积分数82.0%~87.0%,上样液pH值5.3~6.2.验证结果表明模型的预测能力良好,空间内的点满足工艺指标,而空间外的点无法达到生产要求,设计空间稳定.质量源于设计理念的引用可以为中药的分离纯化过程提供稳定可控的操作空间,在确定工艺指标的基础上,灵活设置工艺参数,保证产品质量可控.展开更多
Biocatalysis with nicotinamide adenine dinucleotide phosphate(NADP)-dependent oxidoreductases faces a challenge in improving the efficiency of the costly cofactor utilization.Although enzyme fusion can offer cofactor ...Biocatalysis with nicotinamide adenine dinucleotide phosphate(NADP)-dependent oxidoreductases faces a challenge in improving the efficiency of the costly cofactor utilization.Although enzyme fusion can offer cofactor regeneration,the high-volume input and limited cofactor recyclability still make the enzymatic processes unsustainable.Therefore,it is of great significance to reduce cofactor input in a fusion enzyme(FuE)system,but no successful practice has been reported.Herein,we design a decapeptide bridge,RRRQRRRARR(R10),with high affinity for NADPH to construct fusion oxidoreductases(phenylacetone monooxygenase and phosphite dehydrogenase)for ester synthesis and NADP recycling.The peptide bridge enables electrostatic cofactor channeling that transports NADPH/NADP^(+)across the peptide between the enzymes’NADP-binding pockets,so the fusion enzyme(FuE-R10)presents 2.1-folds and 2.0-folds higher conversions than mixed free enzymes and a flexible linker(GGGGSGGGGS)-fused enzyme,respectively,at NADPH/FuE of 0.1.The fusion enzyme,FuE-R5,bridged by a half-shortened linker,is proved more effective in facilitating cofactor channeling;compared to the mixed free enzymes,FuE-R5 exhibits two orders of magnitude reduction of NADPH input in ester synthesis.The work has thus demonstrated the potential of the cofactor bridging strategy in the development of sustainable cofactor-dependent cascade biocatalysis.展开更多
文摘将质量源于设计(QbD)理念应用于苦参生物碱的纯化工艺,建立稳定可控的操作空间.以苦参总生物碱(TASF)、苦参碱(MT)和氧化苦参碱(OMT)的解吸量为工艺评价指标,静态吸附和解吸试验选取性能最优的大孔树脂作为纯化过程中的吸附剂,单因素和多因素显著性试验(PBD)筛选并确定关键工艺参数(CPPs),中心点复合试验(CCD)进一步优化工艺参数,建立工艺参数和工艺评价指标之间的数学模型,以此获得操作空间,在空间内、外选点,对模型及设计空间进行验证.NK-109型大孔树脂吸附性能良好,为该试验的最优树脂.上样液pH值、洗脱剂乙醇浓度和洗脱流速为该试验的显著性影响因素.关键工艺参数与工艺评价指标之间回归方程的P值均小于0.05,而失拟项均大于0.05,表明回归方程显著.在苦参总生物碱解吸量≥0.87 mg/g,苦参碱解吸量≥32.50μg/g,氧化苦参碱解吸量≥244.10μg/g的工艺指标下,设计空间为:洗脱流速≤25.0 m L/h,洗脱剂乙醇体积分数82.0%~87.0%,上样液pH值5.3~6.2.验证结果表明模型的预测能力良好,空间内的点满足工艺指标,而空间外的点无法达到生产要求,设计空间稳定.质量源于设计理念的引用可以为中药的分离纯化过程提供稳定可控的操作空间,在确定工艺指标的基础上,灵活设置工艺参数,保证产品质量可控.
文摘Biocatalysis with nicotinamide adenine dinucleotide phosphate(NADP)-dependent oxidoreductases faces a challenge in improving the efficiency of the costly cofactor utilization.Although enzyme fusion can offer cofactor regeneration,the high-volume input and limited cofactor recyclability still make the enzymatic processes unsustainable.Therefore,it is of great significance to reduce cofactor input in a fusion enzyme(FuE)system,but no successful practice has been reported.Herein,we design a decapeptide bridge,RRRQRRRARR(R10),with high affinity for NADPH to construct fusion oxidoreductases(phenylacetone monooxygenase and phosphite dehydrogenase)for ester synthesis and NADP recycling.The peptide bridge enables electrostatic cofactor channeling that transports NADPH/NADP^(+)across the peptide between the enzymes’NADP-binding pockets,so the fusion enzyme(FuE-R10)presents 2.1-folds and 2.0-folds higher conversions than mixed free enzymes and a flexible linker(GGGGSGGGGS)-fused enzyme,respectively,at NADPH/FuE of 0.1.The fusion enzyme,FuE-R5,bridged by a half-shortened linker,is proved more effective in facilitating cofactor channeling;compared to the mixed free enzymes,FuE-R5 exhibits two orders of magnitude reduction of NADPH input in ester synthesis.The work has thus demonstrated the potential of the cofactor bridging strategy in the development of sustainable cofactor-dependent cascade biocatalysis.
