• 观点 •
张以恒1,2, 陈雪梅2, 韩平平1,2
收稿日期:
2025-03-20
修回日期:
2025-04-29
出版日期:
2025-05-12
通讯作者:
张以恒
作者简介:
基金资助:
ZHANG Yi-Heng P. Job1,2, CHEN Xuemei2, HAN Pingping1,2
Received:
2025-03-20
Revised:
2025-04-29
Online:
2025-05-12
Contact:
ZHANG Yi-Heng P. Job
摘要:
生物制造是战略新兴产业的典型代表,是生物经济的新质生产力。作者曾提出“道法术器”对工业生物制造的哲学指导意义。为进一步阐述生物制造中“术以立策”的原则,本文首次提出衡量生物催化剂(即“术”)水平的关键经济指标:PE值(Product-to-Enzyme Ratio)与PX值(Product-to-X(Cell) Ratio)。这两个指标具有简单、透明且量化的属性。PE值表示产品与非细胞催化剂(酶分子或多酶分子机器,以下简称“多酶机器”)的重量比值,也可通过技术指标总转换数(Total Turn-over Number,TTN)计算其理论值。PX值应用于细胞工厂发酵,表示产品与细胞催化剂的重量比。基于PE值与PX值,可以快速估算不同生物制造过程中的生物催化剂成本,进而指导降低降本增效的关键路径。作者汇总了生物制造的产业案例及文献数据,展示了酶分子及多酶机器PE值、细胞工厂PX值的特点。研究表明,淀粉酶水解淀粉的PE值是纤维素酶水解纤维素的50-100倍;在固态纤维素水解过程中,纤维素酶的超大用量是非粮生物质糖化与利用的最大经济障碍。最后,本文探讨了PE值的技术改进路径,特别是多酶共固定技术的潜力,并明确了工业酶皇冠——纤维素酶研究的新方向。PE值与PX值的分析将为生物制造战略新兴产业的发展提供了全新的视角,深化了对生物制造中关键“芯片”——生物催化剂成本的理解,为未来技术的发展提供重要参考与指导。
中图分类号:
张以恒, 陈雪梅, 韩平平. 生物制造的PE值与PX值:定义与应用[J]. 合成生物学, DOI: 10.12211/2096-8280.2025-020.
ZHANG Yi-Heng P. Job, CHEN Xuemei, HAN Pingping. PE and PX Values in Biomanufacturing: Definitions and Applications[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2025-020.
比较内容 | 分泌酶 | 胞内酶 |
---|---|---|
代表性菌 | 毕赤酵母、里氏木霉、枯草芽孢杆菌、黑曲霉 | 大肠杆菌、枯草芽孢杆菌 |
代表性酶制剂 | 植酸酶、纤维素酶、木聚糖酶、蛋白酶、液化酶、糖化酶、脂肪酶、果胶酶、漆酶等 | 木糖异构酶、核酸酶、SOD、甲酸脱氢酶、醇脱氢酶等 |
蛋白表达水平 | 200 g/L(最高),一般在10-50 g/L左右 | 5-10 g/L(最高),大多数在1-5 g/L左右 |
蛋白制造成本[ | 约100元/kg酶干重 | 约250-1000元/kg 酶干重 |
酶分离难易程度 | 容易,低成本 | 比较贵 |
目标酶 | 有限制,与底盘细胞的分泌同源酶序列高度相似 | 普适性 |
技术难点 | 基因操作难,要长期(几年以上)系统优化 | 基因操作相当容易 |
蛋白表达的比喻 | 牛产奶 | 杀牛产肉 |
表1 工业酶的分泌与胞内表达生产方法比较
Table 1 Comparison of production methods for industrial enzymes: secretion versus intracellular expression
比较内容 | 分泌酶 | 胞内酶 |
---|---|---|
代表性菌 | 毕赤酵母、里氏木霉、枯草芽孢杆菌、黑曲霉 | 大肠杆菌、枯草芽孢杆菌 |
代表性酶制剂 | 植酸酶、纤维素酶、木聚糖酶、蛋白酶、液化酶、糖化酶、脂肪酶、果胶酶、漆酶等 | 木糖异构酶、核酸酶、SOD、甲酸脱氢酶、醇脱氢酶等 |
蛋白表达水平 | 200 g/L(最高),一般在10-50 g/L左右 | 5-10 g/L(最高),大多数在1-5 g/L左右 |
蛋白制造成本[ | 约100元/kg酶干重 | 约250-1000元/kg 酶干重 |
酶分离难易程度 | 容易,低成本 | 比较贵 |
目标酶 | 有限制,与底盘细胞的分泌同源酶序列高度相似 | 普适性 |
技术难点 | 基因操作难,要长期(几年以上)系统优化 | 基因操作相当容易 |
蛋白表达的比喻 | 牛产奶 | 杀牛产肉 |
图1 微生物发酵与体外生物转化(ivBT)的生物催化剂成本(虚线-细胞催化剂成本(元/吨产品),计算公式为:细胞催化剂成本=细胞制备成本/PX,其中虚线由上至下PX值分别为3、6、15、30,细胞制备成本为30元/kg;实线-酶成本(元/吨产品),计算公式为:酶成本=酶制备成本/PE,其中实线由上至下酶制备成本(E)分别为5000元/kg、1000元/kg、250元/kg、100元/kg;PE value-PE值,计算公式为:PE=产品重量/酶干重; Biocatalyst cost(CNY/tonne of product)-生物催化剂成本(元/吨产品),即细胞催化剂成本或酶成本)
Fig. 1 Biocatalyst costs of microbial fermentation and in vitro biotransformation (ivBT)(Dashed line - Cell cost (CNY/tonne of product), calculated as: Cell cost = Cell preparation cost × 1000 (kg/tonne) / PX, where PX values are 3, 6, 15, and 30 (top to bottom), and the cell preparation cost is 30 CNY/kg dry cell weight. Solid line - Enzyme cost (CNY/tonne of product), calculated as: Enzyme cost = Enzyme preparation cost × 1000 (kg/tonne) / PE, where enzyme preparation costs (E) are 5000, 1000, 250, and 100 CNY/kg (top to bottom). PE value is calculated as: PE = product mass / enzyme dry weight. Biocatalyst cost (CNY/ton of product) refers to either the cell cost or enzyme cost.)
酶类别 | 酶名 | 底物 | 产物 | 酶形式 | 酶数量 | kcat (1/s) | kd (1/s) | TTN | MWP (Da) | MWE (kDa) | PE值① (理论) | PE值② (实际) | 参考文献 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
异构酶 | 葡萄糖磷酸异构酶 | 果糖-6-磷酸 | 葡萄糖-6-磷酸 | 游离 | 1 | 2765 | 8.0E-05 | 3.4E+07 | 260.14 | 48 | 1.9E+05 | NA | [ |
固定化 | 1 | 2198 | 1.0E-06 | 2.2E+09 | 260.14 | 67 | 8.4E+06 | NA | [ | ||||
D-塔格糖-3-差向异构酶 | D-果糖 | D-阿洛酮糖 | 游离 | 1 | 55.1 | 1.7E-04 | 3.3E+05 | 180 | 33 | 1.8E+03 | NA | [ | |
固定化 | 1 | 93.4 | 3.9E-05 | 2.4E+06 | 180 | 33 | 1.3E+04 | 1000 | [ | ||||
木糖异构酶 | D-葡萄糖 | D-果糖 | 游离 | 1 | 30 | 7.7E-06 | 3.9E+06 | 180 | 46 | 1.5E+04 | NA | [ | |
固定化 | 1 | 27 | 1.0E-06 | 2.7E+07 | 180 | 46 | 1.1E+05 | 1.0E+05 | [ | ||||
水解酶 | 青霉素G酰胺酶 | 青霉素G | 6-氨基青霉烷酸 | 游离 | 1 | 44.6 | 1.1E-04 | 4.0E+05 | 216.25 | 88 | 9.8E+02 | NA | [ |
固定化 | 1 | NA | NA | NA | NA | NA | NA | 600 | [ | ||||
脂肪酶 | 油脂+醇类 | 脂肪酸酯 | 游离 | 1 | 500 | 4.1E-05 | 1.2E+07 | 250 | 43 | 6.9E+04 | 150 | [ | |
固定化 | 1 | NA | NA | NA | NA | NA | NA | 1000-5000 | [ | ||||
异构酶 | 核糖磷酸异构酶 | 核酮糖-5-磷酸 | 核糖-5-磷酸 | 游离 | 1 | 540 | 2.7E-06 | 2.0E+08 | 230.1 | 15.9 | 2.9E+06 | NA | [ |
葡萄糖磷酸变位酶 | 葡萄糖-1-磷酸 | 葡萄糖-6-磷酸 | 游离 | 1 | 190 | 2.7E-06 | 7.1E+07 | 260.14 | 64.9 | 2.8E+05 | NA | [ | |
水解酶 | 果糖-1,6-二磷酸酶 | 果糖-1,6-二磷酸 | 果糖-6-磷酸 | 游离 | 1 | 8.57 | 1.4E-07 | 5.9E+07 | 260.14 | 28 | 5.5E+05 | NA | [ |
β-糖苷酶 | 纤维二糖 | 葡萄糖 | 游离 | 1 | 208 | 3.5E-05 | 6.0E+06 | 180 | 62.78 | 1.7E+04 | NA | [ | |
氧化 还原酶 | 肉桂醇 脱氢酶 | 肉桂醇 | 肉桂醛 | 游离 | 1 | 18 | 2.00E-03 | 9.0E+03 | 132.159 | 80 | 15 | NA | [ |
氢酶 | H+ | 氢气 | 游离 | 1 | 1030 | 2.7E-06 | 3.9E+08 | 2 | 110 | 7.0E+03 | NA | [ | |
胺脱氢酶 | NAD+ | NADH | 游离 | 1 | 167 | 1.1E-06 | 1.5E+08 | 663.43 | 45 | 2.1E+06 | NA | [ | |
6-磷酸葡萄糖酸脱氢酶 | 葡萄糖6-磷酸 | 6-磷酸葡萄糖酸 | 游离 | 1 | 325 | 1.4E-06 | 2.4E+08 | 495.3 | 53 | 2.2E+06 | NA | [ | |
转移酶 | 转醛缩酶 | 果糖-6-磷酸, 赤藓糖-4-磷酸 | 景天糖-7-磷酸 | 游离 | 1 | 22.3 | 1.5E-05 | 1.5E+06 | 290.16 | 24 | 1.8E+04 | NA | [ |
混合酶 | 伊沙替韦酶 | 2-乙酰甘油 | 伊沙替韦 | 游离 | 9 | NA | NA | NA | NA | NA | NA | 1 | [ |
PHB合成 | 淀粉 | PHB | 游离 | 19-20 | NA | NA | NA | NA | NA | NA | 1 | [ | |
合成淀粉多酶机器 | CO2+H2 | 合成淀粉 | 游离 | 13 | NA | NA | NA | NA | NA | NA | 1 | [ | |
单萜烯酶 | 葡萄糖 | 单萜烯 | 游离 | 27 | NA | NA | NA | NA | NA | NA | 2 | [ | |
肌醇多酶 机器 | 淀粉 | 肌醇 | 游离 | 4-6 | 1.1 | 2.0E-05 | 5.5E+04 | 180 | 50 | 196 | 13 | [ | |
固定化 | 4-6 | 1.2 | 3.5E-06 | 3.4E+05 | 180 | 50 | 1242 | 60-80 | [ | ||||
D-塔格糖 多酶机器 | 淀粉 | D-塔格糖 | 游离 | 5-6 | NA | NA | NA | NA | NA | NA | 35 | [ | |
固定化 | 4-6 | NA | NA | NA | NA | NA | NA | 103 | [ | ||||
纤维素酶 (混合) | (预处理)生物质 | D-葡萄糖 | 游离 | 3-10 | NA | NA | NA | NA | NA | NA | 50-100 | [ | |
淀粉酶 (混合) | (糊化)淀粉 | D-葡萄糖 | 游离 | 2-3 | NA | NA | NA | NA | NA | NA | 5000 | [ |
表2 酶催化生物制造代表性产品的PE值现状
Table 2 Current PE values for representative products in enzyme-catalyzed biomanufacturing
酶类别 | 酶名 | 底物 | 产物 | 酶形式 | 酶数量 | kcat (1/s) | kd (1/s) | TTN | MWP (Da) | MWE (kDa) | PE值① (理论) | PE值② (实际) | 参考文献 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
异构酶 | 葡萄糖磷酸异构酶 | 果糖-6-磷酸 | 葡萄糖-6-磷酸 | 游离 | 1 | 2765 | 8.0E-05 | 3.4E+07 | 260.14 | 48 | 1.9E+05 | NA | [ |
固定化 | 1 | 2198 | 1.0E-06 | 2.2E+09 | 260.14 | 67 | 8.4E+06 | NA | [ | ||||
D-塔格糖-3-差向异构酶 | D-果糖 | D-阿洛酮糖 | 游离 | 1 | 55.1 | 1.7E-04 | 3.3E+05 | 180 | 33 | 1.8E+03 | NA | [ | |
固定化 | 1 | 93.4 | 3.9E-05 | 2.4E+06 | 180 | 33 | 1.3E+04 | 1000 | [ | ||||
木糖异构酶 | D-葡萄糖 | D-果糖 | 游离 | 1 | 30 | 7.7E-06 | 3.9E+06 | 180 | 46 | 1.5E+04 | NA | [ | |
固定化 | 1 | 27 | 1.0E-06 | 2.7E+07 | 180 | 46 | 1.1E+05 | 1.0E+05 | [ | ||||
水解酶 | 青霉素G酰胺酶 | 青霉素G | 6-氨基青霉烷酸 | 游离 | 1 | 44.6 | 1.1E-04 | 4.0E+05 | 216.25 | 88 | 9.8E+02 | NA | [ |
固定化 | 1 | NA | NA | NA | NA | NA | NA | 600 | [ | ||||
脂肪酶 | 油脂+醇类 | 脂肪酸酯 | 游离 | 1 | 500 | 4.1E-05 | 1.2E+07 | 250 | 43 | 6.9E+04 | 150 | [ | |
固定化 | 1 | NA | NA | NA | NA | NA | NA | 1000-5000 | [ | ||||
异构酶 | 核糖磷酸异构酶 | 核酮糖-5-磷酸 | 核糖-5-磷酸 | 游离 | 1 | 540 | 2.7E-06 | 2.0E+08 | 230.1 | 15.9 | 2.9E+06 | NA | [ |
葡萄糖磷酸变位酶 | 葡萄糖-1-磷酸 | 葡萄糖-6-磷酸 | 游离 | 1 | 190 | 2.7E-06 | 7.1E+07 | 260.14 | 64.9 | 2.8E+05 | NA | [ | |
水解酶 | 果糖-1,6-二磷酸酶 | 果糖-1,6-二磷酸 | 果糖-6-磷酸 | 游离 | 1 | 8.57 | 1.4E-07 | 5.9E+07 | 260.14 | 28 | 5.5E+05 | NA | [ |
β-糖苷酶 | 纤维二糖 | 葡萄糖 | 游离 | 1 | 208 | 3.5E-05 | 6.0E+06 | 180 | 62.78 | 1.7E+04 | NA | [ | |
氧化 还原酶 | 肉桂醇 脱氢酶 | 肉桂醇 | 肉桂醛 | 游离 | 1 | 18 | 2.00E-03 | 9.0E+03 | 132.159 | 80 | 15 | NA | [ |
氢酶 | H+ | 氢气 | 游离 | 1 | 1030 | 2.7E-06 | 3.9E+08 | 2 | 110 | 7.0E+03 | NA | [ | |
胺脱氢酶 | NAD+ | NADH | 游离 | 1 | 167 | 1.1E-06 | 1.5E+08 | 663.43 | 45 | 2.1E+06 | NA | [ | |
6-磷酸葡萄糖酸脱氢酶 | 葡萄糖6-磷酸 | 6-磷酸葡萄糖酸 | 游离 | 1 | 325 | 1.4E-06 | 2.4E+08 | 495.3 | 53 | 2.2E+06 | NA | [ | |
转移酶 | 转醛缩酶 | 果糖-6-磷酸, 赤藓糖-4-磷酸 | 景天糖-7-磷酸 | 游离 | 1 | 22.3 | 1.5E-05 | 1.5E+06 | 290.16 | 24 | 1.8E+04 | NA | [ |
混合酶 | 伊沙替韦酶 | 2-乙酰甘油 | 伊沙替韦 | 游离 | 9 | NA | NA | NA | NA | NA | NA | 1 | [ |
PHB合成 | 淀粉 | PHB | 游离 | 19-20 | NA | NA | NA | NA | NA | NA | 1 | [ | |
合成淀粉多酶机器 | CO2+H2 | 合成淀粉 | 游离 | 13 | NA | NA | NA | NA | NA | NA | 1 | [ | |
单萜烯酶 | 葡萄糖 | 单萜烯 | 游离 | 27 | NA | NA | NA | NA | NA | NA | 2 | [ | |
肌醇多酶 机器 | 淀粉 | 肌醇 | 游离 | 4-6 | 1.1 | 2.0E-05 | 5.5E+04 | 180 | 50 | 196 | 13 | [ | |
固定化 | 4-6 | 1.2 | 3.5E-06 | 3.4E+05 | 180 | 50 | 1242 | 60-80 | [ | ||||
D-塔格糖 多酶机器 | 淀粉 | D-塔格糖 | 游离 | 5-6 | NA | NA | NA | NA | NA | NA | 35 | [ | |
固定化 | 4-6 | NA | NA | NA | NA | NA | NA | 103 | [ | ||||
纤维素酶 (混合) | (预处理)生物质 | D-葡萄糖 | 游离 | 3-10 | NA | NA | NA | NA | NA | NA | 50-100 | [ | |
淀粉酶 (混合) | (糊化)淀粉 | D-葡萄糖 | 游离 | 2-3 | NA | NA | NA | NA | NA | NA | 5000 | [ |
产物 | 细胞工厂 | PX 值 |
---|---|---|
重组蛋白 | 大肠杆菌 | 0.1-0.2 |
全蛋白(可食用) | SCP生产菌 | 0.4-0.65 |
α-淀粉酶 | 枯草芽孢杆菌 | 0.5-2 |
糖化酶 | 黑曲霉 | 0.5-2 |
植酸酶 | 毕赤酵母 | 1 |
青霉素 | 产黄青霉 | 1-2 |
蛋白酶 | 枯草芽孢杆菌 | 1-3 |
丁二酸 | 大肠杆菌 | 2-3 |
纤维素酶 | 里氏木霉 | 2-4 |
赖氨酸 | 大肠杆菌 | 2-4 |
谷氨酸 | 谷棒杆菌 | 3-4 |
乙醇 | 酿酒酵母 | 5-10 |
乳酸 | 大肠杆菌 | 5-10 |
柠檬酸 | 黑曲霉 | 8-10 |
L-丙氨酸 | 大肠杆菌 | ~10 |
表3 细胞工厂生物制造代表性产品的PX值现状
Table 3 Current PX values for representative products in cell factory biomanufacturing
产物 | 细胞工厂 | PX 值 |
---|---|---|
重组蛋白 | 大肠杆菌 | 0.1-0.2 |
全蛋白(可食用) | SCP生产菌 | 0.4-0.65 |
α-淀粉酶 | 枯草芽孢杆菌 | 0.5-2 |
糖化酶 | 黑曲霉 | 0.5-2 |
植酸酶 | 毕赤酵母 | 1 |
青霉素 | 产黄青霉 | 1-2 |
蛋白酶 | 枯草芽孢杆菌 | 1-3 |
丁二酸 | 大肠杆菌 | 2-3 |
纤维素酶 | 里氏木霉 | 2-4 |
赖氨酸 | 大肠杆菌 | 2-4 |
谷氨酸 | 谷棒杆菌 | 3-4 |
乙醇 | 酿酒酵母 | 5-10 |
乳酸 | 大肠杆菌 | 5-10 |
柠檬酸 | 黑曲霉 | 8-10 |
L-丙氨酸 | 大肠杆菌 | ~10 |
图2 酶催化产品的代表性PE值与底物-酶的可溶性(Soluble enzyme-游离酶;Water-soluble substrate-水溶性底物;Immobilized enzyme-固定化酶;Solid substrate-固态底物;Enzymatic starch hydrolysis-淀粉酶水解;Immobolized xylose isomerase-葡萄糖-果糖异构酶;Enzymatic cellulose hydrolysis-纤维素酶水解;Immobilized cellulase on cellulose-固定化纤维素酶水解纤维素,数据来源参见表2)
Fig. 2 Representative PE values of enzyme-catalyzed products and the solubility of substrate-enzyme(Data source: refer to Table 2)
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