Biotechnology Bulletin ›› 2020, Vol. 36 ›› Issue (11): 173-180.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0123
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CAI Yu-zhen(), BAI Qiao-yan, SU Min, TANG Liang-hua()
Received:
2020-02-11
Online:
2020-11-26
Published:
2020-11-20
Contact:
TANG Liang-hua
E-mail:2968128035@qq.com;biotlh@fjnu.edu.cn
CAI Yu-zhen, BAI Qiao-yan, SU Min, TANG Liang-hua. Strategies and Advances in the Molecular Modification of Substrate Binding Pocket of Lipase[J]. Biotechnology Bulletin, 2020, 36(11): 173-180.
改造策略 | 脂肪酶 | 酶性质的改变 | 突变位点 | 突变结果 | 参考文献 | |
---|---|---|---|---|---|---|
理性设计 | 改变氨基酸 侧链大小 | Malassezia globosalipase(MgMDL2) | 位置选 择性 | F278 E282 | F278A和E282A获得水解三酰甘油的能力 | [3] |
Candida Antarcticalipase A(CALA) | 底物链 长偏好 | G237 | G237V 和G237Y对pNP-C4:0和pNP-C6:0水解活性显著提高,其中G237Y对pNP-C6:0的水解活性高WT的3倍 | [5] | ||
Lipase from Streptomyces sp. strain W007(MAS1) | 底物链 长偏好 | H108 F153 V233 | H108A、F153A和V233A对pNP-C8的kcat/Km分别是WT的2.3、2.1、1.4倍;对pNP-C16的比活分别比WT提高了3.0、2.2、2.0倍 | [6] | ||
Candida antarcticalipase B(CALB) | 立体选 择性 | W104 | 对heptan-4-ol和 nonan-5-ol的kcat/Km分别提高了270倍和5 500倍 | [8] | ||
改变底物结合 口袋的亲、 疏水性 | Rhizopus chinensislipase(RCL) | 底物链长、不饱和脂肪酸偏好 | H284 L285 | 突变体HQL(H284 和L285之间插入Q)和L285Q对p-NPP(C16)的催化活性分别为WT的2.72倍和1.5倍;HQL水解不饱和脂肪酸的活性是饱和脂肪酸1.45倍,而WT只有1.10倍 | [7] | |
Geobacillus zalihaelipase(T1 lipase) | 对映选 择性 | Q114 | Q114M催化(R,S)-布洛芬与油酸醇的选择性酯化反应中E值为WT的3.2倍 | [9] | ||
Geobacillus zalihae lipase(T1 lipase) | 催化活性 | Q114 | Q114L催化的丁酸薄荷酯的酯化反应的转化率能够达到92% | [10] | ||
增加π环体系的非经典相互作用 | Rhizomucor mieheilipase(RML) | 催化活性 | P209 L258 | 对硝基苯酯(C6)的比活提高了3.98倍 | [14] | |
半理性设计 | 定点饱和诱变 | Candida rugosalipase(LIP2) | 底物链 长偏好 | L132 | L132A和L132I的底物特异性表现出向中长链的脂肪酸转移 | [15] |
组合活性中心饱和突变(CAST)/简并密码子NDT | Candida Antarcticalipase A(CALA) | 对映选 择性 | F233 | 以2-苯基丙酸对硝基苯基酯为底物的催化反应中,F233G的E值高达259 | [16] | |
迭代饱和突变(ISM) | Bacillus subtilislipase(Lip A) | 热稳定性 | R33/D34/K35 K112/ M134 Y139/I157 | 以4-硝基苯基辛酸酯底物测得两个最佳突变体(M134D/I157M/Y139C/K112D/R33Q/D34N/K35D)和(M134D/I157M/Y139C/K112D /R33G)的T5060分别为89℃和93℃,远高于WT(T5060为48℃) | [17] | |
ISM/简并密码子NDT | Candida antarcticalipase B(CALB) | 立体选 择性 | W104/L144 V149/V154 I189/V190 A281/A282 | (V149D/I189V/V190C/A281G/A282V)对2-苯基丙酸对硝基苯酯的kcat / Km为WT的近277倍,突变体(W104C/L144Y/V149I/V154I/A281C/A282F)对R-2-苯基丙酸对硝基苯酯的活性比WT高15倍 | [18] | |
理性聚焦迭代定点诱变(FRISM) | Candida antarcticalipase B(CALB) | 对映选 择性 | W104/ D134 Q157/I189 V190/ A281 A282 | 突变体(A281G/A282V/V190C)、(Q157L/I189A)、 (W104/I189)、(W104A/I189M/V190C/D134L)对各自的最佳对映体的选择性分别高达95%、94%、95%和91% | [19] |
改造策略 | 脂肪酶 | 酶性质的改变 | 突变位点 | 突变结果 | 参考文献 | |
---|---|---|---|---|---|---|
理性设计 | 改变氨基酸 侧链大小 | Malassezia globosalipase(MgMDL2) | 位置选 择性 | F278 E282 | F278A和E282A获得水解三酰甘油的能力 | [3] |
Candida Antarcticalipase A(CALA) | 底物链 长偏好 | G237 | G237V 和G237Y对pNP-C4:0和pNP-C6:0水解活性显著提高,其中G237Y对pNP-C6:0的水解活性高WT的3倍 | [5] | ||
Lipase from Streptomyces sp. strain W007(MAS1) | 底物链 长偏好 | H108 F153 V233 | H108A、F153A和V233A对pNP-C8的kcat/Km分别是WT的2.3、2.1、1.4倍;对pNP-C16的比活分别比WT提高了3.0、2.2、2.0倍 | [6] | ||
Candida antarcticalipase B(CALB) | 立体选 择性 | W104 | 对heptan-4-ol和 nonan-5-ol的kcat/Km分别提高了270倍和5 500倍 | [8] | ||
改变底物结合 口袋的亲、 疏水性 | Rhizopus chinensislipase(RCL) | 底物链长、不饱和脂肪酸偏好 | H284 L285 | 突变体HQL(H284 和L285之间插入Q)和L285Q对p-NPP(C16)的催化活性分别为WT的2.72倍和1.5倍;HQL水解不饱和脂肪酸的活性是饱和脂肪酸1.45倍,而WT只有1.10倍 | [7] | |
Geobacillus zalihaelipase(T1 lipase) | 对映选 择性 | Q114 | Q114M催化(R,S)-布洛芬与油酸醇的选择性酯化反应中E值为WT的3.2倍 | [9] | ||
Geobacillus zalihae lipase(T1 lipase) | 催化活性 | Q114 | Q114L催化的丁酸薄荷酯的酯化反应的转化率能够达到92% | [10] | ||
增加π环体系的非经典相互作用 | Rhizomucor mieheilipase(RML) | 催化活性 | P209 L258 | 对硝基苯酯(C6)的比活提高了3.98倍 | [14] | |
半理性设计 | 定点饱和诱变 | Candida rugosalipase(LIP2) | 底物链 长偏好 | L132 | L132A和L132I的底物特异性表现出向中长链的脂肪酸转移 | [15] |
组合活性中心饱和突变(CAST)/简并密码子NDT | Candida Antarcticalipase A(CALA) | 对映选 择性 | F233 | 以2-苯基丙酸对硝基苯基酯为底物的催化反应中,F233G的E值高达259 | [16] | |
迭代饱和突变(ISM) | Bacillus subtilislipase(Lip A) | 热稳定性 | R33/D34/K35 K112/ M134 Y139/I157 | 以4-硝基苯基辛酸酯底物测得两个最佳突变体(M134D/I157M/Y139C/K112D/R33Q/D34N/K35D)和(M134D/I157M/Y139C/K112D /R33G)的T5060分别为89℃和93℃,远高于WT(T5060为48℃) | [17] | |
ISM/简并密码子NDT | Candida antarcticalipase B(CALB) | 立体选 择性 | W104/L144 V149/V154 I189/V190 A281/A282 | (V149D/I189V/V190C/A281G/A282V)对2-苯基丙酸对硝基苯酯的kcat / Km为WT的近277倍,突变体(W104C/L144Y/V149I/V154I/A281C/A282F)对R-2-苯基丙酸对硝基苯酯的活性比WT高15倍 | [18] | |
理性聚焦迭代定点诱变(FRISM) | Candida antarcticalipase B(CALB) | 对映选 择性 | W104/ D134 Q157/I189 V190/ A281 A282 | 突变体(A281G/A282V/V190C)、(Q157L/I189A)、 (W104/I189)、(W104A/I189M/V190C/D134L)对各自的最佳对映体的选择性分别高达95%、94%、95%和91% | [19] |
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