三.定向进化的选择策略 关键突变和筛选 突变—采用回交法 筛选蛋白酶选择平板初选,配合活性染色和Ⅹ 光片消化分析
– 关键突变和筛选 – 突变——采用回交法 – 筛选蛋白酶选择平板初选，配合活性染色和X 光片消化分析
Ancestral gene Natural evolution Species 4 Species 3 Species 2 Species 1 DNA shuffling Hybrid gene DNA shuffling
DNA shuffling
DNA shunning Methodology Famdy of related DNA sequences Pool of random DNA fragments Reassemble 体外定向进化 Large labrary of elect or screen Select positive Remove negative combinations of mutations combinations of mutations
体外定向进化
expression mutagenesIs library of bacteia mutant genes colonies on in a test agar plate ∝ screening colony for picking enantiose(x lectivity xlx_xo bacterial produc ing visualization of mutant enz ymes grown posi tive mutants in nutrient broth
Increasing the ee-value of the lipase catalyed hydrolysis of the chiral ester 75 81% 57% 31 2% 0 4 mutant generations
Increasing the ee-value of the lipasecatalyed hydrolysis of the chiral ester 2% 31% 57% 75% 81% 0% 0 1 2 3 4 mutant generations % e e