Universal strategy for efficiently improving enzyme thermodynamics stability

A thermodynamic and stable technology, applied in the direction of enzymes, hydrolases, biochemical equipment and methods, etc., can solve the problems of loss of catalytic reaction function, easy inactivation and denaturation of LIP1, weak stability restricting application, etc., and achieve half-life improvement. , the effect of improving stability and optimum temperature

Active Publication Date: 2016-11-09
JIAOHONG BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, the weak stability of LIP1 limits its application under industrial transformation conditions such as high temperature and high pressure (Chang, S.W. et al. Codon optimization of Candida rugosa LIP1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase[J]. Journal of Agricultural and Food Chemistry, 2006, 54(3): 815-822.)
LIP1 with poor stability is easy to inactivate and denature, and loses the function of catalytic reaction

Method used

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  • Universal strategy for efficiently improving enzyme thermodynamics stability
  • Universal strategy for efficiently improving enzyme thermodynamics stability
  • Universal strategy for efficiently improving enzyme thermodynamics stability

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, the selection of mutation site

[0040] The wild-type (WT) amino acid sequence of Candida rugosa lipase 1 involved in the present invention is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 9.

[0041] The crystal structure of LIP1 was searched in the PDB database, and the structure with the highest resolution was selected as the research object. Since LIP1 has a helical segment called "lid" above the active pocket, resulting in two structures of LIP1 in solution: Open and Closed, the two structures were analyzed simultaneously when selecting the target site. Both are centered on the catalytic residue Ser209, and the surrounding All amino acids within (use PyMOL command: PyMOL->select AA, polymer within 10 of Ser209 to find). Then use the B-FITTER software to analyze the B-factor values ​​of these amino acids, arrange them in order of value from large to small, and select the top 11 amino acids with the largest B-factor in eac...

Embodiment 2

[0045] Example 2, site-directed saturation mutation construction gene mutation library

[0046] The primers for constructing the saturation mutation library are shown in Table 2.

[0047] Table 2 is used to construct the primer of saturation mutant library

[0048]

[0049]

[0050] Under the action of PrimerSTAR max DNA polymerase (TaKaRa Company), the whole plasmid was amplified using the wild-type gene of LIP1 as a template. PCR reaction conditions: pre-denaturation at 98°C for 5 min, each cycle of denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 2.5 min, a total of 30 cycles; the final extension at 72°C for 5 min. After the PCR product was purified, it was directly transformed into Escherichia coli DH5α competent, heat-shocked at 42°C for 90s, and incubated at 37°C for 1h. Then spread it on the LB plate containing 25μg / ml Zeocin resistance. After the clones grow out, directly wash all the clones from the plate with sterile water, coll...

Embodiment 3

[0051] Embodiment 3, the preparation of Pichia pastoris competent cell

[0052] Pick a single colony of Pichia pastoris from the YPD plate, inoculate it in 4ml of YPD liquid medium, and cultivate it with shaking at 30°C overnight for about 24-48 hours, and the OD is about 6-8. Dispense the culture solution into 1.5ml EP tubes, centrifuge at 4°C, 4000rpm for 5min, and discard the supernatant. The precipitate was washed with ice-cold sterile water, centrifuged at 4°C and 4000 rpm for 5 min, and the supernatant was discarded. The precipitate was treated with 1ml of lithium acetate solution, and after the precipitate was resuspended, it was placed at 30°C for 30min. Then centrifuge at 4°C, 4000rpm for 5min, and discard the supernatant. (Lithium acetate solution: 10mM pH7.5 Tris-HCL; 10mM DTT; 100mM LiAC) The precipitate was washed with ice-cold sterile water, centrifuged at 4000rpm for 5min at 4°C, and the supernatant was discarded. The precipitate was washed with ice-cold 1M D...

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Abstract

The invention discloses a universal strategy for efficiently improving enzyme thermodynamics stability. The strategy is specifically implemented in the way of taking a catalytic residue of zymophore as the center, selecting a high-flexibility residue 10 angstroms away from the center at most as a target locus according to the B-factor value in an enzyme crystal structure, and obtaining a mutant with enzyme thermodynamics stability improved through mutation screening. Under the guidance of the strategy, the thermal stability of candida rugosa lipase 1 with a highly complicated structure is improved remarkably. By means of stability change results of protein (LipA) with the smallest structure and moderately complicated protein (CalB), it is found that the success rate of enzyme mutation can be increased remarkably through mutation of residues with high B-factor 6-10 angstroms away from the catalytic residue. Meanwhile, through system analysis of a mutant structure-function relationship, a foundation is laid for deep research of enzyme region stabilization law and development of enzyme stabilization technique.

Description

technical field [0001] The present invention relates to a general strategy for efficiently improving the thermodynamic stability of enzymes. Analysis of enzyme catalytic residues by Pymol and B-FITTER software Amino acids with high flexibility within; mutants with improved thermodynamic stability were screened with the help of a saturated mutation library. Specifically, it relates to a mutant of Candida rugosa lipase 1, and the structure and function analysis of the mutant. Background technique [0002] Enzyme is a highly efficient biocatalyst. Compared with chemical catalysts, enzyme molecules have the advantages of high efficiency, high specificity, mild reaction conditions and no pollution. They have important application value in scientific research and industrial production. However, under harsh conditions such as high pressure, high temperature, and extreme pH, enzymes are prone to disintegration and loss of activity, which severely limits the wide application of en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N9/20C12Q1/00C12Q1/34
CPCC12N9/00C12N9/20C12Q1/00C12Q1/34
Inventor 冯雁张小飞杨广宇张勇谢渊
Owner JIAOHONG BIOTECHNOLOGY (SHANGHAI) CO LTD
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