Deacetoxycephalosporin C hydroxylase mutants, DNA encoding the mutants, method for utilizing the mutants and application thereof

In order to remove the ampicillin resistance gene from pRSET-A vector (Invitrogen), VF and VR primers (table 1) were designed based on pRSET-A sequence.

In order to obtain the kanamycin resistance gene from pET-28b vector (Novogen), KF and KR primers (table 1) were designed based on the kanamycin resistance gene sequence of pET-28b.

Specifically, DNA fragment PR was amplified using primers VF and VR and the pRSET-A plasmid as template. DNA fragment KAN was amplified using primers KF and KR and the pET-28b plasmid as template.

PCR amplification conditions for PR are as follows: 1 μg of plasmid pRSET-A, 0.1 μg of primers (VF+VR), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols for PR are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 3 mins, 72° C. for 10 mins at the end.

After 1% (w/v) agarose gel electrophoresis, a 2036 bp PCR product of PR was extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.).

PCR amplification conditions for KAN are as follows: 1 μg of plasmid pET-28b, 0.1 μg of primers (KF+KR), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols for KAN are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 3 mins, 72° C. for 10 mins at the end. After 1% (w/v) agarose gel electrophoresis, a 816 bp PCR product of KAN was extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.).

Both PR and KAN DNA fragments were ligated using T4 DNA ligase (NEB) and plasmid PK was obtained. The plasmid was transformed into E. coli BL21 competent cells (Novagen). The transformed cells were incubated on LB plates with 50 mg/L kanamycin at 37° C. PK plasmids were obtained from single colony and extracted using DNA-spin plasmid DNA purification kit (Intron Biotechnology). The correct sequence was confirmed with DNA sequencing (sequence list SEQ ID NO.:12).

The deacetoxycephalosporin C hydroxylase gene DNA sequence was obtained from DNA sequence database (GenBank: M63809). The DNA sequence was optimised according to host cell codon usage bias using software Gene Designer 2.0. Primers HF and HR (table 1) were designed based on the gene sequence.

The aforementioned DNA sequence was synthesized and plasmid Hd-pUC was obtained (synthesized and ligated to vector pUC57 by GenScript corp.). A 957 bp PCR amplification product was obtained using HF and HR as primers and plasmid Hd-pUC as template.

PCR amplification conditions are as follows: 1 μg of plasmid Hd-pUC, 0.1 μg of primers (HF+HR), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 3 mins, 72° C. for 10 mins at the end.

The amplified wild-type deacetoxycephalosporin C hydroxylase gene DNA were purified with 1% (w/v) agarose gel electrophoresis, a 957 bp PCR product of hd was extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.). The fragment was ligated to pGEMT-Easy (Promega) using T4 ligase (NEB) through TA cloning to create plasmid pGEMT-hd. The plasmid was transformed into E. coli BL21 competent cells (Novagen). The transformed cells were incubated on LB plates with 50 mg/L ampicillin at 37° C. pGEMT-hd plasmids were obtained from single colony and extracted using DNA-spin plasmid DNA purification kit (Intron Biotechnology). The correct sequence was confirmed with DNA sequencing.

Example 3: Amino Acid Site-Directed Mutagenesis of Deacetoxycephalosporin C Hydroxylase at Position 41

“PCR Protocols (John M. S. Bartlett and David Stirling. Totowa, N.J.: Humana Press, 2003)” was used as reference for site-directed mutagenesis techniques.

The plasmid pGEMT-hd (example 2) was used as template to design primers 41TF and 41TR (table 1). The Glycine (Gly) at position 41 in the original amino acid sequence was substituted into Threonine (Thr), to create mutant HD-G41T.

Specifically, DNA fragment 41T1 was amplified using primers HF and 41TR and plasmid pGEMT-hd as template. DNA fragment 41T2 was amplified using primers 41TF and HR. PCR amplification conditions are as follows: 1 μg of plasmid pGEMT-hd, 0.1 μg of primers (HF+41TR) (for fragment 41T1 amplification), or 0.1 μg of primers (41TF+HR) (for fragment 41T2 amplification), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 60 sec, 72° C. for 10 mins at the end. The amplified 41T1 and 41T2 DNA fragments were purified with 1% (w/v) agarose gel electrophoresis and were extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.), and then were used to amplify the full-length gene using primers HF and HR. PCR amplification conditions are as follows: 50 ng of DNA fragment 41T1 and 50 ng of DNA fragment 41T2, 0.1 μg of primers (HF+HR), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 2 mins, 72° C. for 10 mins at the end.

The full length mutated gene was purified with 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.) to obtain a 957 bp full-length mutated gene HD-G41T.

Mutants HD-G29H, HD-A40L, HD-R182D and HD-T272R were constructed using similar methods mentioned above, with primers referred in table 1.

The mutant DNA HD-G41T and the vector PK were digested by restriction enzymes NdeI+BglII (NEB) and purified by DNA gel electrophoresis in 1% (w/v) agarose gel. The restriction enzyme digested products were extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.). They were ligated using T4 ligase (NEB) and transformed into E. coli HB101 competent cells (Bio-Rad). The transformed cells were incubated on LB plates with 50 mg/L kanamycin at 37° C. PK-HD-G41T plasmids were obtained from single colony and extracted using DNA-spin plasmid DNA purification kit (Intron Biotechnology). The correct sequence was confirmed with DNA sequencing.

The plasmids PK-HD-G29H, PK-HD-A40L, PK-HD-R182D and PK-HD-T272R were constructed using similar methods mentioned above.

Example 5: Construction of Deacetoxycephalosporin C Hydroxylase Plasmid PK-HD-29H41T with Two Mutations Combination

“PCR Protocols (John M. S. Bartlett and David Stirling. Totowa, N.J.: Humana Press, 2003)” was used as reference for site-directed mutagenesis techniques.

The plasmid PK-HD-G41T (example 4) was used as template to perform substitution of Glycine (Gly) at position 29 with Histidine (His), to create mutant HD-29H41T.

Specifically, PK-HD-G41T was used as template to amplify fragment 29H41T1 using primers HF and 29HR, and fragment 29H41T2 using primers 29HF and HR. PCR amplification conditions are as follows: 1 μg of plasmid PK-HD-G41T, 0.1 μg of primers (HF+29HR) (for fragment 29H41T1 amplification), or 0.1 μg of primers (29HF+HR) (for fragment 29H41T2 amplification), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 60 sec, 72° C. for 10 mins at the end.

The amplified 29H41T1 and 29H41T2 DNA fragments were purified with 1% (w/v) agarose gel electrophoresis and were extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.), and then were used to amplify the full-length gene using primers HF and HR. PCR amplification conditions are as follows: 50 ng of DNA fragment 29H41T1 and 50 ng of DNA fragment 29H41T2, 0.1 μg of primers (HF+HR), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 2 mins, 72° C. for 10 mins at the end.

The amplified full length mutated gene were purified with 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.) to obtain a 957 bp full-length mutated gene HD-29H41T.

NdeI and BglII (NEB) were used to digest the mutant gene HD-29H41T and vector PK. The digested products were purified by 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.). Ligated using T4 ligase (NEB) and transformed into E. coli HB101 competent cells (Bio-Rad). The transformed cells were incubated on LB plates with 50 mg/L kanamycin at 37° C. PK-HD-29H41T plasmids were obtained from single colony and extracted using DNA-spin plasmid DNA purification kit (Intron Biotechnology). The correct sequence was confirmed with DNA sequencing.

Example 6: Construction of Deacetoxycephalosporin C Hydroxylase Plasmid PK-HD-Sp3 with Three Mutations Combination

“PCR Protocols (John M. S. Bartlett and David Stirling. Totowa, N.J.: Humana Press, 2003)” was used as reference for site-directed mutagenesis techniques.

The plasmid PK-HD-29H41T (example 5) was used as template to perform substitution of Threonine (Thr) at position 272 with Arginine (Arg), to create mutant HD-sp3.

Specifically, PK-HD-29H41T was used as template to amplify fragment sp3-1 using primers HF and 272RR, and fragment sp3-2 using primers 272RF and HR. PCR amplification conditions are as follows: 1 μg of plasmid PK-HD-29H41T, 0.1 μg of primers (HF+272RR) (for fragment sp3-1 amplification), or 0.1 μg of primers (272RF+HR) (for fragment sp3-2 amplification), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 60 sec, 72° C. for 10 mins at the end.

The amplified sp3-1 and sp3-2 DNA fragments were purified with 1% (w/v) agarose gel electrophoresis and were extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.), and then were used to amplify the full-length gene using primers HF and HR. PCR amplification conditions are as follows: 50 ng of DNA fragment sp3-1 and 50 ng of DNA fragment sp3-2, 0.1 μg of primers (HF+HR), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 2 mins, 72° C. for 10 mins at the end.

The amplified full length mutated gene were purified with 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.) to obtain a 957 bp full-length mutated gene HD-sp3.

NdeI and BglII were used to digest the mutant gene HD-sp3 and the vector PK. The digested products were purified by 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.). Ligated using T4 ligase (NEB) and transformed into E. coli HB101 competent cells (Biorad). The transformed cells were incubated on LB plates with 50 mg/L kanamycin at 37° C. PK-HD-sp3 plasmids were obtained from single colony and extracted using DNA-spin plasmid DNA purification kit (Intron Biotechnology). The correct sequence was confirmed with DNA sequencing.

Example 7: Construction of Deacetoxycephalosporin C Hydroxylase Plasmid PK-HD-Sp4 with Four Mutations Combination

“PCR Protocols (John M. S. Bartlett and David Stirling. Totowa, N.J.: Humana Press, 2003)” was used as reference for site-directed mutagenesis techniques.

The plasmid PK-HD-sp3 (example 6) was used as template to perform substitution of Alanine (Ala) at position 40 with Leucine (Leu), to create mutant HD-sp4.

Specifically, PK-HD-sp3 was used as template to amplify fragment sp4-1 using primers HF and 40LR, and fragment sp4-2 using primers 40LF and HR. PCR amplification conditions are as follows: 1 μg of plasmid PK-HD-sp3, 0.1 μg of primers (HF+40LR) (for fragment sp4-1 amplification), or 0.1 μg of primers (40LF+HR) (for fragment sp4-2 amplification), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 60 sec, 72° C. for 10 mins at the end.

The amplified sp4-1 and sp4-2 DNA fragments were purified with 1% (w/v) agarose gel electrophoresis and were extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.), and then were used to amplify the full-length gene using primers HF and HR. PCR amplification conditions are as follows: 50 ng of DNA fragment sp4-1 and 50 ng of DNA fragment sp4-2, 0.1 μg of primers (HF+HR), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 2 mins, 72° C. for 10 mins at the end.

The full length mutated gene were purified with 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.) to obtain a 957 bp full-length mutated gene HD-sp4.

NdeI and BglII were used to digest the mutant gene HD-sp4 and vector PK. The digested products were purified by 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.). Ligated using T4 ligase (NEB) and transformed into E. coli HB101 competent cells (Biorad). The transformed cells were incubated on LB plates with 50 mg/L kanamycin at 37° C. PK-HD-sp4 plasmids were obtained from single colony and extracted using DNA-spin plasmid DNA purification kit (Intron Biotechnology). The correct sequence was confirmed with DNA sequencing.

Example 8: Construction of Deacetoxycephalosporin C Hydroxylase Plasmid PK-HD-Sp5 with Five Mutations Combination

“PCR Protocols (John M. S. Bartlett and David Stirling. Totowa, N.J.: Humana Press, 2003)” was used as reference for site-directed mutagenesis techniques.

The plasmid PK-HD-sp4 (example 7) was used as template to perform substitution of Arginine (Arg) at position 182 with Aspartic acid (Asp), to create mutant HD-sp5.

Specifically, PK-HD-sp4 was used as template to amplify fragment sp5-1 using primers HF and 182DR, and fragment sp5-2 using primers 182DF and HR. PCR amplification conditions are as follows: 1 μg of plasmid PK-HD-sp4, 0.1 μg of primers (HF+182DR) (for fragment sp5-1 amplification), or 0.1 μg of primers (182DF+HR) (for fragment sp5-2 amplification), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 60 sec, 72° C. for 10 mins at the end.

The amplified sp5-1 and sp5-2 DNA fragments were purified with 1% (w/v) agarose gel electrophoresis and were extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.), and then were used to amplify the full-length gene using primers HF and HR. PCR amplification conditions are as follows: 50 ng of DNA fragment sp5-1 and 50 ng of DNA fragment sp5-2, 0.1 μg of primers (HF+HR), 5 μl of 10× buffer (200 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100), 4% DMSO, 4 μl of 2.5 mM dNTP, 1 U of LA Taq DNA polymerase (TaKaRa), sterilized water to make up to 50 μl reaction.

PCR amplification protocols are as follows: 96° C. for 5 mins, 30 cycles of: 94° C. for 45 sec, 53° C. for 45 sec and 72° C. for 2 mins, 72° C. for 10 mins at the end.

The full length mutated gene were purified with 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.) to obtain a 957 bp full-length mutated gene HD-sp5.

NdeI and BglII (NEB) were used to digest the mutant gene HD-sp5 and vector PK. The digested products were purified by 1% (w/v) agarose gel electrophoresis, extracted and purified using E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc.). Ligated using T4 ligase (NEB) and transformed into E. coli HB101 competent cells (Biorad). The transformed cells were incubated on LB plates with 50 mg/L kanamycin at 37° C. PK-HD-sp5 plasmids were obtained from single colony and extracted using DNA-spin plasmid DNA purification kit (Intron Biotechnology). The correct sequence was confirmed with DNA sequencing.

The wild-type deacetoxycephalosporin C hydroxylase plasmid and various deacetoxycephalosporin C hydroxylase mutants plasmids mentioned above were transformed into E. coli BL21 (Novagen), respectively, and incubated on LB plates with 50 mg/L kanamycin at 37° C. The transformed cells from single colony were inoculated to 3 ml LB broth with 50 mg/L kanamycin and incubated at 37° C., 250 rpm for 8 hours, followed by taking 1 ml and inoculated to 50 ml LB broth with 50 mg/L kanamycin and incubated at 37° C., 250 rpm for 18 hours. The cells were centrifuged and resuspended in 10 mM pH 7.4 sodium phosphate buffer, followed by cell sonication (50W) lysis with sonication time for 5 sec, 30 times. Cell debris was removed by centrifugation and supernatant were collected as liquid enzyme. The liquid enzyme was tested for deacetoxycephalosporin C hydroxylase enzymatic activity and for enzyme expression by SDS-PAGE. The following are the details:

900 μl of substrate (10 mM G-7-ADCA, 20 mM sodium α-ketoglutarate, 4 mM sodium L-ascorbate and 1.8 mM ferrous sulfate heptahydrate, 6 mM sodium phosphate buffer (pH 7.5)) were added to 1.5 ml eppendorf tube, followed by adding 100 μl of the above mentioned liquid enzyme. The reaction solution was mixed well and shaken at 200 rpm in a thermoshaker at 30° C. for 30 mins. 10 μl supernatant was taken and added into 990 μl H2O, mixed, followed by UPLC analysis for G-D-7ACA concentration and the enzymatic activity was calculated. The activities of wild-type deacetoxycephalosporin C hydroxylase and various deacetoxycephalosporin C hydroxylase mutants were compared as shown in table 2.

UPLC analysis conditions were as follows: Acquity UPLC column: Acquity UPLC BEH C18 column (Waters Corporation, 1.7 μm, guard column: 2.1 mm×5 mm, column: 2.1 mm×50 mm); mobile phase: (A) 50 mM KH2PO4/K2HPO4 (pH 7.0), 6% acetonitrile, (B) 60% acetonitrile; column temperature: 40° C.; flowrate: 0.3 ml/min; detection wavelength: 260 nm.

Liquid enzymes of wild-type deacetoxycephalosporin C hydroxylase and the various deacetoxycephalosporin C hydroxylase mutants were prepared according to example 9. 200 μl liquid enzymes were added into 1.5 ml eppendorf tubes and were placed in 42° C. water bath for 10 mins of heat treatment. The samples were centrifuged and supernatant were collected for enzymatic activity assay according to example 9. The remaining enzymatic activity of wild-type deacetoxycephalosporin C hydroxylase and of various deacetoxycephalosporin C hydroxylase mutants were calculated by dividing activity of heat treated deacetoxycephalosporin C hydroxylase by activity of untreated deacetoxycephalosporin C hydroxylase and presented it as percentage. The percentage of increased enzyme thermostability was calculated by the following formula. Table 3 in the following indicated the remaining enzymatic activity of wild-type and various deacetoxycephalosporin C hydroxylase mutants after heat treatment.

A =( B−C )/ C× 100%=( B− 2%)/2%×100%

Where:

A—Percentage of increased thermostability

B—The remaining activity of deacetoxycephalosporin C hydroxylase mutant after the heat treatment

C—the remaining activity of wild-type after the heat treatment

Substrate was prepared with the following final concentration of components: 10 mM G-7-ADCA, 20 mM sodium α-ketoglutarate, 4 mM sodium L-ascorbate and 1.8 mM ferrous sulfate heptahydrate were dissolved in 90 ml 6 mM pH 7.4 sodium phosphate buffer. 1M NaOH was used to adjust the pH to 6, followed by adding 10 ml liquid enzyme. The reaction was placed on magnetic stir plate and stirred at high speed. The reaction was maintained at 30° C. and pH 6.4 for 150 mins. 1 ml sample was taken at 30, 60, 90, 120 and 150 mins from the reaction. The reaction samples were centrifuged at 13000 rpm for 1 min and 10 μl supernatant was taken and added into 990 μl H2O, mixed, followed by UPLC analysis for G-D-7ACA concentration.

UPLC analysis conditions were as follows: Acquity UPLC column: Acquity UPLC BEH C18 column (Waters Corporation, 1.7 μm, guard column: 2.1 mm×5 mm, column: 2.1 mm×50 mm); mobile phase: (A) 50 mM KH2PO4/K2HPO4 (pH 7.0), 6% acetonitrile, (B) 60% acetonitrile; column temperature: 40° C.; flow rate: 0.3 ml/min; detection wavelength: 260 nm.

This invention is not limited by the detailed description provided in the text above. Various modifications can be made and these modifications should also be regarded as being within the scope of the invention, which is defined by the claims.