Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (3): 121-129.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0471
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FU Ya-li(), PENG Wan-li, LIN Shuang-jun, DENG Zi-xin, LIANG Ru-bing()
Received:
2021-04-11
Online:
2022-03-26
Published:
2022-04-06
Contact:
LIANG Ru-bing
E-mail:fuyali_1502@163.com;icelike@sjtu.edu.cn
FU Ya-li, PENG Wan-li, LIN Shuang-jun, DENG Zi-xin, LIANG Ru-bing. Gene Cloning and Enzymatic Properties of the Short Chain Dehydrogenase SDR-X1 from Pseudomonas citronellolis SJTE-3[J]. Biotechnology Bulletin, 2022, 38(3): 121-129.
Fig. 1 Multiple sequence alignment,advanced structural model and evolutionary analysis of SDR-X1 protein A:Multiple sequence alignment of short chain dehydrogenase from different microorganisms. Protein SDR-X1(WP_043267487.1)from P. citronellol SJTE-3,protein CAD85559.1 from N. europaea ATCC19718,protein WP_010595991.1 and WP_010595922.1 from R. ruber P14,protein WP_061563290.1 from P. citronellolis P3B5,protein WP_008027336.1 from P. putida B6-2,protein ABQ79984.1 from Pseudomonas putida F1,protein WP_012271104.1 from Pseudomonas putida GB-1,protein WP_014754112.1/ANI02794.1/ANI04816.1 from P. putida SJTE-1,protein WP_057091786.1 from C.testosteroni P19,and protein WP_010128053.1 from Sphingomonas KC8. The spiral marks the α-helix and the arrow marks the β-sheet. B:Having 4iqg.1.A as a model,and a three-dimensional model of protein obtained from homologous modeling of protein SDR-X1 with SWISSMODEL. C:Evolutionary analysis of the protein SDR-X1
Fig. 2 Transcription levels of the gene sdr-x1 in strain SJTE-3 with different steroid hormones as carbon sources The carbon source is 10 μg/mL of steroid hormones(E1,E2,EE2 or TES)or 0.1% ethanol,and the transcription level of the gene sdr-x1 in ethanol as carbon source medium is set as 1.0. The results of fluorescence quantitative PCR amplification were processed by 2-∆∆Ct method. Three parallel experiments were set for each group and repeated three times,and based on which the mean and standard error were calculated
Fig. 3 Efficacy determination of recombinant strain BL21-SDR-X1 for transforming E2,purification of recombinant protein SDR-X1 and its transformation reaction to E2 A:The efficacy determination of recombinant strain BL21-SDR-X1 for transforming E2. The conversion efficiency of strain BL21-SDR-X1 to 10 μg/mL 17β-estradiol at 3,6,12,18 and 24 h is detected by HPLC. The control group is BL21(DE3)strain with plasmid pET28a. Three parallel experiments are set in each group and repeated for three times,and based on it the mean and standard error are calculated. B:Electrophoresis detection of affinity-purified recombinant protein SDR-X1. Lane 1:protein maker;lane 2:cell lysis solution before induction;lane 3:cell lysis solution after induction;lane 4:centrifugal precipitation of the crushed cells;lane 5:centrifugal supernatant of the crushed cells;lane 6-7:the solution after column loading;lane 8-9:the impurities;lane 10:protein eluent. C:Kinetic curve of recombinant protein SDR-X1 transforming E2. Using E2 as substrate,the effect of protein SDR-X1 on different concentration(0.0100,0.0125,0.0150,0.0200,0.0250,0.0300 0,0.0500 0,0.075,and 0.1 mmol/L)substrate is determined. Three parallel experiments are set for each group and repeated three times,and the mean and standard error are calculated
Fig. 4 Determination of the enzymatic properties of E2 transformed by recombinant enzyme SDR-X1 A:Determination of optimal reaction temperature. The conversion efficiency of recombinase SDR-X1 at different temperature(30,40,50,and 60℃)was detected with E2 as substrate. B:Temperature tolerance was measured. The conversion efficiency of recombinase SDR-X1 treated with different temperature(37,42,and 50℃)and time(30,60,and 90 min)was detected with E2 as substrate. C:Optimal reaction pH determination. The conversion efficiency of recombinase SDR-X1 at different pH value(pH4-11)was detected with E2 as substrate. D:Determination of influence of divalent metal ions. The conversion efficiency of recombinase SDR-X1 under the action of different metal ion(Zn2+,Mn2+,Mg2+,Ni2+,Ca2+,and Cu2+)at 1 mmol/L was determined. Three experiments were set in parallel for each group and repeated for three times. The mean value and standard error are calculated
Fig. 5 HPLC analysis of reaction products of conversion of E2 by recombinase SDR-X1 A:The HPLC map of 50 μg/mL E2. B:The HPLC map of 50 μg/mL E1. C:The HPLC map of the products of E2 convered by recombinase SDR-X1. The reaction solution is 0.1 mmol/L E2,0.2 mmol/L NAD+ and 100 μg/mL protein SDR-X1 in a 200 μL reaction system at 42℃ for 15 min. Then adding 1/3 volume of acetonitrile to the reaction solution,swirling and mixing,50 μL sample is taken to detect degradation of E2 by HPLC
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