Enhanced permeability of recombinant Escherichia coli with deep - PowerPoint PPT Presentation

Enhanced permeability of recombinant Escherichia coli with deep eutectic solvent for conversion of rutin extracted from grapefruit peel Fan Zhang, Fang ‐ Qin Wang, Jin ‐ Zheng Wang, Chang ‐ Tong Zhu, Jun Wang* School of Biotechnology, Jiangsu University of Science and Technology Sericultural Research Institute, Chinese Academy of Agricultural Sciences Zhenjiang 212018, PR China E ‐ mail: wangjun@just.edu.cn 6th International Conference on Sustainable Solid Waste Management, Naxos Island, Greece, 13–16 June 2018

Content Background Present study Conclusions Acknowledgments

Background Resource waste Flavonoids sources Grapefruit peel Rutaceae plants are rich in flavonoids, In 2017, the annual output of grapefruit such as rutin, naringin and hesperidin. in China was about 4.8 million tons Method of reutilization should be found

antihypertensive Isoquercitrin hypotensive

Biocatalysts for biotransformation Inseparable Easily to lost Free No separation and purification enzyme Reduce enzyme activity loss Whole cell catalyst Immobilized Biocatalyst Non ‐ recyclable Enzyme Activity lost

RhaB1 as catalyst 115kDa (A)The recombinant plasmid pET21a ‐ rhaB1 was extracted from transformed E.coli. Lines: M, 10 kb DNA marker; (B) pET21a ‐ rhaB1 was digested by SalI and HindIII. M ： 10 kb DNA marker; (C) SDS ‐ PAGE of whole cell lysates. Lanes: M, protein size markers; 1, purified RhaB1 protein; 2, whole cell lysates of recombinant BL21 ‐ pET21a ‐ rhaB1; 3, whole cell lysates of BL21 ‐ pET21a; (D) Western blot of whole cell lysates. Lanes: M, protein size markers; 1, purified RhaB1 protein; 2, whole cell lysates of recombinant BL21 ‐ pET21a ‐ rhaB1; 3, whole cell lysates of BL21 ‐ pET21a. Western blot analysis of the RhaB1 protein was performed using an anti ‐ His HRP ‐ conjugated antibody and goat anti ‐ mouse IgG. Wang F, He S, et al. Journal of Chemical Technology and Biotechnology Doi ： 10.1002 / jctb.5621.

Present study HOW? Problem: Cell membranes hinder intracellular and extracellular mass transfer

Table 1. Improvement strategies of cell permeability. Methods Process Mechanism Effect Refs Formation of micropores in Physical treatment Electrical treatment Electric field [1] phospholipids bilayer Chemical Solvent medium Hydrophobic interaction Total lipid content reduced by 8.3% [2] regulation Molecular biology Change the membrane Gene mutation/knockout Overexpression of ydeD [3] regulation structure and composition [1] Ritter A, Mahmoudi A E, Esser A, et al. Bioem (2016). [2] Shen Yanbing, Wang Lifang and Liang Jingting. Microbial Cell Factories 15 :118 ‐ 128 (2016). [3] Ohtsu Iwao , Li Zhao Di, Applied Microbiology Biotechnology 81 :903 ‐ 913 (2009).

Biocompatible Environmental friendly Good thermodynamic stability Enlarge cell permeability ILs/DESs E.Coli BL21 ‐ pET21a ‐ rhaB1

Table 2. The ionic liquids used in the present study. M W /g ∙ mol ‐ 1 Name Abbreviation Molecular Formula 1 ‐ butyl ‐ 3 ‐ methylimidazolium [BMIM][Tf 2 N] C 10 H 15 F 6 N 3 O 4 S 2 419.36 bis[(trifluoromethyl)sulfonyl]imide 1 ‐ butyl ‐ 3 ‐ methylimidazolium [BMIM][PF 6 ] C 8 H 15 N 2 F 6 P 284.20 hexafluorophosphate 1 ‐ Butyl ‐ 3 ‐ methylimidazolium [BMIM][BF 4 ] C 8 H 15 N 2 BF 4 226.02 tetrafluoroborate 1 ‐ Hexyl ‐ 3 ‐ methylimidazolium [HMIM][PF 6 ] C 10 H 19 F 6 N 2 P 312.24 hexafluorophosphate 1 ‐ Ethyl ‐ 3 ‐ methylimidazolium [EMIM][PF 6 ] C 6 H 11 F 6 N 2 P 256.13 hexafluorophosphate

Table 3. The deep eutectic solvent used in the present study. Name Abbreviation T f / ° C T m / ° C Choline chloride/urea (1:2) ChCl/U 12 25 Choline chloride/glycerin (1:2) ChCl/GI ‐ 35 ‐‐ Choline chloride/malonic acid (1:1) ChCl/MA 10 ‐‐ Choline chloride/ethylene glycol (1:2) ChCl/EG ‐ 66 ‐‐ Choline chloride/acetamide (1:2) ChCl/A 51 ‐‐

cell Strain Strain cell pH=5.0, Temperature: 35 ° C, Rutin concentration 0.02g/L Speed 180rpm, Time 3h. Fig. 1 Effect of ILs on growth of E. coli and catalytic ability of cultured cells (A: culturing strain; B: cultured cell). Effect of DESs on growth of E. coli and catalytic ability of cultured cells (C: culturing strain; D: cultured cell). Fermentation medium: 10 g/L of tryptone and NaCl, and 5 g/L yesat extract

Effects of different ChCl/U on the whole ‐ cell catalyst E. coli BL21 ‐ pET21a ‐ rhaB1 Fig. 2 Effects of different contents of ChCl/U on the permeability of E. coli membrane (A) and isoquercitrin yield (B). Absorbance was measured at 260 and 280 nm after centrifugation of the supernatant. Catalytic conditions: rutin concentration: 0.02g/L (pH 5.0), reaction temperature: 35 ° C, rotation speed: 180rpm, time: 3h.

Fig. 3 SEM of E.coli cells before (A) and after (B) treatment with ChCl/U. ChCl/U content: 6%; whole cells concentration: 0.02g/ml; incubation temperature and time: 35 ° C, 30min.

Fig. 4 TEM images of whole cells treated with 0% (A), 6% (B) and 10% (C) ChCl/U .

Effects of factors on the enzymatic hydrolysis of rutin to synthesize isoquercitrin Fig.5 Effect of different factors on the enzymatic hydrolysis of rutin to synthesize isoquercitrin. Reaction conditions: pH (A) 5.0 ‐ 7.0, rutin concentration: 0.02g/L, temperature: 35 ° C. Rutin concentration (B): 0.02 ‐ 0.2g/L, pH: 6.5, reaction temperature: 35 ° C. Temperature (C): 30 ‐ 50 ° C, pH: 6.5, rutin concentration: 0.05g/L. All reactions were reacted in a batch reactor ,180 rpm for 3 hours .

Operational stability of whole ‐ cell catalyst Fig.6 Effect of reusability of ChCl/U ‐ treated whole ‐ cell catalyst on the yield of isoquercitrin. Reaction conditions: catalyst concentration: 0.02g/ml; ChCl/U amount: 6%; substrate concentration: 0.05g/L; temperature: 40 ° C; rotational speed: 180rpm, each cycle reacted for 2 hours.

Table4. Comparison of enzyme rhaB1and whole ‐ cell catalyst expressing rhaB1 t opt /(h) Substrate concentration opt Catalyst type pH opt T opt / ° C Yield (%) (g/L) Whole ‐ cell a 6.5 40 2 0.05 93.05 ± 1.3% Crude rhaB1 b 5.0 35 10 0.01 98.3 ± 3.8% a Reaction condition: rutin concentration 0.05 g/L, reaction temperature 40 ° C, 180 rpm for 2 h, whole ‐ cell catalyst (0.04g/mL) treated with 0.06 g/mL ChCl/U for 30min. b Reaction condition: rutin concentration 0.01 g/L, reaction temperature 35 ° C, 180 rpm for 10 h, crude rhaB1 30mg/mL, 0.02 g/mL. [Toma][Tf 2 N] ‐ buffer (pH 5.0) as the reaction medium.

Conclusions 1. ChCl/U selected from five ILs ([BMIM][Tf2N], [BMIM][PF 6 ], [BMIM][BF 4 ], [HMIM][PF 6 ], [EMIM][PF6]) and DES (ChCl/U, ChCl/GI, ChCl/MA, ChCl/EG, ChCl/A) showed the best solvent to improve the catalytic ability of whole ‐ cell catalyst BL21 ‐ pET21a ‐ rhaB1 and the optimum ChCl/U content was 6% (v: v). 2. The optimal reaction condition (temperature, pH and rutin concentration) was investigated in a shaking Bath. ChCl/U ‐ treated cells were more tolerated than crude rhaB1, because the optimum pH and temperature of the whole ‐ cell catalyst were 6.5 and 40 o C, respectively, which all higher than that of crude rhaB1 (pH5.0, 35 o C) 3. The ChCl ‐ treated catalyst promoted the reaction process by 4/5 compared with the crude enzyme , and the catalyst could be reused for 6 times and the enzyme activity remained above 52%. 4. The ChCl/U ‐ treated whole ‐ cell catalyst can effectively improve the permeability of the cell membrane. Thus, the application of DESs in the whole ‐ cell biotransformation of rutin extracted from pomelo skin provided an effective way to recycle flavonoids in rutaceae plants.

Acknowledgments We are grateful to Collaborators: • Prof. Dr. Wolfgang Streit, UH, Germany • Dr. Ulrich Rabausch, UH, Germany Group students: • Fan Zhang, JUST, China Fangqin Wang, JUST, China • • Changtong Zhu, JUST, China Fundings: • National Natural Science Foundations of China (Grant No.21676130, 2017 ‐ 2020) • Major Program of the National Science Foundation of Jiangsu Province (Grant No.16KJA530002, 2016 ‐ 2019) • Six Talent Peaks Project of Jiangsu Province (Grant No. 2015 ‐ NY ‐ 018, 2015 ‐ 2018) • Young Scholars Program of Jiangsu University of Science and Technology (2015 ‐ 2019)

Thank you for your kind attention! Jinshan Temple (1600 years old) Zhenjiang City Please feel free to ask any questions… 6th International Conference on Sustainable Solid Waste Management, Naxos Island, Greece, 13–16 June 2018