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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

Present study Conclusions Acknowledgments Background

Flavonoids sources Resource waste Method of reutilization should be found

Grapefruit peel In 2017, the annual output of grapefruit in China was about 4.8 million tons

Background

Rutaceae plants are rich in flavonoids, such as rutin, naringin and hesperidin.

Isoquercitrin

antihypertensive hypotensive

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

Biocatalyst

Biocatalysts for biotransformation

Wang F, He S, et al. Journal of Chemical Technology and Biotechnology Doi：10.1002 / jctb.5621.

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

• f 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.

RhaB1 as catalyst

Problem: Cell membranes hinder intracellular and extracellular mass transfer

Present study

HOW?

Methods Process Mechanism Effect Refs Physical treatment Electrical treatment Electric field Formation of micropores in phospholipids bilayer [1] Chemical regulation Solvent medium Hydrophobic interaction Total lipid content reduced by 8.3% [2] Molecular biology regulation Change the membrane structure and composition Gene mutation/knockout Overexpression of ydeD [3]

Table 1. Improvement strategies of cell permeability.

[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 Enlarge cell permeability Good thermodynamic stability

E.Coli BL21‐pET21a‐rhaB1

ILs/DESs

Name Abbreviation Molecular Formula MW/g∙mol‐1 1‐butyl‐3‐methylimidazolium bis[(trifluoromethyl)sulfonyl]imide [BMIM][Tf2N] C10H15F6N3O4S2 419.36 1‐butyl‐3‐methylimidazolium hexafluorophosphate [BMIM][PF6] C8H15N2F6P 284.20 1‐Butyl‐3‐methylimidazolium tetrafluoroborate [BMIM][BF4] C8H15N2BF4 226.02 1‐Hexyl‐3‐methylimidazolium hexafluorophosphate [HMIM][PF6] C10H19F6N2P 312.24 1‐Ethyl‐3‐methylimidazolium hexafluorophosphate [EMIM][PF6] C6H11F6N2P 256.13

Table 2. The ionic liquids used in the present study.

Table 3. The deep eutectic solvent used in the present study.

Name Abbreviation Tf/°C Tm/°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 ‐‐

• 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
• f 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

pH=5.0, Temperature: 35 °C, Rutin concentration 0.02g/L Speed 180rpm, Time 3h. Strain Strain cell cell

• 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.

Effects of different ChCl/U on the whole‐cell catalyst E. coli BL21‐pET21a‐rhaB1

• 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.

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.

Effects of factors on the enzymatic hydrolysis of rutin to synthesize isoquercitrin

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.

Operational stability of whole‐cell catalyst

• Table4. Comparison of enzyme rhaB1and whole‐cell catalyst expressing rhaB1

Catalyst type pHopt Topt/°C topt/(h) Substrate concentration opt (g/L) Yield (%) 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][Tf2N]‐buffer (pH 5.0) as the reaction medium.

Conclusions

• 1. ChCl/U selected from five ILs ([BMIM][Tf2N], [BMIM][PF6], [BMIM][BF4], [HMIM][PF6], [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 40oC, respectively, which all higher than that

• f crude rhaB1 (pH5.0, 35oC)
• 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.

We are grateful to

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) 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

Acknowledgments

Thank you for your kind attention!

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