SUPPORTING INFORMATION

Synthesis of Lipase Polymer Hybrids with Retained or Enhanced Activity using the Grafting-From Strategy

Marina Kovaliovab Michael L Allegrezzac Bertram Richterb Dominik Konkolewiczc Saadyah Averickab

aNeuroscience Disruptive Research Lab Allegheny Health Network Research Institute Allegheny General Hospital Pittsburgh Pennsylvania 15212 USAbNeuroscience Institute Allegheny Health Network Allegheny General Hospital Pittsburgh Pennsylvania 15212 USAcDepartment of Chemistry and Biochemistry Miami University Oxford Ohio 45056 United States

Author to whom correspondence should be addressed Prof Saadyah Averick Tel+1-412-359-4943 e-mail SaadyahAverickahnorg

S1

Experimental

Materials

All materials were used without further purification Carbon disulfide (99+ ) and triethylamine (TEA 99) were purchased from Alfa Aesar N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideHCl (EDCHCl 98) were purchased from Carbosynth Candida antarctica lipase B (CalB) and Thermomyces lanuginosa lipase (TL) were purchased from Novozymes N-(Isobutoxymethyl)acrylamide (MIBMA) and eosin Y were purchased from Sigma Aldrich N-[3-(NN-Dimethylamino)propyl] acrylamide (DMAPA) was purchased from Polysciences Inc Solvents and deuterated solvents were obtained from Worldwide Medical Products SDS-poly(acrylamide) gel electrophoresis was performed using Bio-Rad Mini PROTEAN TGX 4-20 gradient gels GelCode Blue protein stain was obtained from Thermo Scientific Unmodified protein concentrations were determined using a direct-detect spectrophotometer (Millipore) Polymerizations were conducted using two commercially available blue LED light strips (SuperbrightLEDscom 100 cm 09 Wft 16459 mcdft light intensity at reaction 300 Lux) wound inside a 1000 mL beaker wrapped in aluminum foil1 Water was deionized with a Millipore system as a Milli-Q grade Monomers were passed over a column of basic alumina prior to use to remove the inhibitor

1timesPBS (Phosphate buffered saline) = 2 mgmL KCl 2 mgmL KH2PO4 80 mgmL NaCL 115

mgmL Na2HPO4

Instrumentation

NMR spectroscopy 1H NMR spectra were recorded on a Bruker Advance 500 MHz NMR spectrometer using the residual solvent signal as a reference

UV-visible spectroscopy UV-visible spectra were obtained on a BioTek Cytation 3 plate reader Measurements were conducted on clear 96-well polystyrene microplates (Nunc) UV-vis spectra were collected on a DeNovix DS-11 spectrophotometer

Gel permeation chromatography (GPC) Aqueous GPC analyses were conducted in 005 M aqueous Na2SO4acetonitrile (8020) at a flow rate of 1 mLmin (Viscotek GPCMax VE 2001 module columns TSKgel PWXL guard column (Tosoh) + TSKgel G4000PWXL analytical column (Tosoh 78 mm times 30 cm 10 μm particle size))

Matrix-Assisted Laser DesorptionIonization Mass Specrtometry (MALDI-MS) MALDI-TOF data acquisition was performed on a Bruker AutoFlexIII MALDI-TOF mass spectrometer All samples were mixed with the sinapinic acid (SA) matrix Samples were analyzed in the positive ion linear mode to detect [M+H]+ ions In general 30 μL of sample (1 mgmL) was mixed with

S2

30 μL of saturated sinapinic acid solution (01 TFA 40 acetonitrile) and 1 μL was spotted directly on the target plate and allowed to dry at room temperature

Dynamic Light Scattering (DLS) A NanoPlus3 from Micromeritics was used for nanoparticles measurement Samples were prepared at ~1 mgml in water

Gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were cast and run using Mini-PROTEANreg systems (Bio-Rad) TGX 4-20 gradient gels Samples were prepared in SDS-PAGE sample buffer containing 1 DTT and heated 10 min to denature proteins prior to running at 100 V for 1 h to separate the proteins which were visualized with GelCodetrade Blue staining

Methods

Synthesis of trithiocarbonate CTA The chain transfer agent 1 2-(((ethylthio)-carbonothioyl)thio)propanoic acid was used in the synthesis of polymers by RAFT polymerization2

Potassium hydroxide (1460 g 0260 mol) was dissolved in 15 mL of DI water and added dropwise to a solution of ethanethiol (1603 g 0260 mol) in acetone 150 mL while stirring at 0 degC Carbon disulfide (2165 g 0284 mol) was added to the reaction mixture and stirred on ice for 30 minutes The solution was removed from the ice bath and 2-bromopropionic acid (3740 g 0245 mol) was added dropwise The reaction mixture was left to stir overnight at room temperature Excess solvent was removed under reduced pressure and the remaining solution was dissolved in 200 mL of ether The solution was washed seven times with 200 mL of DI water and 200 mL of brine Solvent was then removed via rotovap to recover product The resulting product 1 (327 g 0155 mol 59) is a viscous yellow liquid which can be frozen to solidify 1H-NMR (300 MHz CDCl3) ppm 487 (1H q J = 74 Hz CH3CH(S)COOH) 338 (2H q J = 74 Hz CH3CH2S) 163 (3H d J = 74 Hz CH3CH(S)COOH) 136 (3H t J =74 Hz CH3CH2S)

S3

Synthesis of N-hydroxy succinmide CTA (NHS-CTA) 2

S S

S

O

OH

N

O

O

OH

EDC-HClDCM

S S

S

ON

O

OO

1 2

Compound 1 (299 g 00138 mmol) was dissolved in 50 ml of DCM EDC-HCl (397 g 0021 mmol) and NHS (23 g 0021 mmol) were added to the solution of PAETC and the reaction was stirred for 12 hours Ethyl acetate (100 mL) and water (100 mL) were added and the mixture was stirred for 10 min The mixture was transferred to a separatory funnel the organic layer was extracted washed with brine and dried with anhydrous magnesium sulfate The solvent was removed under reduced pressure to give 2 that was used without further purification and stored at -20˚C 1H-NMR (300 MHz CDCl3) δ ppm 514 (1H q J = 74 Hz CH3CH(S)CO) 338 (2H qd J = 74 06 Hz CH3CH2S) 286-281 (4H m COCH2CH2CO) 174 (3H d J = 74 Hz CH3CH(S)CO) 136 (3H t J = 74 Hz CH3CH2S)

Determination of an average number of CTA moieties for CalB-CTA and TL-CTA using UV-Vis Spectroscopy

To determine the average number of CTA modifications we used UV-Vis spectroscopy The CTA trithiocarbonate group has absorption peak at ~310 nm lipase and lipase-polymer hybrid after purification in addition to 280 nm peak has a small shoulder due to the attachment of CTA group at ~310 nm Maximum absorbance values of native TL and CalB (280 nm) and TL-CTA TL-pDMAPA-S and CalB-CTA CalB-pDMAPA-S capped with a trithiocarbonate group (308 nm (CalB) and 310 nm (TL)) were determined using UV-Visible Spectroscopy A calibration was created with increasing ratios of protein to polymer (11 13 16 19 112 115) However a shift in maximum absorbance was observed with increasing ratios of polymer This shift was not observed at 270nm and a calibration was set up involving the ratio of the absorbance at 310 nm (TL) and 308 (CalB) to the absorbance at 270 nm (Figure S1)

Determination of the experimental Mn (Mnexpt) by UV-Vis Spectroscopy and DirectDetect IR spectrophotometer

The experimental Mn was determined by measuring the total concentration of the bio hybrid in solution using a Direct Detect IR spectrometer The Direct Detect instrument used to measure the concentration of the amide bond from the IR peak (both the polymers and proteins have amide bonds in their backbones) The polymer concentration was obtained by subtracting the mass of protein in solution

S4

(1) Wpolymera = [PPH]b ndash [protein]c = [polymer] (mgmL)

aMass fraction polymer bConcentration of protein polymer hybrid was determined using DirectDetect IR spectrophotometer cConcentration determined using UV-vis absorption at 280 nm

The mass of the protein in solution was determined using UV-vis spectroscopy the polymer does not absorb at 280 nm in the UV-vis spectrum Once the mass of the polymer in solution was obtained the amount of moles of both polymer and protein were determined and moles polymermoles protein 3 CTAs per protein gave the average degree of polymerization (DP) for each grafted polymer

(2) npolymer = [polymer]Mwmonomer (mmol)

nprotein = [polymer]MwCalBTL (mmol)

(3) DP(per CTA) = npolymer nprotein3cta

Multiplying the DP by the molecular weight of the monomer gives the number average molecular weight

(4) Mn = DPtimesMwmonomer

Assay of lipolytic activity and thermal stability of lipase and lipase ndashprotein conjugates Lipolytic activity was determined spectrophotometrically by using p-nitrophenyl palmitate (p-NPP) as substrate

NO2

O

O14

Lipase-polymerconjugate

OH

O14

NO2

HO

=405 nm

p-NPP p-NP

S5

Supplemental Figures and Tables

Figure S1 Plot of peak height normalized Ultraviolet-visible (UV-Vis) spectra of CalB (a) TL (b) with increasing molar ratio of trithiocarbonate (CTA) capped polymer (polyacrylamide 2K) to protein and UV-Vis spectra of modified TL-CTA and CalB-CTA for comparison (blue spectrum) Calibration curve of CalB (c) and TL (d) with increasing molar ratio of trithiocarbonate capped polymer to protein with presentation of the ratio of TI-pDMAPA-S and CalB-pDMAPA-S (red lines indicate the ratio point) (see Methods section)

Table S1 Summary of UV-Vis spectra this table provides approximate number of CTA modification of lipase (TL and CalB)

Sample Absorbance Ratio (270 nm 310) for TL and (270 nm

308) for CalB

Approximate number of CTA groups

TL 1 012 -CalB 1 019 -

TL-CTA 1 036 3CalB-CTA 1 037 3

TL-pDMAPA-S 1 035 3CalB-pDMAPA-S 1 039 3

S6

Figure S2 MALDI-TOF MS data for CalB and CalB-CTA

Figure S3 MALDI-TOF MS data for TL and TL-CTA

S7

Figure S4 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate TL-pDMAPA)

S8

Figure S5 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate CalB-pDMAPA)

S9

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

30 μL of saturated sinapinic acid solution (01 TFA 40 acetonitrile) and 1 μL was spotted directly on the target plate and allowed to dry at room temperature

Dynamic Light Scattering (DLS) A NanoPlus3 from Micromeritics was used for nanoparticles measurement Samples were prepared at ~1 mgml in water

Gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were cast and run using Mini-PROTEANreg systems (Bio-Rad) TGX 4-20 gradient gels Samples were prepared in SDS-PAGE sample buffer containing 1 DTT and heated 10 min to denature proteins prior to running at 100 V for 1 h to separate the proteins which were visualized with GelCodetrade Blue staining

Methods

Synthesis of trithiocarbonate CTA The chain transfer agent 1 2-(((ethylthio)-carbonothioyl)thio)propanoic acid was used in the synthesis of polymers by RAFT polymerization2

Potassium hydroxide (1460 g 0260 mol) was dissolved in 15 mL of DI water and added dropwise to a solution of ethanethiol (1603 g 0260 mol) in acetone 150 mL while stirring at 0 degC Carbon disulfide (2165 g 0284 mol) was added to the reaction mixture and stirred on ice for 30 minutes The solution was removed from the ice bath and 2-bromopropionic acid (3740 g 0245 mol) was added dropwise The reaction mixture was left to stir overnight at room temperature Excess solvent was removed under reduced pressure and the remaining solution was dissolved in 200 mL of ether The solution was washed seven times with 200 mL of DI water and 200 mL of brine Solvent was then removed via rotovap to recover product The resulting product 1 (327 g 0155 mol 59) is a viscous yellow liquid which can be frozen to solidify 1H-NMR (300 MHz CDCl3) ppm 487 (1H q J = 74 Hz CH3CH(S)COOH) 338 (2H q J = 74 Hz CH3CH2S) 163 (3H d J = 74 Hz CH3CH(S)COOH) 136 (3H t J =74 Hz CH3CH2S)

S3

Synthesis of N-hydroxy succinmide CTA (NHS-CTA) 2

S S

S

O

OH

N

O

O

OH

EDC-HClDCM

S S

S

ON

O

OO

1 2

Compound 1 (299 g 00138 mmol) was dissolved in 50 ml of DCM EDC-HCl (397 g 0021 mmol) and NHS (23 g 0021 mmol) were added to the solution of PAETC and the reaction was stirred for 12 hours Ethyl acetate (100 mL) and water (100 mL) were added and the mixture was stirred for 10 min The mixture was transferred to a separatory funnel the organic layer was extracted washed with brine and dried with anhydrous magnesium sulfate The solvent was removed under reduced pressure to give 2 that was used without further purification and stored at -20˚C 1H-NMR (300 MHz CDCl3) δ ppm 514 (1H q J = 74 Hz CH3CH(S)CO) 338 (2H qd J = 74 06 Hz CH3CH2S) 286-281 (4H m COCH2CH2CO) 174 (3H d J = 74 Hz CH3CH(S)CO) 136 (3H t J = 74 Hz CH3CH2S)

Determination of an average number of CTA moieties for CalB-CTA and TL-CTA using UV-Vis Spectroscopy

To determine the average number of CTA modifications we used UV-Vis spectroscopy The CTA trithiocarbonate group has absorption peak at ~310 nm lipase and lipase-polymer hybrid after purification in addition to 280 nm peak has a small shoulder due to the attachment of CTA group at ~310 nm Maximum absorbance values of native TL and CalB (280 nm) and TL-CTA TL-pDMAPA-S and CalB-CTA CalB-pDMAPA-S capped with a trithiocarbonate group (308 nm (CalB) and 310 nm (TL)) were determined using UV-Visible Spectroscopy A calibration was created with increasing ratios of protein to polymer (11 13 16 19 112 115) However a shift in maximum absorbance was observed with increasing ratios of polymer This shift was not observed at 270nm and a calibration was set up involving the ratio of the absorbance at 310 nm (TL) and 308 (CalB) to the absorbance at 270 nm (Figure S1)

Determination of the experimental Mn (Mnexpt) by UV-Vis Spectroscopy and DirectDetect IR spectrophotometer

The experimental Mn was determined by measuring the total concentration of the bio hybrid in solution using a Direct Detect IR spectrometer The Direct Detect instrument used to measure the concentration of the amide bond from the IR peak (both the polymers and proteins have amide bonds in their backbones) The polymer concentration was obtained by subtracting the mass of protein in solution

S4

(1) Wpolymera = [PPH]b ndash [protein]c = [polymer] (mgmL)

aMass fraction polymer bConcentration of protein polymer hybrid was determined using DirectDetect IR spectrophotometer cConcentration determined using UV-vis absorption at 280 nm

The mass of the protein in solution was determined using UV-vis spectroscopy the polymer does not absorb at 280 nm in the UV-vis spectrum Once the mass of the polymer in solution was obtained the amount of moles of both polymer and protein were determined and moles polymermoles protein 3 CTAs per protein gave the average degree of polymerization (DP) for each grafted polymer

(2) npolymer = [polymer]Mwmonomer (mmol)

nprotein = [polymer]MwCalBTL (mmol)

(3) DP(per CTA) = npolymer nprotein3cta

Multiplying the DP by the molecular weight of the monomer gives the number average molecular weight

(4) Mn = DPtimesMwmonomer

Assay of lipolytic activity and thermal stability of lipase and lipase ndashprotein conjugates Lipolytic activity was determined spectrophotometrically by using p-nitrophenyl palmitate (p-NPP) as substrate

NO2

O

O14

Lipase-polymerconjugate

OH

O14

NO2

HO

=405 nm

p-NPP p-NP

S5

Supplemental Figures and Tables

Figure S1 Plot of peak height normalized Ultraviolet-visible (UV-Vis) spectra of CalB (a) TL (b) with increasing molar ratio of trithiocarbonate (CTA) capped polymer (polyacrylamide 2K) to protein and UV-Vis spectra of modified TL-CTA and CalB-CTA for comparison (blue spectrum) Calibration curve of CalB (c) and TL (d) with increasing molar ratio of trithiocarbonate capped polymer to protein with presentation of the ratio of TI-pDMAPA-S and CalB-pDMAPA-S (red lines indicate the ratio point) (see Methods section)

Table S1 Summary of UV-Vis spectra this table provides approximate number of CTA modification of lipase (TL and CalB)

Sample Absorbance Ratio (270 nm 310) for TL and (270 nm

308) for CalB

Approximate number of CTA groups

TL 1 012 -CalB 1 019 -

TL-CTA 1 036 3CalB-CTA 1 037 3

TL-pDMAPA-S 1 035 3CalB-pDMAPA-S 1 039 3

S6

Figure S2 MALDI-TOF MS data for CalB and CalB-CTA

Figure S3 MALDI-TOF MS data for TL and TL-CTA

S7

Figure S4 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate TL-pDMAPA)

S8

Figure S5 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate CalB-pDMAPA)

S9

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Synthesis of N-hydroxy succinmide CTA (NHS-CTA) 2

S S

S

O

OH

N

O

O

OH

EDC-HClDCM

S S

S

ON

O

OO

1 2

Compound 1 (299 g 00138 mmol) was dissolved in 50 ml of DCM EDC-HCl (397 g 0021 mmol) and NHS (23 g 0021 mmol) were added to the solution of PAETC and the reaction was stirred for 12 hours Ethyl acetate (100 mL) and water (100 mL) were added and the mixture was stirred for 10 min The mixture was transferred to a separatory funnel the organic layer was extracted washed with brine and dried with anhydrous magnesium sulfate The solvent was removed under reduced pressure to give 2 that was used without further purification and stored at -20˚C 1H-NMR (300 MHz CDCl3) δ ppm 514 (1H q J = 74 Hz CH3CH(S)CO) 338 (2H qd J = 74 06 Hz CH3CH2S) 286-281 (4H m COCH2CH2CO) 174 (3H d J = 74 Hz CH3CH(S)CO) 136 (3H t J = 74 Hz CH3CH2S)

Determination of an average number of CTA moieties for CalB-CTA and TL-CTA using UV-Vis Spectroscopy

To determine the average number of CTA modifications we used UV-Vis spectroscopy The CTA trithiocarbonate group has absorption peak at ~310 nm lipase and lipase-polymer hybrid after purification in addition to 280 nm peak has a small shoulder due to the attachment of CTA group at ~310 nm Maximum absorbance values of native TL and CalB (280 nm) and TL-CTA TL-pDMAPA-S and CalB-CTA CalB-pDMAPA-S capped with a trithiocarbonate group (308 nm (CalB) and 310 nm (TL)) were determined using UV-Visible Spectroscopy A calibration was created with increasing ratios of protein to polymer (11 13 16 19 112 115) However a shift in maximum absorbance was observed with increasing ratios of polymer This shift was not observed at 270nm and a calibration was set up involving the ratio of the absorbance at 310 nm (TL) and 308 (CalB) to the absorbance at 270 nm (Figure S1)

Determination of the experimental Mn (Mnexpt) by UV-Vis Spectroscopy and DirectDetect IR spectrophotometer

The experimental Mn was determined by measuring the total concentration of the bio hybrid in solution using a Direct Detect IR spectrometer The Direct Detect instrument used to measure the concentration of the amide bond from the IR peak (both the polymers and proteins have amide bonds in their backbones) The polymer concentration was obtained by subtracting the mass of protein in solution

S4

(1) Wpolymera = [PPH]b ndash [protein]c = [polymer] (mgmL)

aMass fraction polymer bConcentration of protein polymer hybrid was determined using DirectDetect IR spectrophotometer cConcentration determined using UV-vis absorption at 280 nm

The mass of the protein in solution was determined using UV-vis spectroscopy the polymer does not absorb at 280 nm in the UV-vis spectrum Once the mass of the polymer in solution was obtained the amount of moles of both polymer and protein were determined and moles polymermoles protein 3 CTAs per protein gave the average degree of polymerization (DP) for each grafted polymer

(2) npolymer = [polymer]Mwmonomer (mmol)

nprotein = [polymer]MwCalBTL (mmol)

(3) DP(per CTA) = npolymer nprotein3cta

Multiplying the DP by the molecular weight of the monomer gives the number average molecular weight

(4) Mn = DPtimesMwmonomer

Assay of lipolytic activity and thermal stability of lipase and lipase ndashprotein conjugates Lipolytic activity was determined spectrophotometrically by using p-nitrophenyl palmitate (p-NPP) as substrate

NO2

O

O14

Lipase-polymerconjugate

OH

O14

NO2

HO

=405 nm

p-NPP p-NP

S5

Supplemental Figures and Tables

Figure S1 Plot of peak height normalized Ultraviolet-visible (UV-Vis) spectra of CalB (a) TL (b) with increasing molar ratio of trithiocarbonate (CTA) capped polymer (polyacrylamide 2K) to protein and UV-Vis spectra of modified TL-CTA and CalB-CTA for comparison (blue spectrum) Calibration curve of CalB (c) and TL (d) with increasing molar ratio of trithiocarbonate capped polymer to protein with presentation of the ratio of TI-pDMAPA-S and CalB-pDMAPA-S (red lines indicate the ratio point) (see Methods section)

Table S1 Summary of UV-Vis spectra this table provides approximate number of CTA modification of lipase (TL and CalB)

Sample Absorbance Ratio (270 nm 310) for TL and (270 nm

308) for CalB

Approximate number of CTA groups

TL 1 012 -CalB 1 019 -

TL-CTA 1 036 3CalB-CTA 1 037 3

TL-pDMAPA-S 1 035 3CalB-pDMAPA-S 1 039 3

S6

Figure S2 MALDI-TOF MS data for CalB and CalB-CTA

Figure S3 MALDI-TOF MS data for TL and TL-CTA

S7

Figure S4 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate TL-pDMAPA)

S8

Figure S5 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate CalB-pDMAPA)

S9

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

(1) Wpolymera = [PPH]b ndash [protein]c = [polymer] (mgmL)

aMass fraction polymer bConcentration of protein polymer hybrid was determined using DirectDetect IR spectrophotometer cConcentration determined using UV-vis absorption at 280 nm

The mass of the protein in solution was determined using UV-vis spectroscopy the polymer does not absorb at 280 nm in the UV-vis spectrum Once the mass of the polymer in solution was obtained the amount of moles of both polymer and protein were determined and moles polymermoles protein 3 CTAs per protein gave the average degree of polymerization (DP) for each grafted polymer

(2) npolymer = [polymer]Mwmonomer (mmol)

nprotein = [polymer]MwCalBTL (mmol)

(3) DP(per CTA) = npolymer nprotein3cta

Multiplying the DP by the molecular weight of the monomer gives the number average molecular weight

(4) Mn = DPtimesMwmonomer

Assay of lipolytic activity and thermal stability of lipase and lipase ndashprotein conjugates Lipolytic activity was determined spectrophotometrically by using p-nitrophenyl palmitate (p-NPP) as substrate

NO2

O

O14

Lipase-polymerconjugate

OH

O14

NO2

HO

=405 nm

p-NPP p-NP

S5

Supplemental Figures and Tables

Figure S1 Plot of peak height normalized Ultraviolet-visible (UV-Vis) spectra of CalB (a) TL (b) with increasing molar ratio of trithiocarbonate (CTA) capped polymer (polyacrylamide 2K) to protein and UV-Vis spectra of modified TL-CTA and CalB-CTA for comparison (blue spectrum) Calibration curve of CalB (c) and TL (d) with increasing molar ratio of trithiocarbonate capped polymer to protein with presentation of the ratio of TI-pDMAPA-S and CalB-pDMAPA-S (red lines indicate the ratio point) (see Methods section)

Table S1 Summary of UV-Vis spectra this table provides approximate number of CTA modification of lipase (TL and CalB)

Sample Absorbance Ratio (270 nm 310) for TL and (270 nm

308) for CalB

Approximate number of CTA groups

TL 1 012 -CalB 1 019 -

TL-CTA 1 036 3CalB-CTA 1 037 3

TL-pDMAPA-S 1 035 3CalB-pDMAPA-S 1 039 3

S6

Figure S2 MALDI-TOF MS data for CalB and CalB-CTA

Figure S3 MALDI-TOF MS data for TL and TL-CTA

S7

Figure S4 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate TL-pDMAPA)

S8

Figure S5 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate CalB-pDMAPA)

S9

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Supplemental Figures and Tables

Figure S1 Plot of peak height normalized Ultraviolet-visible (UV-Vis) spectra of CalB (a) TL (b) with increasing molar ratio of trithiocarbonate (CTA) capped polymer (polyacrylamide 2K) to protein and UV-Vis spectra of modified TL-CTA and CalB-CTA for comparison (blue spectrum) Calibration curve of CalB (c) and TL (d) with increasing molar ratio of trithiocarbonate capped polymer to protein with presentation of the ratio of TI-pDMAPA-S and CalB-pDMAPA-S (red lines indicate the ratio point) (see Methods section)

Table S1 Summary of UV-Vis spectra this table provides approximate number of CTA modification of lipase (TL and CalB)

Sample Absorbance Ratio (270 nm 310) for TL and (270 nm

308) for CalB

Approximate number of CTA groups

TL 1 012 -CalB 1 019 -

TL-CTA 1 036 3CalB-CTA 1 037 3

TL-pDMAPA-S 1 035 3CalB-pDMAPA-S 1 039 3

S6

Figure S2 MALDI-TOF MS data for CalB and CalB-CTA

Figure S3 MALDI-TOF MS data for TL and TL-CTA

S7

Figure S4 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate TL-pDMAPA)

S8

Figure S5 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate CalB-pDMAPA)

S9

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S2 MALDI-TOF MS data for CalB and CalB-CTA

Figure S3 MALDI-TOF MS data for TL and TL-CTA

S7

Figure S4 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate TL-pDMAPA)

S8

Figure S5 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate CalB-pDMAPA)

S9

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S4 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate TL-pDMAPA)

S8

Figure S5 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate CalB-pDMAPA)

S9

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S5 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with DMAPA at different time points (The NMR spectra indicate the disappearance of the monomer DMAPA and the formation of the polymer conjugate CalB-pDMAPA)

S9

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S6 NMR spectra of RAFT-PET polymerization reaction mixture of TL-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer conjugate could not be observed due to the formation of insoluble conjugate)

S10

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S7 NMR spectra of RAFT-PET polymerization reaction mixture of CalB-CTA with NIBMA at 0 and 15 min (The NMR spectra indicate the disappearance of the monomer NIBMA unfortunately the polymer-conjugate could not be observed due to the formation of insoluble conjugate)

Figure S8 GPC trace for TL protein-polymer hybrids (TL-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

S11

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S9 GPC trace for CalB protein-polymer hybrids (CalB-pDMAPA) GPC trace recorded at using UV detector set at 280 nm (a) and 360 nm (b)

Figure S10 DLS data for TL(a) and TL-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (TL diameter 33 nm plusmn 11 and TL-pNIBMA diameter 3962 nm polydispersity index 0352)

S12

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S11 DLS data for CalB(a) and CalB-pNIBMA (b) Volume intensity distribution in 1timesPBS (01 mgmL) (CalB diameter 46 nm plusmn 11 and CalB-pNIBMA diameter 9364 nm polydispersity index 0412)

Table S2 Lipolytic activity of lipase derivatives

Lipase activity (micromol p-NP min-1 mg-1)CalB 3177 plusmn 506CalB-CTA 3017 plusmn 281CalB-pDMAPA-S 1889 plusmn 191CalB-pDMAPA-M 1484 plusmn 303CalB-pDMAPA-L 1856 plusmn 187CalB- pNIBMA 16205 plusmn 2733TL 12700 plusmn 1149TL-CTA 16032 plusmn 182TL-pDMAPA-S 19237 plusmn 1303TL-pDMAPA-M 20813 plusmn 573TL-pDMAPA-L 28921 plusmn 362TL-pNIBMA 32212 plusmn 840

S13

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S12 Images of TL- pNIBMA (A) and TL and TL- pNIBMA painted onto glass slides before (B) and after washing (C) (note some traces of buffer can be observed in the intial TL- pNIBMA)

Figure S13 Images of TL and TL- pNIBMA paint reaction after drying

S14

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

S16

Figure S14 Concentration of p-NP formed from the hydrolysis of p-NPP catalyzed by native TL lipase (17 mgml enzyme) glass coating and TL-pNIMBA conjugate (12 mgml enzyme) after 15 min (before and after wash)

S15

References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

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References

1 B S Tucker M L Coughlin C A Figg and B S Sumerlin ACS Macro Lett 2017 6 452-4572 R Falatach C McGlone M S Al-Abdul-Wahid S Averick R C Page J A Berberich and D

Konkolewicz Chem Commun 2015 51 5343-5346

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