Supplementary Information

Identification and comparison of cutinases for synthetic polyester degradation

Peter James Baker1, Christopher Poultney2, Zhiqiang Liu3, Richard Gross1 and Jin Kim Montclare1,4

1Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, Brooklyn, New York 11201, 2Courant Institute of Mathematical Sciences, New York University, New York, NY 10003, 3Institute of Bioengineering,Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China, 4Department of Biochemistry, SUNY-Downstate Medical Center, Brooklyn, New York, 11203

Corresponding Author:

Jin Kim Montclare

Gene Synthesis

The Protein Data Bank (PDB) and National Center for Biotechnology Information (NCBI) databases were used to obtain amino acid sequences of different cutinases. The gene sequences encoding cutinases were determined using Gene designer (DNA 2.0, Menlo Park, USA.). The codons were optimized for Pichia pastoris in order to increase expression yields. The cutinase genes were synthesized by DNA 2.0 (Menlo Park, USA), and cloned into the pPICZα (Invitrogen, Carlsbad, USA) vector. Recombinant plasmids were amplified in E. coli, and then extracted and linearized using SacI for transformation. Competent cells and transformation were completed using Pichia EasycompTM kit (Invitrogen, Carlsbad, USA).

Enzyme Expression

The cutinase genes were expressed inP. pastoris, and recombinant cutinases were produced by using thestrong methanol-induced AOX1 promoter (Juge et al. 1996; Raschke et al. 1996). Single colonies were cultured in 250 mL of BMGY medium composed of 0.5 % yeast extract, 1.0 % peptone, supplemented with 250 mM potassium phosphatebuffer pH 6.0, 0.17 % yeast nitrogen base, 38 mM ammonium sulfate, and 2 % glycerol. Precultures of P. pastoris harboring cutinase genes were incubated at 30 oC, 200 rpm, for 3 days. After centrifugation at 4,000 X g for 10 min, cells were transferred into a parallel fermentor (DASGIP, Germany) containing 1 L basal salt medium composed of 4 % glycerol, 6.6 mM calcium sulfate, 85 mM potassium sulfate, 97 mM magnesium sulfate, 68 mM ammonium sulfate, 12 mL hexametaphosphate trace salts (24 mM cupric sulfate, 0.5 mM sodium iodide, 25 mM manganese sulfate, 1 mM sodium molybdate, 0.3 mM boric acid, 2.1 mM cobalt chloride, 147 mM zinc chloride, 232 mM ferrous sulfate, 0.8 mM biotin). The constant dissolved oxygen was set as 40%. The glycerol (50%, v/v) feeding time was 6 h. The rate of methanol feeding was 5 mL/h.

Enzyme Purification

Fermentation broth was centrifuged at 8,000 rpm for 10 min at 4oC, and the supernatant was concentrated approximately 10 fold using an ultrafiltration unit (Millipore TFE system, Millipore, Billerica, USA) with a 10 KD membrane. Cutinaseswere purified by fast perfusion liquid chromatography (FPLC) using ÄKTA Workstation (GE Healthcare, Piscataway, USA) with a HiTRAP IMAC FF 5 mL column, where the metal affinity site on the column was saturated with cobalt chloride solution according to operating instructions of column. The starting buffer was comprised of 50 mM sodium phosphate with 0.5 mM imidazole (pH 8.0) and the elution buffer contained 50 mM sodium phosphate with500 mM imidazole (pH 8.0). Approximately 150 mL of concentrated sample was loaded onto the column using 150 mL Superloop (GE Healthcare, Piscataway, USA). Several column volumes of starting buffer were run over the column to remove proteins that were bound non-specifically to the column. Each cutinase was eluted by using a linear gradient, from 0.5 mM to 500 mM imidazole, run over 20 column volumes at a flow rate of 5 mL/min. The purity of the proteins was determined by SDS-PAGE analysis. The purified proteins were then run over a desalting column and subjected to buffer exchange using a HiPrep 26/10 (GE Healthcare, Piscataway, USA). The proteins were eluted in deionized water and then subjected to lyophilization for storage at -80 oC.

Differential Scanning Calorimetry

Calorimetric scans were performed using a high-sensitivity DSC (Nano-DSC, TA Instruments, New Castle, USA). The sample and the reference cells were filled with 300 µL of enzyme (1 mg/mL) in either 20 mM sodium phosphate buffer at pH 3.0, 20 mM sodium acetate buffer at pH 5.0 or 20 mM sodium phosphate buffer at pH 8.0. The cells were heated at a rate of 1 oC/min from 4 oC to 80 oC. Calorimetric measurements were conducted in a pressurized cell at 3 atm. Buffer tracings were obtained under the same conditions and subtracted from sample curves. The observed thermograms were baseline corrected and normalized data were analyzed using NanoAnalyze software. These experiments were conducted in triplicate and the data is reported as the average of those three trials.

Supplementary Fig. 1Lineweaver-Burke plots of A = FsC, B = AoC, C = AbC, D = AfC, E = HiC for pNPB. Plots represent an average of at least 3 trials in which the error bars signify the standard deviation

Supplementary Fig 2 CD wavelength scans of A = FsC, B = AoC, C = AbC, D = AfC, E = HiC in 14.5 mM Tris-HCl, 0.75% glycerol, pH 7.5 (straight line), in the presence of 2.5% methanol (dashed line). All scans were performed at 25 oC

Supplementary Fig. 3 Backbone homology models of cutinases sequences used in these experiments. FsC = purple, AoC = green, AbC = navy, AfC = red, HiC = orange. Shown in yellow are the disulphide bonds. Highlighted in white are the residues of the catalytic triad and highlighted in cyan are the residues of the oxyanion hole

Supplementary Fig. 4 Differential scanning calorimetry thermograms of cutinase. A = FsC, B = AoC, C = AbC, D = AfC and E = HiC. Solid line represents the initial scan and the dashed line shows the secondary scan

Cutinase Sequences:

FsC (accession number K02640):

MKFFALTTLLAATASALPTSNPAQELEARQLGRTTRDDLINGNSASCRDVIFIYARGSTETGNLGTLGPSIASNLESAFGKDGVWIQGVGGAYRATLGDNALPRGTSSAAIREMLGLFQQANTKCPDATLIAGGYSQGAALAAASIEDLDSAIRDKIAGTVLFGYTKNLQNRGRIPNYPADRTKVFCNTGDLVCTGSLIVAAPHLAYGPDARGPAPEFLIEKVRAVRGSA

AoC (accession number BAE55151.1):

MHLRNIVIALAATAVASPVDLQDRQLTGGDELRDGPCKPITFIFARASTEPGLLGISTGPAVCNRLKLARSGDVACQGVGPRYTADLPSNALPEGTSQAAIAEAQGLFEQAVSKCPDTQIVAGGYSQGTAVMNGAIKRLSADVQDKIKGVVLFGYTRNAQERGQIANFPKDKVKVYCAVGDLVCLGTLIVAPPHFSYLSDTGDASDFLLSQLG

AfC (accession number XP_755273):

MKFALLSLAAMAVASPVAIDVRQTAITGDELRTGPCEPITFIFARGSTEPGLLGITTGPGVCNALKLSRPGQVACQGVGPAYIADLASNFLPQGTSQVAIDEAAGLFKLAASKCPDTKIVAGGYSQGAAVMHGAIRNLPSNVQNMIKGVVLFGDTRNKQDGGRIPNFPTDRTKIYCAFGDLVCDGTLIITPAHLSYGDDVPSATSFLLSKV

AbC (accession number AAA03470.1):

MMNLNLLLSKPCQASTTRNELETGSSDACPRTIFIFARGSTEAGNMGALVGPFTANALESAYGASNVWVQGVGGPYTAGLVENALPAGTSQAAIREAQRLFNLAASKCPNTPITAGGYSQGAAVMSNAIPGLSAAVQDQIKGVVLFGYTKNLQNGGRIPNFPTSKTTIYCETGDLVCNGTLIITPAHLLYSDEAAVQAPTFLRAQIDSA

HiC(accession number ABC06408.1):

GAIENGLESGSANACPDAILIFARGSTEPGNMGITVGPALANGLESHIRNIWIQGVGG PYDAALATNFLPRGTSQANIDEGKRLFALANQKCPNTPVVAGGYSQGAALIAAAVSELSGAVKEQVKGVALFGYTQNLQRGGIPNYPRERTKVFCNVGDAVCTGTLIITPAHLSYTIEARGEAARFLRDRIR