Identification, expression and characterization of a R- -transaminase from Capronia semiimmersa.

Transcription

1 Applied Microbiology and Biotechnology Supporting Information for Identification, expression and characterization of a R- -transaminase from Capronia semiimmersa. César Iglesias 1,2, Paola Panizza 1,2 and Sonia Rodríguez Giordano* 1,2 1 Cátedra de Microbiología, DEPBIO, Facultad de Química, Universidad de la República, Gral. Flores 2124, Montevideo, Uruguay. 2 Laboratorio de Biocatálisis y Biotransformaciones, DEPBIO-DQO, Facultad de Química, Universidad de la República, Gral. Flores 2124, Montevideo, Uruguay. *Author for correspondence: Sonia Rodríguez. Tel: , soniar@fq.edu.uy

2 R-TA Capronia gene sequence optimized for E. coli > accession number: BankIt Synthetic KY GAATTCATGACCACGATGGAAAAAATTTTTAGCGCGTATC AGGCCCGCGTTTCTACCCTGACGGCATCACGTGCTTCGAATCCGT TCGCAGATGGTATCGCTTGGGTCCAAGGCCGCGTGACCCCGATTC ATGAAGCGCAGATCCCGATGCTGGACCAAGGTTTCCTGCACAGTG ATCTGACCTACGACGTGCCGTCCGTTTGGGATGGCCGTTTCTTTC GCCTGGATGACCATCTGGAACGTCTGGAACGCAGTTGCGCAAAAA TGCGTCTGCGTTGTCCGCTGCCGCGTGCTCAGGTTCGCCATACCC TGTGTGCCATGCTGGCACGTAGCGGTATTCGTGATGCATTTGTTG AACTGATTGTCACCCGTGGCCTGCGCGGTGTCCGTGGCCTGTCAG CGGCCGAAGTGGATGCGCTGCCGAACTCGCTGTATATGTGGATTG TTCCGTACGTGTGGGTTATGGAACCGGCCGTGCAGCTGGCGGGTA GCGGTTCTGCAATCGTGGCTCGCACCGTTCATCGTACGCCGCCGG TGTGTATGGATCCGACCGTTAAAAATCTGCAATGGGGTGACCTGA CGCGCGGCATGTTTGAAGCGAACGATCGTGGCGCCGGTTATCCGT TCCTGACCGATCGCAGTATTTCCGAAGAAACCGCAGACGCTAATA TTACGGAAGGCTCTGGTTTTAACATCGTGGTTGTCAAAGATGGTA CCCTGCACACGCCGAAACGCGGTGTCCTGGAAGGCGTGACCCGTG AAAGCGTTTTCGAATGTTGCCGTCGCCTGGGCGTCCCGTATGCAC TGGACACCGTCCCGGTGCGTCTGGCCCTGGAAGCAGATGAAATCT TTATGTGCACCACGGCGGGCGGTATTATGCCGATCACCACGCTGG ATGGTAAACCGGTTGGTGATGGTGCGGTTGGTCCGATTACCCGTC AGATCTGGGATGTGTATTGGCGTCTGCATTACGAAGACGGTTTTA GCTTCGCCGTTGATTATGAAGACGAAACCGGTCTGGAAGGCGTGG CAAACGGTAATGCTAACGGCACGGTTTCTGTCAATGGCAAAGCAC ATCACCATCACCATCACTAAAAGCTT Purification of R-TA Capronia Figure S1 - SDS-PAGE purification of R-TACap; lane 1: AI TB 37C 16hs Soluble; lane 2: AI TB 37C 16hs Insoluble; lane 3: AI TB 28C 24hs soluble; lane 4: AI TB 28C 24hs Insoluble; lane 5 : flow-through of crude extract during column loading; lane 6: fractions of RTA Cap protein post HisTag purification. (AI TB = Autoinduction terrific broth)

3 Analysis of insertion present in R-TA Capronia compared to other R-TA It can be observed that five different fungal potential R-TA presented an insertion at the loop identified by Guang et al as important for defyning substrate specificity. R-TA Cap was the only one that presented a second insertion at position 213. Figure S2 Analysis of insertions in R-TA Cap compared to other R-TA selected in the same study. a insertion at the loop (L130,S131) (Guang 2015), b second insertion found only in R-TA Cap (residues ) Determination of biochemical parameters Figure S3 a. Michaelis Menten plot (4,5 µg R-TA Cap enzyme, 2,5 mm Pyruvate). b. Lineweaver-Burke plot.

4 Enzyme Km Reference Arthrobacter sp (R- TA) 2,62 Iwasaki 2011 Halomonas elongate (S-TA) 2,57 Cerioli 2015 Capronia semiimmersa. (R-TA) 0,30 This study Table S1 - a. Km reported for different TAs with methylbenylamine. Figure S4 Determination of optimal Temperature for R-TA Cap Analytical conditions for the different substrates and products GC analysis: Enantiomeric excess values were determined by chiral GC on Shimadzu 2010 chromatograph equipped with a Megadex DET-TBS (25 m, 0.25 mm) column (MEGA, Italy) and a FID detector. Temperature program: 80 ºC (2 min)/ 1.25 ºC/min/125 ºC/ 40 ºC/min/210 ºC (5 min). TSPLIT: 230 ºC, TFID: 230 ºC. HPLC analysis: Chiral normal phase HPLC was performed on an Agilent system (Santa Clara, CA, USA) equipped with a G1379A degasser, G1312A binary pump, a G1367A well plate autosampler unit, a G1316A temperature controlled column compartment and a G1315C diode array detector. CHIRALCEL OD-H Analytical (Daicel (Osaka, Japan), 250 mm length, 4.6 mm diameter, 5 µm particle size) column was used. The typical injection

5 volume was 15 µl and chromatograms were monitored at 265 nm. All solvent mixtures are given in Hexane:Isopropanol (+0.1%DEA) (v/v) ratios. Table S2 Analytical conditions and retention times (Rt) for the different substrates and products. Entry Method Column Substrate Rt Product Rt 1 GC MEGA DET-TBS 1a a HPLC ODH 95:5 1 1b b GC MEGA DET-TBS 1c c GC MEGA DET-TBS 1d d HPLC ODH 90:10 1e e HPLC ODH 95:5 1f f GC MEGA DET-TBS 1g g HPLC ODH 95:5 1h h Column and solvent mixture (hexane:isopropanol)