Biocatalytic cascade to polysaccharide amination

Abstract

Background: Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated.

Results: Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure.

Conclusions: Using oxidase-amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme.

Keywords: Aminated polysaccharide; Amine transaminases; Enzymatic cascade; Transaminase activity assay.

Fig. 1

The proposed oxidation–transamination biocatalytic cascade adopted from Aumala et al. [13]. The amination of d-galactose at the C-6 position is shown as an example. Aminations of other monosaccharides and positions are possible in theory by selecting different carbohydrate oxidoreductases

Fig. 2

A Mechanism of red precipitate formation in the NPEA assay and the Q-NPEA assay. B Workflow of the Q-NPEA assay when performed in wells of a transparent microtiter plate

Fig. 3

The standard curve established in this study to convert optical absorbance at 440 nm to NPEA depletion. Pyruvate with concentrations from 0.1 to 1.0 mM was used as the amino acceptor, and 10 mM NPEA was used as the amino donor. The microtiter plate was sealed with transparent and adhesive film and was incubated at 37 °C and 600 rpm for 24 h with the film side facing down. The absorbance of reaction wells was measured at 440 nm using a spectrophotometer, and depletion of pyruvate in all reactions was confirmed using HPLC-RI (Additional file 1: Fig. S4) (n = 4, error bars indicate standard errors)

Fig. 4

Conversions of FgrGaOx-oxidized carbohydrates through amination by CvATA (blue) and SpATA (orange) measured using the Q-NPEA assay. The oxidation and transamination reactions were performed simultaneously in one pot. The inserted line graph shows the amination of oxidized galactose with a linear time scale to underscore the higher initial activity of CvATA. Reactions (200 µL) comprised 50 mM HEPES buffer (pH 7.5), 29.8 µg/mL FgrGaOx, 12.8 µg/mL catalase, 1.8 µg/mL HRP, 150 µg/mL ATA, 20 µM PLP, 10 mM NPEA and carbohydrates containing 5 mM galactose and were carried out at 37 °C and 600 rpm. (n = 4, error bars indicate standard errors)

Fig. 5

ESI-Q-TOF MS spectra (positive ionization mode) of aminated galactose produced via CvATA and SpATA treatments with simultaneous FgrGaOx oxidation. The relative abundance of aminated galactose was quantified using HEPES as an internal reference. Reactions (1 mL) comprised 50 mM HEPES (pH 7.5), 29.8 µg/mL FgrGaOx, 1.8 µg/mL HRP, 12.8 µg/mL catalase, 0.15 mg/mL ATA, 20 µM PLP, 5 mM (S)-1-phenylethylamine and 5 mM galactose were carried out at 37 °C and 600 rpm for 5 h

Fig. 6

The XPS spectra showing nitrogen profiles of CvATA and SpATA aminated galactomannan with oxidized and non-oxidized galactomannan as references. The spectra were adjusted by scaling amide peaks to the same height so the amine peaks could be directly compared. The intensity of peaks is shown using a linear scale. Reactions (500 µL) comprised 50 mM potassium phosphate buffer (pH 7.5), 20 µM PLP, 29.8 µg/mL FgrGaOx, 12.8 µg/mL catalase, 1.8 µg/mL HRP, 0.15 mg/mL ATA, 5 mM (S)-1-PEA and 0.29% galactomannan containing 5 mM galactose. Reactions were carried out at 37 °C and 600 rpm for 24 h in 1.5 mL centrifuge tubes. The oxidized reference did not contain ATA, and the non-oxidized reference did not contain any enzymes. Enzymes (SpATA, FgrGaOx, catalase, and HRP) were added to both references after reactions to the concentrations stated above. Samples were desalted immediately using 100 kDa centrifuge filters before freeze–drying and XPS analyses

Fig. 7

Conversions of galactomannan amination catalyzed by FgrGaOx with reduced doses and SpATA measured using the Q-NPEA assay. Precipitate formation were measured separately from the transparent adhesive film and the liquid suspension. Reactions (200 µL) comprised 50 mM HEPES buffer (pH 7.5), 20 µM PLP, 10 mM NPEA, 0.15 mg/mL SpATA, 0.29% galactomannan equivalent to 5 mM galactose and FgrGaOx at various doses. The 1.0 × FgrGaOx dose corresponds to 29.8 µg/mL FgrGaOx, 12.8 µg/mL catalase, and 1.8 µg/mL HRP. All reactions were carried out at 37 °C and 600 rpm on 96-well plates (n = 4, error bars indicate standard errors)

Fig. 8

Conversions of galactomannan amination catalyzed by FgrGaOx and SpATA with increasing SpATA dose measured using the Q-NPEA assay. The dotted lines represent the best-fitted exponential functions. Reactions (200 µL) comprised 50 mM HEPES buffer (pH 7.5), 20 µM PLP, 10 mM NPEA, 29.8 µg/mL FgrGaOx, 12.8 µg/mL catalase, 1.8 µg/mL HRP, 0.29% galactomannan which contains 5 mM galactose and SpATA at concentrations shown in the figure legend. All reactions were carried out at 37 °C and 600 rpm on 96-well plates (n = 4, error bars indicate standard errors)

Fig. 9

Four hydrophilic amino acids on the SpATA surface blocking the binding of galactomannan oligosaccharide to the predicted polysaccharide binding cleft. The polysaccharide binding cleft is indicated by the yellow shade on the enzyme surface. Amino acids in green and cyan are from different polypeptide chains. The SpATA structure was obtained from the RCSB PDB database (PDB ID: 3HMU) and the sulfate ions in active sites were replaced with PMP to construct the holoenzyme. A galactomannan oligosaccharide with an 11-mannose backbone and one galactose side chain on the fifth mannose unit connected via α-(1→6)-linkage was used in the docking simulation

Fig. 10

Conversions of galactomannan amination catalyzed by FgrGaOx and SpATA mutants measured using the Q-NPEA assay. The dashed line indicates the conversion achieved by the wild-type SpATA in 30 h, and percentages indicate how 30-h conversions achieved by SpATA variants compare to the wild-type SpATA. Reactions (200 µL) consisted of 50 mM HEPES buffer (pH 7.5), 29.8 µg/mL FgrGaOx, 12.8 µg/mL catalase, 1.8 µg/mL HRP, 0.15 mg/mL ATA, 10 mM NPEA, 20 µM PLP and 0.29% galactomannan containing 5 mM galactose. Reactions were conducted at 37 °C and 600 rpm on a 96-well plate (n = 4, error bars indicate standard errors)

Fig. 11

The postulated ATA deactivation mechanism reproduced from Börner et al. [31]