Characterization of in silico modeled synthetic protein enriched with branched-chain amino acids expressed in Pichia pastoris.

We have designed earlier the 3-dimensional structure of protein enriched with 56% branched-chain amino acids (BCAA) based on an α-helical coiled-coil structure. The chemically synthesized DNA (BCAA51 gene) was expressed in Pichia pastoris and confirmed by SDS-PAGE and western blot analysis. In the present study, the purified recombinant protein was characterized using circular dichroism and data revealed that the secondary structure contained 53.5% α-helix, 3.2% ß-strand, and 43.3% turns, which is in concurrence with the overall structure predicted by in silico modeling. The LC-ESI-MS/MS spectra revealed that three peptide masses showed similarity to peptides like EQLTK, LEIVIR, and ILDK, of the modeled BCAA51 protein with the sequence coverage of ~16% from N-terminal region. The N-terminal sequence of the first seven amino acid residues (EQLTKLE) was exactly matching with the in silico designed protein. In vitro digestibility of the protein using SGF and SIF showed the disappearance of ~11 kDa band and appearance of low molecular weight peptides, which indicated that the protein was easily digestible and non-allergenic, which is the overall objective of this study. Further in vivo digestibility and toxicology studies are required to conclusively utilize this protein as a supplement for the treatment of chronic liver diseases.

Improvement, cloning, and expression of an in silico designed protein enriched with large neutral amino acids in Pichia pastoris for possible application in phenylketonuria.

Phenylketonuria (PKU) is an inborn disease caused by defective phenylalanine hydroxylase, which consequently results in the accumulation of phenylalanine in the brain leading to further complications. One of the promising approaches in dietary treatment is the supplementation of large neutral amino acid (LNAA). The LNAA compete with phenylalanine for the common L-type LNAA transporter across the blood-brain barrier, and decrease phenylalanine levels in the brain. In this study, the earlier LNAA-enriched protein model was improved (Protein Model-66) and validated in silico. The reverse translated and codon-optimized synthetic LNAA66 gene was cloned into pPICZαC and expressed in Pichia pastoris. The expressed protein was purified by His Select affinity chromatography. SDS-PAGE and Western blotting analysis showed a band at an expected molecular weight of 12 kDa, confirming the expression of the modeled protein. To our knowledge, this is the first report showing the cloning and expression of an in silico designed LNAA-enriched protein. PRACTICAL APPLICATIONS: One of the promising dietary treatment of phenylketonuria (PKU) is the supplementation of large neutral amino acid (LNAA), wherein high levels of LNAA compete with phenylalanine for the same L-type LNAA transporter, and consequently decrease phenylalanine accumulation in the brain, thereby decreasing neurological complications. For the first time, here, we are showing that an in silico designed and validated Protein Model-66, rich in LNAA, can be successfully cloned and expressed in Pichia pastoris. The complete biochemical and structural characterization of this protein will give a clear insight into its potential application for PKU treatment. The protein can be potentially used as a supplement to treat PKU to those who are non-adherent to the restricted, non-palatable, and expensive diet. Furthermore, this novel and effective strategy of in silico designing, cloning and expression can be exploited to develop proteins for various applications of industrial, food, medical, and academic relevance.

Purification and Characterization of a Salt-Dependent Pectin Methylesterase from Carica papaya Fruit Mesocarp-Exocarp Tissue.

Pectin methylesterase (PME) is a ubiquitous cell wall enzyme, which de-esterifies and modifies pectins for food applications. Functional properties of pectin rely on molecular weight and degree of esterification, and thus de-esterification by PME influences the pectin functionality. The main aim of the study is to purify and biochemically characterize PME from the outer mesocarp-exocarp tissue of unripe Carica papaya L. fruit. The ion-exchange and gel-permeation chromatography purified enzyme exhibited a specific activity of 2363.1 ± 92.8 units/mg protein, with a fold purification of 10.6, and final recovery of 9.0%. The PME showed a low apparent mass of 27 kDa by SDS-PAGE. The optimal activity of purified PME was found at pH 7.0, and at 60 °C. The enzyme is fairly stable at 60 °C for 10 min, retaining 60% activity. The optimum activity was found with 0.25 mol/L monovalent salts indicating that this PME is salt-dependent. The Km of PME was 0.22 mg/mL, and the Vmax value was 1289.15 ± 15.9 units/mg. The increase in the calcium sensitivity of the PME-treated pectin indicated a blockwise mode of action. The PME significantly differs from other known plant PMEs in their biochemical properties. Manual inspection and MASCOT searching of generated tryptic peptides confirmed no homology to known papaya PME sequences. The preliminary results indicate that the papaya PME can be potentially utilized to modify pectin functionality at elevated temperature. However, further investigation is required to understand the usefulness of this enzyme for the modification of pectins for various food applications. PRACTICAL APPLICATION: In this work, a small, 27 kDa papaya PME was purified by ion-exchange and gel-permeation chromatography and biochemically characterized. The papaya PME significantly differs from other known plant PMEs in their biochemical properties. The preliminary results like fair thermostability coupled with high temperature optimum indicate that the papaya PME can be potentially utilized to modify pectin functionality at high temperature. Modification of pectin functionality at elevated temperatures is advantageous since it evades the detrimental action of other pectinolytic enzymes.

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation.

We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6-9) and moderate thermostability (optimum ca. 70°C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure.

Enzymatic modification of a model homogalacturonan with the thermally tolerant pectin methylesterase from Citrus: 1. Nanostructural characterization, enzyme mode of action, and effect of pH.

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ∼10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).