Recombinant Lignin Peroxidase Research Articles

Recombinant Lignin Peroxidase with Superior Thermal Stability and Melanin Decolorization Efficiency in a Typical Human Skin-Mimicking Environment.

Recently, the desire for a safe and effective method for skin whitening has been growing in the cosmetics industry. Commonly used tyrosinase-inhibiting chemical reagents exhibit side effects. Thus, recent studies have focused on performing melanin decolorization with enzymes as an alternative due to the low toxicity of enzymes and their ability to decolorize melanin selectively. Herein, 10 different isozymes were expressed as recombinant lignin peroxidases (LiPs) from Phanerochaete chrysosporium (PcLiPs), and PcLiP isozyme 4 (PcLiP04) was selected due to its high stability and activity at pH 5.5 and 37 °C, which is close to human skin conditions. In vitro melanin decolorization results indicated that PcLiP04 exhibited at least 2.9-fold higher efficiency than that of well-known lignin peroxidase (PcLiP01) in a typical human skin-mimicking environment. The interaction force between melanin films measured by a surface forces apparatus (SFA) revealed that the decolorization of melanin by PcLiP04 harbors a disrupted structure, possibly interrupting π-π stacking and/or hydrogen bonds. In addition, a 3D reconstructed human pigmented epidermis skin model showed a decrease in melanin area to 59.8% using PcLiP04, which suggests that PcLiP04 exhibits a strong potential for skin whitening.

Medium optimization for enhanced production of recombinant lignin peroxidase in Pichia pastoris.

Different cultivation conditions and parameters were evaluated to improve the production and secretion of a recombinant Phanerochaete chrysosporium lipH8 gene in Komagataella phaffii (Pichia pastoris). The recombinant lipH8 gene with its native secretion signal was successfully cloned and expressed in Komagataella phaffii (Pichia pastoris) under the control of the alcohol oxidase 1 promoter (PAOX1). The results revealed that co-feeding with sorbitol and methanol increased rLiP secretion by 5.9-fold compared to the control conditions. The addition of 1mM FeSO4 increased LiP activity a further 6.0-fold during the induction phase. Moreover, the combination of several optimal conditions and parameters yielded an extracellular rLiP activity of 20.05Ul-1, which is more than ten-fold higherrelative to standard growth conditions (BMM10 medium, pH 6 and 30°C). Extracellular activity of a recombinant LiP expressed in P. pastoris increased more than ten-fold when co-feeding sorbitol and methanol as carbon sources, together with urea as nitrogen source, FeSO4 supplementation, lower pH and lowercultivation temperature.

Expression of Pleurotus eryngii versatile peroxidase in Escherichia coli and optimisation of in vitro folding

Heterologous expression of Pleurotus eryngii versatile peroxidase (VP) in Escherichia coli was investigated. The cDNA encoding the mature sequence of the allelic variant VPL2 was cloned into the expression vector pFLAG1 and expressed in E. coli W3110. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant polypeptide (VPL2∗) was found to be the major protein located in inclusion bodies. In vitro folding of VPL2∗ was initially performed under conditions previously determined for folding of recombinant lignin peroxidase from Phanerochaete chrysosporium (LiPH8∗) but a very low yield of active enzyme was obtained (<0.1% of the total protein in the folding reaction). The influence of different parameters in VPL2∗ folding was investigated and the results compared with those obtained for other peroxidases. Up to 7% folding yield was achieved with VPL2∗ using optimised conditions which included: 0.15 M urea, 5 mM Ca2+, 20 μM haemin, a 4:1 oxidised-glutathione/reduced-glutathione ratio and 0.1 mg/ml protein concentration at pH 9.5, a yield twice as high as previously obtained for other peroxidases from Classes II or III. The enzyme presented spectral and kinetic properties identical to those of the fungally derived protein. It was fully functional in both Mn-mediated and Mn-independent peroxidase assays.

Rapid Colorimetric Assay and Yeast Surface Display for Screening of Highly Functional Fungal Lignin Peroxidase.

Rapid screening method using color reaction was developed to quantitatively evaluate the lignin peroxidase (LiP) activity. For 2,4-dichlorophenol (DCP) degradation activity, this method correlated well with the HPLC method. The lignin peroxidase H2 gene was isolated from Phanerochaete chrysosporium, a white-rot fungus. The molecular weight of the recombinant LiP was ca. 38 kDa, which corresponded to the predicted mature LiP H2 gene. We performed cloning into pYD vector for yeast cell surface display of the gene. The surface expression was checked by the rapid, colorimetric screening method. The supernatant did not change the color, but the washed yeast cells changed the color from white to red, which confirmed the enzymes were located on the cell surface. Five colonies expressing the LiP H2 gene at high levels were selected. The average amount of 2,4-DCP degradation was about 23%. It was approximately 63% degradation level of the LiP from the wild type fungus. This screening and selection protocol is being applied to the DNA shuffling effort to screen LiPs with improved functionality and stability.

Oxidation of a Tetrameric Nonphenolic Lignin Model Compound by Lignin Peroxidase

The present study maps the active site of lignin peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta-O-4 linkages. The fungal and recombinant wild type lignin peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that lignin peroxidase is able to attack any of the C(alpha)-C(beta) linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant lignin peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.

Engineering of a Manganese-Binding Site in Lignin Peroxidase Isozyme H8 from Phanerochaete chrysosporium

A Mn2+-binding site was created in the recombinant lignin peroxidase isozyme H8 from Phanerochaete chrysosporium. In fungal Mn peroxidase, the Mn-binding site is composed of Glu35, Glu39, and Asp179. We generated a similar site in lignin peroxidase by generating an anionic binding site. We generated three mutations: Asn182Asp, Asp183Lys, and Ala36Glu. Its activity, veratryl alcohol, and Mn2+ oxidation were compared to those of native recombinant enzyme and to fungal Mn peroxidase isozyme H4, respectively. The mutated enzyme was able to oxidize Mn2+ and still retain its ability to oxidize veratryl alcohol. Steady-state results indicate that the enzyme's ability to oxidize veratryl alcohol was lowered slightly. The Km for Mn2+ was determined to be 1.57 mM and the kcat = 5.45 s−1. These results indicate that the mutated lignin peroxidase is less effective in Mn2+ oxidation that the wild type fungal enzyme. The pH optima of veratryl alcohol and Mn oxidation were altered by the mutation. They are one unit of pH value higher than those of recombinant H8 and wild type fungal Mn peroxidase isozyme H4.

The role of tryptophan residues in radiation sensitivity of enzymes as exemplified in horseradish peroxidase

The stability of recombinant wild-type horseradish peroxidase and its tryptophan-less mutant Trp117Phe toward γ-radiation was studied. The absence of tryptophan in the enzyme molecule results in a certain stabilization, which is manifested as the absence of the initial drop in activity and appearance of a lag period for doses of up to 45 Gy. Contrary to the wild-type enzyme, the dose response of the mutant is almost independent of the nature of the substrate used to measure the catalytic activity; this indirectly indicates that Trp117 participates in the oxidation of substrates. Pretreatment of the wild-type recombinant enzyme with hydrogen peroxide destabilizes the enzyme towards irradiation, while the same procedure for the mutant enzyme has virtually no effect on the dose response curve. This suggests the modification of Trp117 in the oxidation of the native enzyme with H2O2 in the absence of electron-donor substrates, which is the modification of Trp171 in the recombinant lignin peroxidase.

Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium.

The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter was used to drive expression of lip2, the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of Phanerochaete chrysosporium. The expression vector, pUGL, also contained the Schizophyllum commune ura1 gene as a selectable marker. pUGL was used to transform a P. chrysosporium Ura11 auxotroph to prototrophy. Ura+ transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPh8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.

Addition of Veratryl Alcohol Oxidase Activity to Manganese Peroxidase by Site-Directed Mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungusPhanerochaete chrysosporium.Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed inEscherichia coli.The kcatfor veratryl alcohol oxidation was 11 s−1, Kmfor veratryl alcohol ∼0.49 mM, and Kmfor hydrogen peroxide ∼25 μM at pH 2.3. The Kmfor veratryl alcohol was higher and Kmfor hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcatwas ∼2.6 × 102s−1at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.

Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus

Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (p I 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone λML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The K m of the recombinant enzyme for veratryl alcohol and H 2O 2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn 2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the p I of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification.

Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus

The cDNA clone λML-1 encoding one of the extracellular lignin peroxidases from the white rot fungus, Phanerochaete chrysosporium, was heterologously expressed in an active form using a recombinant baculovirus system. The glycosylated extracellular form of the recombinant protein contained the ferriprotoporphyrin IX moiety and was capable of oxidizing both iodide and the model lignin compound, veratryl alcohol. In comparative peroxidase assays using guaiacol and Mn(II), the recombinant lignin peroxidase did not appear to be Mn(II) dependent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the heterologously expressed peroxidase had an apparent molecular weight similar to that of the native fungal isozyme H8. The elution profile of the active recombinant enzyme derived by ion-exchange chromatography and immunoblot analysis using an anti-H8 monoclonal antibody provided further evidence that the λML-1 DNA encodes the lignin peroxidase H8.