Screening and Efficacy Evaluation of High-Yielding Manganese Peroxidase Strain

Abstract

Background: Lignocellulose resources are abundant, but the utilization of lignocellulose is seriously hindered by the existence of lignin. Microorganisms rely on lignin-degrading enzymes to degrade lignin efficiently, so screening strains with high-yielding lignin-degrading enzymes will surely lay the foundation for the full utilization of lignocellulose. Objective: Therefore, this study expects to screen strains with high production of manganese peroxidase by effective means, and conduct preliminary evaluation of their efficacy. Methods: First, the strains with high production of manganese peroxidase were first screened by aniline blue medium; then some strains that produced decolorized transparent circles were screened again by enzyme activity assay, so as to screen out the strains with high production of manganese peroxidase (Take P. chrysosporium as the control); observe the growth state of the strain and identify the strain; the enzyme activity was further improved by optimizing the fermentation medium; finally, the enzyme properties were analyzed to evaluate the stability of manganese peroxidase. Results: In this study, a strain (L7) with high production of manganese peroxidase was successfully screened through primary screening and re-screening. After 6 days of fermentation, its enzyme activity was as high as 39.63 U/mL. On PDA solid medium, the strain was a light brown, round colony with brittle and slender mycelia, and it was identified as Fusarium proliferatum. The optimization test of fermentation medium showed that the highest activity of manganese peroxidase was 46.06 IU/mL under the conditions of glucose 10 g, beef extract 10 g, pH 6.5 and rotation speed 100 r/min. The analysis of enzymatic properties showed that the optimum reaction temperature of manganese peroxidase in this study was 20–40 ℃; When the pH is between 4.8–5.6, the stability of manganese peroxidase is good; Cu²⁺, Fe²⁺, Mn²⁺ can promote the activity of manganese peroxidase in this study, while K²⁺ has obvious inhibition, which can reach 95.346%. The strain screened in this study had not only high manganese peroxidase activity and enzyme production efficiency, but also had stable properties, therefore it is very valuable and worthy for further development and utilization. As one of the key enzymes in lignin degradation, it will certainly provide new ideas for the efficient utilization of biomass resources also.

Appendix

Support Material 1

Sup 1. Sample Collection

12 samples of rotten wood and rotten wood leaves used for the screening of high-yielding manganese peroxidase strains were collected in the mountain forest region of Bijie City, Guizhou Province, in order to enrichment of high-yielding manganese peroxidase strains.

Sup 2. Strain Identification

The genomic DNA of the strain was extracted, selected the universal primers of ITS sequence of fungus, the specific sequence was amplified by the universal primers of strain identifying, it was compared with the known sequence in GenBank, and then the classification of the strain was determined. The strain was identified as Fusarium, presumably Fusarium proliferatum.

Description	Max score	Total score	Query cover	E value	Per. Ident	Accession
Fusarium proliferatum isolate RS_79 small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	MK332493.1
Fusarium proliferatum strain LCBPF06 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	KX553873.1
Fusarium proliferatum strain C118 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	KU377463.1
Fusarium proliferatum strain A1 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	KU184608.1
Fusarium sp. BAB-4794 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	KU571528.1
Fusarium proliferatum strain ECBR-1A small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	MW686898.1
Fusarium sp. Isolate YZ7-4 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	MW369617.1
Fusarium fujikuroi isolate RIZ5-2 small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	MW260108.1
Gibberella intermedia strain DG-10-4-3-2 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	JN889714.1
Gibberella intermedia strain DG-10-5-1-1 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence	985	985	100%	0.0	100.00%	JN889713.1