Characterization and Functional Analysis of Lytic Polysaccharide Monooxygenase TtLPMO9I from Thermothelomyces thermophilus

doi:10.13560/j.cnki.biotech.bull.1985.2023-0877

Abstract

Abstract:

【Objective】 To explore a novel lytic polysaccharide monooxygenase（LPMO）enzyme and to elucidate its crucial role in cellulose degradation.【Method】 A new LPMO enzyme TtLPMO9I was cloned from Thermothelomyces thermophilus genome, and its sequence and structure were systematically analyzed to determine its evolutionary characteristics. The enzymatic properties of TtLPMO9I were characterized using the DNS method; the impact of different concentrations of ascorbic acid on the activity of TtLPMO9I was investigated by adding external electron donors to the reaction system. By detecting the production of reducing sugars, the synergistic effect between TtLPMO9I and cellulase was calculated when pretreated corn straw and Avicel were used as substrates. 【Result】 TtLPMO9I presented the highest activity at 60℃ and pH 5.0. After 12 h of incubation at 60℃, approximately 54% of its activity remained. TtLPMO9I still maintained initial enzyme activity at pH 6.0-8.0 for up to 12 h. Adding an external electron donor, ascorbic acid, to the reaction system, the activity of TtLPMO9I increased to 184%. In addition, TtLPMO9I has shown promising synergistic effects with cellulase in the degradation of pretreated corn straw and Avicel. Adding 50-200 μg of TtLPMO9I into the degradation system increased reducing sugar yields by 34%-142% and 6%-46% respectively. 【Conclusion】 TtLPMO9I not only has excellent thermal and pH stability, but also has outstanding performance in degrading lignocellulose, providing potential high-quality enzyme resources for industrial production.

Key words: lytic polysaccharide monooxygenase（LPMO）, heterologous expression, enzymatic properties, co-degradation

ZHENG Fei, YANG Jun-zhao, NIU Yu-feng, LI Rui-lin, ZHAO Guo-zhu. Characterization and Functional Analysis of Lytic Polysaccharide Monooxygenase TtLPMO9I from Thermothelomyces thermophilus[J]. Biotechnology Bulletin, 2024, 40(2): 289-299.

Figures/Tables 7

Table 1 Protein property prediction of the LPMO AA9 family from T. thermophilus

序号No.	蛋白 Protein	氨基酸数目 Number of amino acids	分子量 Molecular weight/kD	参考文献 Reference
1	MYCTH_112089	232	26.1	[19]
2	MtLPMO9B	323	32.4	[20]
3	MtLPMO9D	255	27.0	[19,21]
4	MtLPMO9A	225	24.4	[22]
5	AEO59955.1	235	24.4	未报道
6	AEO54509.1	303	31.5	未报道
7	MtLPMO9	342	34.9	[23]
8	TtLPMO9I	306	31.4	本文研究材料
9	MtLPMO9J	246	26.0	[24-25]
10	AEO59836.1	227	24.1	未报道
11	AEO59482.1	198	21.5	未报道
12	AEO58921.1	340	35.8	未报道
13	AEO58412.1	338	35.2	未报道
14	AEO61257.1	241	25.9	未报道
15	AEO56498.1^a	151	16.2	未报道
16	AEO59823.1	254	28.4	未报道
17	AEO55776.1	444	46.6	未报道
18	AEO61305.1	230	25.0	未报道
19	AEO55082.1	226	24.5	未报道
20	MtLPMO9C	237	24.9	[20]
21	AEO56547.1	245	26.5	未报道
22	AEO55652.1	242	25.5	未报道

Fig. 1 Phylogenetic tree of LPMO proteins in the AA9 family of T. thermophilus

Fig. 2 Bioinformatics analysis of TtLPMO9I A: Multiple sequence alignment of TtLPMO9I with LPMO in crystal structure. B: 3D structural simulation diagram of TtLPMO9I

Fig. 3 Validation of the recombinant protein TtLPMO9I A: SDS-PAGE analysis of TtLPMO9I. B: MALDI-TOF mass spectrometry analysis results diagram for TtLPMO9I. Marker: Protein molecular weight standard. 1: Purified protein of TtLPMO9I

Fig. 4 Effect of ascorbic acid concentration on the enzyme activity of TtLPMO9I ns: Not significance, the same below

Fig. 5 Enzymatic properties of TtLPMO9I A: Effect of temperature on the cellulase activity of TtLPMO9I. B: Effect of temperature on the stability of TtLPMO9I. C: Effect of pH on the cellulase activity of TtLPMO9I. D: Effect of pH on the viability stability of TtLPMO9I

Fig. 6 Synergistic reaction between TtLPMO9I and cellulase（Novozymes 188）for the degradation of pretreated corn straw and Avicel A: Pretreated corn straw was used as substrate for 12 h. B: Pretreated corn straw was used as substrate for 24 h. C: Reaction with Avicel as substrate for 12 h. D: Reaction with Avicel as substrate for 24 h. The blue block in the figure above indicates the amount of reducing sugar produced by adding 50, 100, and 200 μg TtLPMO9I, respectively. The green block indicates the amount of reducing sugar produced by adding 100 μL cellulase. The orange block indicates the amount of reducing sugar produced by 50 μg TtLPMO9I+100 μL cellulase, 100 μg TtLPMO9I+100 μL cellulase, and 200 μg TtLPMO9I+100 μL cellulase

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