Molecular Characterization of Novel D-lyxose Isomerases from a Hot Spring Metagenome

doi:10.13523/j.cb.2205014

China Biotechnology

2022, Vol. 42

Issue (9): 27-38 DOI: 10.13523/j.cb.2205014

Molecular Characterization of Novel D-lyxose Isomerases from a Hot Spring Metagenome

SU Ya-li¹,CHEN Feng-zhen¹,SHI Xian-ai^1,²,WANG Guo-zeng^1,^2,^**(

)

1. College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China
2. Fujian Key Laboratory of Medical Instrument and Pharmaceutical Technology, Fuzhou University, Fuzhou 350108, China

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Abstract

Objective: To explore new thermostable D-lyxose isomerases that can efficiently catalyze the synthesis of rare sugars from the metagenomes of high temperature hot springs. Methods: Metagenomic DNA was extracted from the sediment of Jifei Hot Spring in Changning,Yunnan and subjected to high-throughput sequencing. The D-lyxose isomerase genes were identified by gene annotation and sequence alignment,and heterologous expression vectors were constructed and induced in E. coli. Two recombinant D-lyxose isomerases were purified by affinity chromatography and were characterized. Results: Eight D-lyxose isomerase genes were identified from the metagenomic sequencing results of the Jifei Hot Spring sediment. Four genes were selected for heterologous expression,among which JF-LI1 and JF-LI4 were successfully expressed in E. coli and their enzymatic activities were detected. The optimum temperatures of recombinant JF-LI1 and JF-LI4 were 70℃ and 75℃,respectively. JF-LI4 has a wide action temperature and high thermal stability,which retained more than 40% of its enzymatic activity in the temperature range of 30℃ to 100℃. The optimum pH of recombinant JF-LI1 and JF-LI4 were 7.0 and 7.5,respectively,and they have high activity and stability under neutral and slightly acidic conditions. Recombinant JF-LI1 and JF-LI4 have a broad substrate spectrum and are active on L-ribose,L-ribulose,D-fructose and D-mannose in addition to their most active effect on D-lyxose. The catalytic efficiencies of recombinant JF-LI1 and JF-LI4 for L-ribose were 0.56 and 0.61 L/(mmol·s^-1),respectively,which were the highest among the known D-lyxose isomerases. Conclusion: Eight new D-lyxose isomerase genes were obtained from the high temperature hot spring metagenome,and two of them were heterologously expressed and characterized. They have high pH stability,strong thermal stability and wide substrate specificity,which enable them to have important application potential in pharmaceutical industry, food, cosmetics and other industrial fields.

Key words： D-lyxose isomerase High temperature hot springs Metagenome Rare sugars Functional sugars

Received: 06 May 2022 Published: 10 October 2022

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Corresponding Authors: Guo-zeng WANG E-mail: wanggz@fzu.edu.cn

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	Ya-li SU
	Feng-zhen CHEN
	Xian-ai SHI
	Guo-zeng WANG

Cite this article:

SU Ya-li,CHEN Feng-zhen,SHI Xian-ai,WANG Guo-zeng. Molecular Characterization of Novel D-lyxose Isomerases from a Hot Spring Metagenome. China Biotechnology, 2022, 42(9): 27-38.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2205014 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I9/27

Table 1 Primers used in this study

Table 2 Protein sequence analysis of the eight D-lyxose isomerases from the Jifei hot spring metagenome

Fig.1 Phylogenetic tree and heatmap of sequence identities of the eight D-lyxose isomerases from the Jifei hot spring metagenome and those have been characterized Cl-LI: C. laevoribosii(ABI93960.1); Ps-LI: P. stuartii(EDU58657); Bl-LI: B. licheniformis LI(AAU22106.1); Bs-LI: B. subtilis(AIY91703); Dt-LI: D. turgidum(YP_002352606.1); Ts-LI: Thermofilum sp. ex4484_79(OYT29093.1); Cp-LI: C. polysaccharolyticus(WP_026486773.1); Td-LI: T. dichotomicum(WP_175482550); Pb-LI: P. bacterium 1109(KLU40859.1); Ta-LI: T. archaeon(RLE72808.1); To-LI: T. oceani(ADL08607.1); Sp-LI: S. proteamaculans LI(BAJ07463.1); Ka-LI: K. aerogenes LI(WP_154105566.1); Ec-LI: E. coli LI(Q8X5Q7.1)

Fig.2 Protein sequence alignment of the eight D-lyxose isomerases from the Jifei hot spring metagenome and those have been characterized Metal ion binding sites are marked with diamonds and those residues involved in the substrate binding are marked with triangles. The source and GenBank accession numbers are the same as in Fig. 1

Fig.3 SDS-PAGE analysis of the purified recombinant JF-LI1 and JF-LI4 M: Molecular weight protein marker; 1: Uninduced cell lysate; 2: Supernate of induced transformed cell lysate; 3: Precipitate of induced transformed cell lysate; 4: Purified protein

Table 3 Relative activity of recombinant JF-LI1 and JF-LI4 towards various sugars

Fig.4 Effect of pH on the activity (a) and stability (b) of the recombinant JF-LI1 and JF-LI4

Fig.5 Effect of temperature on the activity (a) and stability (b) of the recombinant JF-LI1 and JF-LI4

Table 4 Comparison of biochemical properties of D-lyxose isomerases from various source

Fig.6 Effect of various metal ions on the activity of JF-LI1 and JF-LI4

Fig.7 Kinetic parameters of JF-LI1and JF-LI4 towards D-lyxose and L-ribose (a) Kinetic parameters of JF-LI1 towards D-lyxose (b) Kinetic parameters of JF-LI1 towards L-ribose (c) Kinetic parameters of JF-LI4 towards D-lyxose (d) Kinetic parameters of JF-LI4 towards L-ribose


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