Purification and Characterization of a Novel β-1,3–1,4-Glucanase (Lichenase) from Thermophilic <i>Rhizomucor miehei</i> with High Specific Activity and Its Gene Sequence

Tang, Yanbin; Yang, Shaoqing; Yan, Qiaojuan; Zhou, Peng; Cui, Jian; Jiang, Zhengqiang

doi:10.1021/jf2049799

Cited by 58 publications

(50 citation statements)

References 32 publications

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“…Besides, NfEG12A showed higher activity toward ␤-1,3-1,4-glucans than CMC-Na, which makes it different from GH16 and GH17 glucanases. For example, McLic1 from Malbranchea cinnamomea (20), RmLic16A from Rhizomucor miehei (21), and ␤-glucanase from A. niger US368 (22), belonging to GH16, exhibited strict substrate specificity for mixed ␤-1,3-1,4-glucans (barley ␤-glucan and lichenin), whereas they displayed no activity on CMC. In contrast, GH17 glucanases hydrolyze internal ␤-1,3 glycosidic linkages in ␤-glucan oligo-and polysaccharides (23).…”

Section: Discussionmentioning

confidence: 99%

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Yang

Shi

Liu

et al. 2017

Appl Environ Microbiol

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Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger. Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (k cat /K m , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (k cat /K m ) of these enzymes (in descending order, NfEG12A ). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (k cat ). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the Ϫ1 or ϩ1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (k cat ). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.KEYWORDS Neosartorya fischeri, GH12 ␤-endoglucanase, loop, catalytic efficiency, hydrogen bond I n recent decades, bioethanol as a clean energy source has become a hot spot of research and industrial application (1). The bioconversion processes of producing fermentable sugars from biomass need a few glycoside hydrolases (GH), especially the

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Section: Discussionmentioning

confidence: 99%

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Yang

Shi

Liu

et al. 2017

Appl Environ Microbiol

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“…There was no significant differences between the native and the recombinant proteins. The recombinant protein exhibited maximal enzyme activity at 65°C with oat β-glucan as substrate, and showed a higher thermoresistance than previously reported for β-1,3-1,4-glucanases, such as from B. subtilis MA139 (40°C) (Qiao et al, 2009), B. licheniformis EGW039 (40°C) (Teng et al, 2006), B. amyloliquefaciens ATCC 23350 (50°C) (Sun et al, 2012), and thermophilic Rhizomucor miehei (60°C) (Tang et al, 2012). For the thermostability assay, the native and recombinant proteins were stable at 65°C for 1.5 h and had residual activity of 82.7 and 90.77%, respectively.…”

Section: Discussionmentioning

confidence: 70%

Purification, characterization, and heterologous expression of an antifungal protein from the endophytic Bacillus subtilis strain Em7 and its activity against Sclerotinia sclerotiorum

Wang¹,

Gao²,

Yan³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. An antifungal protein exhibiting a high activity against Sclerotinia sclerotiorum in vivo was purified by ammonium sulfate precipitation, hydrophobic chromatography, and gel filtration chromatography from the culture filtrate of the endophytic Bacillus subtilis strain Em7. The protein was characterized as a β-1,3-1,4-glucanase according to amino acid analysis, and showed excellent properties in thermal stability and acid resistance. At the same time, the antifungal protein was cloned and heterologously expressed in Escherichia coli BL21. The recombinant protein was purified and showed similar enzymatic properties to the native protein, exhibiting strong inhibitory activity against S. sclerotiorum. This shows that the β-1,3-1,4-glucanase may play a very important role in B. subtilis Em7 biocontrol function. In addition, many physiochemical properties of the native and purified recombinant protein were compared, including the effect of pH, temperature, metal cations, substrate specificity, and kinetic parameters. All parameters were similar between the native and recombinant pu- rified protein, indicating that the purified recombinant protein has potential for industrial applications.

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“…4967) was isolated from a pile of high-temperature hay due to anaerobic respiration in Sanmenxia city of Henan province, China. R. miehei CAU432 was maintained on potato dextrose-agar (PDA) plate as described by Tang et al [11]. The strain was grown in rich medium (2% oat flour, 1% tryptone, 1% yeast extract, 5% KH 2 PO 4 , 0.3% MgSO 4 · 7H 2 O, 0.3% CaCl 2 ) at 50°C for 2 days in a shaker with a rotation speed of 200 rpm.…”

Section: Methodsmentioning

confidence: 99%

“…Comparative genomic analyses of three thermophilic ascomycete species, Thermomyces lanuginosus [20], Thielavia terrestris and Myceliophthora thermophila suggest that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to be manipulated using classical and molecular genetics [23]. A thermophilic fungus R. miehei strain CAU432, newly isolated from self-heating hay in Henan Province of China, has been found to be a good producer of aspartic proteases and β-1,3-1,4-glucanase [11]. It exhibits a broad growth temperature ranging from 25–55°C and the optimum growth temperature is at 50°C.…”

Section: Introductionmentioning

confidence: 99%

Genome sequence and transcriptome analyses of the thermophilic zygomycete fungus Rhizomucor miehei

et al. 2014

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BackgroundThe zygomycete fungi like Rhizomucor miehei have been extensively exploited for the production of various enzymes. As a thermophilic fungus, R. miehei is capable of growing at temperatures that approach the upper limits for all eukaryotes. To date, over hundreds of fungal genomes are publicly available. However, Zygomycetes have been rarely investigated both genetically and genomically.ResultsHere, we report the genome of R. miehei CAU432 to explore the thermostable enzymatic repertoire of this fungus. The assembled genome size is 27.6-million-base (Mb) with 10,345 predicted protein-coding genes. Even being thermophilic, the G + C contents of fungal whole genome (43.8%) and coding genes (47.4%) are less than 50%. Phylogenetically, R. miehei is more closerly related to Phycomyces blakesleeanus than to Mucor circinelloides and Rhizopus oryzae. The genome of R. miehei harbors a large number of genes encoding secreted proteases, which is consistent with the characteristics of R. miehei being a rich producer of proteases. The transcriptome profile of R. miehei showed that the genes responsible for degrading starch, glucan, protein and lipid were highly expressed.ConclusionsThe genome information of R. miehei will facilitate future studies to better understand the mechanisms of fungal thermophilic adaptation and the exploring of the potential of R. miehei in industrial-scale production of thermostable enzymes. Based on the existence of a large repertoire of amylolytic, proteolytic and lipolytic genes in the genome, R. miehei has potential in the production of a variety of such enzymes.

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Purification and Characterization of a Novel β-1,3–1,4-Glucanase (Lichenase) from Thermophilic Rhizomucor miehei with High Specific Activity and Its Gene Sequence

Cited by 58 publications

References 32 publications

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Purification, characterization, and heterologous expression of an antifungal protein from the endophytic Bacillus subtilis strain Em7 and its activity against Sclerotinia sclerotiorum

Genome sequence and transcriptome analyses of the thermophilic zygomycete fungus Rhizomucor miehei

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