Heterologous expression and characterization of thermostable chitinase and β-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-d-glucosamine

Qin, Xing; Xin, Yanzhe; Su, Xiaoyun; Wang, Xiaolu; Zhang, Jie; Tu, Tao; Wang, Yaru; Yao, Bin; Huang, Huoqing; Luo, Huiying

doi:10.1016/j.ijbiomac.2021.09.204

Cited by 14 publications

(4 citation statements)

References 44 publications

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“…To date, the heterologous expression of well-performing chitinolytic enzymes at a high level and in a cost-effective manner is essential to optimizing the enzymatic production of GlcNAc [ 46 ]. Ike et al [ 47 ] reported that the chitinase Chi46 derived from T. reesei could hydrolyze colloidal chitin to yield mainly (GlcNAc) 2 , and Chen et al [ 48 ] found that the GlcNAcase NAG1 derived from T. reesei could hydrolyze (GlcNAc) 2 to produce GlcNAc.…”

Section: Resultsmentioning

confidence: 99%

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

Wang

Xie

Chang

et al. 2023

Biotechnol Biofuels

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Background Filamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications. Results A novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%. Conclusions This PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications.

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Section: Resultsmentioning

confidence: 99%

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

Wang

Xie

Chang

et al. 2023

Biotechnol Biofuels

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“…25 In order to evaluate the contribution of BbHex1 to chitin and insect cuticle degradation, the dinitrosalicylic acid (DNS) method was used to measure the degradation product, reducing sugar. 26,27 Briefly, 2 μg purified BbHex1 in 10 μL 0.02 M PBS (pH 7.2) or/and 10 μL of 1 mg mL −1 chitinase (from Streptomyces griseus; Shanghai Yuanye Bio-Technology Co., Ltd, China) in sterile water was mixed with 0.5% (w/v) colloidal chitin or insect cuticle in 80 μL 50 mM sodium acetate-acid acetate (NaAc-Ac) buffer (pH 6.0), and incubated at 37 °C for 1 h. The reaction mixtures were terminated by addition of 200 μL DNS buffer (182 g L −1 C 4 H 4 KNaO 6 , 6.3 g L −1 C 7 H 4 N 2 O 7 , 21 g L −1 NaOH, 5.1 g L −1 C 6 H 5 OH, 5.1 g L −1 Na 2 SO 4 ), which were heated at 100 °C for 5 min and then cooled down on an ice bath for 3 min. The absorbance at 540 nm (OD 540 ) of the supernatant was photometrically determined with glucose (reducing sugar) as the standard as described previously.…”

Section: Heterologous Expression and Characterization Of Bbhex1mentioning

confidence: 99%

A fungal pathogen secretes a cell wall‐associated β‐N‐acetylhexosaminidase that is co‐expressed with chitinases to contribute to infection of insects

Lu,

Zhu,

Bai

et al. 2024

Pest Management Science

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BACKGROUNDβ‐N‐acetylhexosaminidases (HEXs) are widely distributed in fungi and involved in cell wall chitin metabolism and utilization of chitin‐containing substrates. However, details of the fungal pathogens‐derived HEXs in the interaction with their hosts remain limited.RESULTSAn insect nutrients‐induced β‐N‐acetylhexosaminidase, BbHex1, was identified from the entomopathogenic fungus Beauveria bassiana, which was involved in cell wall modification and degradation of insect cuticle. BbHex1 was localized to cell wall and secreted, and displayed enzyme activity to degrade the chitinase‐hydrolyzed product (GlcNAc)2. Disruption of BbHex1 resulted in a significant decrease in the level of cell wall chitin in the presence of insect nutrients and during infection of insects, with impaired ability to penetrate insect cuticle, accompanying downregulated cell wall metabolism‐involved and cuticle‐degrading chitinase genes. However, the opposite phenotypes were examined in the gene overexpression strain. Distinctly altered cell wall structures caused by BbHex1 mutation and overexpression led to the easy activation and evasion (respectively) of insect immune response during fungal infection. As a result, BbHex1 contributed to fungal virulence. Bioinformatics analysis revealed that promoters of some co‐expressed chitinase genes with the BbHex1 promoter shared conserved transcription factors Skn7, Msn2 and Ste12, and CreA‐binding motifs, implying co‐regulation of those genes with BbHex1.CONCLUSIONThese data support a mechanism that the fungal pathogen specifically expresses BbHex1, which is co‐expressed with chitinases to modify cell wall for evasion of insect immune recognition and to degrade insect cuticle, and contributes to the fungal virulence against insects. © 2024 Society of Chemical Industry.

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“…Dimeric N -acetyl chitobiose (GlcNAc) 2 , a highly valuable N -acetyl chitooligosaccharide (NCOS) with anti-oxidant, antiviral, metabolic regulatory, lipid-lowering, chelating, and hypoglycemic effects, can also be used to produce bioactive oligomers [ 8 , 9 , 10 ]. As potential functional materials in the agricultural, food, pharmaceutical, cosmetic, and other industries, the growing interest in these products has led to an increased desire for GlcNAc and (GlcNAc) 2 [ 11 , 12 ]. However, chitin has a crystalline structure with numerous intra- and inter-molecular hydrogen bonds, making it insoluble in aqueous media.…”

Section: Introductionmentioning

confidence: 99%

Heterologous Expression and Characterization of a pH-Stable Chitinase from Micromonospora aurantiaca with a Potential Application in Chitin Degradation

Guo,

Wang,

Yang

et al. 2024

Marine Drugs

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To promote the bioconversion of marine chitin waste into value-added products, we expressed a novel pH-stable Micromonospora aurantiaca-derived chitinase, MaChi1, in Escherichia coli and subsequently purified, characterized, and evaluated it for its chitin-converting capacity. Our results indicated that MaChi1 is of the glycoside hydrolase (GH) family 18 with a molecular weight of approximately 57 kDa, consisting of a GH18 catalytic domain and a cellulose-binding domain. We recorded its optimal activity at pH 5.0 and 55 °C. It exhibited excellent stability in a wide pH range of 3.0–10.0. Mg2+ (5 mM), and dithiothreitol (10 mM) significantly promoted MaChi1 activity. MaChi1 exhibited broad substrate specificity and hydrolyzed chitin, chitosan, cellulose, soluble starch, and N-acetyl chitooligosaccharides with polymerization degrees ranging from three to six. Moreover, MaChi1 exhibited an endo-type cleavage pattern, and it could efficiently convert colloidal chitin into N-acetyl-D-glucosamine (GlcNAc) and (GlcNAc)2 with yields of 227.2 and 505.9 mg/g chitin, respectively. Its high chitin-degrading capacity and exceptional pH tolerance makes it a promising tool with potential applications in chitin waste treatment and bioactive oligosaccharide production.

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Heterologous expression and characterization of thermostable chitinase and β-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-d-glucosamine

Cited by 14 publications

References 44 publications

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

A fungal pathogen secretes a cell wall‐associated β‐N‐acetylhexosaminidase that is co‐expressed with chitinases to contribute to infection of insects

Heterologous Expression and Characterization of a pH-Stable Chitinase from Micromonospora aurantiaca with a Potential Application in Chitin Degradation

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