High-level expression of β-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc

Jiang, Shun; Jiang, Hongying; Zhou, Yanmin; Jiang, Sijing; Zhang, Guimin

doi:10.1007/s00449-018-02067-5

Cited by 18 publications

(21 citation statements)

References 33 publications

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“…All these researches used the crude enzyme to obtain GlcNAc. Shun Jiang et al reported that commercial chitinase CtnSg can hydrolyze 3% colloidal chitin into N, N'-diacetylchitobiose, and then hydrolyze into GlcNAc by a heterologous expressed β-N-acetylglucosaminidase BsNagZ [25]. In our case, when combined with BsNagZ, ChiA could hydrolyze 30% colloidal chitin to the unique product GlcNAc.…”

Section: Discussionmentioning

confidence: 54%

“…β- N -acetylglucosaminidase BsNagZ has been identified to cut the colloidal chitin from the terminal to obtain GlcNAc at low efficiency [ 25 ]. Here, a mixture of 600 µL of 30% (w/v) colloidal chitin and 200 µL ChiA (18 U/mL) was incubated at 50 °C for 12 h, and then 200 µL BsNagZ (5 U/mL) was added and the mixture was kept at 60 °C for another 20 min.…”

Section: Resultsmentioning

confidence: 99%

“…BsNagZ is β- N -acetylglucosaminidase from B. subtilis , which has been identified to cut the colloidal chitin from the terminal to obtain GlcNAc with low efficiency, combining commercial chitinase can improve the yield of GlcNAc efficiently in our previous work [ 25 ]. Here, a mixture of 600 µL of 30% (w/v) colloidal chitin and 200 µL ChiA (18 U/mL) was kept at 50 °C for 12 h, and 200 µL BsNagZ (5 U/mL) was added and the mixture further incubated at 60 °C for another 20 min to obtain GlcNAc.…”

Section: Methodsmentioning

confidence: 99%

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Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc

et al. 2020

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Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N′-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.

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Section: Discussionmentioning

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc

et al. 2020

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“…Several NAGases were already expressed inside the cells of E. coli, which required cell lysis during the purification (Zhou et al, 2016;Du et al, 2019). For ease of the process, BsNagZ secretory expression in Pichia pastoris with multiple copy strategy has been conducted, which takes 14 days per fermentation (Jiang et al, 2019). Since the activity of BsNagZ is low, we need to find new NAGases with higher activity and better thermal stability.…”

Section: Introductionmentioning

confidence: 99%

“…NAGase from Bacillus licheniformis (BlNagZ) was expressed. Although P. pastoris has the advantage of secretory expression, the fermentation period is too long (Jiang et al, 2019). Hence, we try to express BlNagZ in E. coli to shorten the fermentation period.…”

Section: Introductionmentioning

confidence: 99%

High-Level Extracellular Expression of a New β-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc

Jiang

et al. 2021

Front. Microbiol.

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N-acetyl-β-D glucosamine (GlcNAc) is wildly used in cosmetics, nutraceuticals and pharmaceuticals. The traditional chemical process for GlcNAc production from chitin causes serious acidic pollution. Therefore, the enzymatic hydrolysis becomes a great promising and alternative strategy to produce GlcNAc. β-N-acetylglucosaminidase (NAGase) can hydrolyze chitin to produce GlcNAc. Here, a GH3 family NAGase encoding gene BlNagZ from Bacillus licheniformis was expressed extracellularly in Escherichia coli guided by signal peptide PelB. The recombinant BlNagZ presented the best activity at 60°C and pH 5.5 with a high specific activity of 13.05 U/mg. The BlNagZ activity in the fermentation supernatant can reach 13.62 U/mL after optimizing the culture conditions, which is 4.25 times higher than optimization before. Finally, combining BlNagZ with chitinase ChiA we identified before, chitin conversion efficiency to GlcNAc can reach 89.2% within 3.5 h. In all, this study provided not only a high active NAGase, and a secreted expression strategy to reduce the cost of production, which is conducive to the industrial application.

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A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high‐level expression in Pichia pastoris

et al. 2020

Biotech and App Biochem

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Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713 bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60 °C and over a broad pH range between 5.0 and 9.0, and was stable at 50 °C and over the pH range 4.0–9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of β‐N‐acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

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High-level expression of β-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc

Cited by 18 publications

References 33 publications

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc

High-Level Extracellular Expression of a New β-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc

A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high‐level expression in Pichia pastoris

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