Biochemical characterization of a β-N-acetylhexosaminidase from Streptomyces alfalfae and its application in the production of N-acetyl-d-glucosamine

Zhou

et al. 2022

Preprint

Background N-acetyl glucosamine (GlcNAc), possesses many specific bioactivities and has been widely used in biomedical, food, and chemical industries. Alternatively, enzymatic hydrolysis of chitin into GlcNAc using chitinolytic enzymes was shown to be a more attractive approach in recent years, because of the green process and the excellent bioactivity of product. Therefore, it is of economic and environmental value to realize the efficient production of GlcNAc from abundant chitin resources. Results In this study, a gene encoding β-N-acetylglucosaminidase, designated NAGaseA, was cloned from Chitinibacter sp. GC72 and subsequently functional expressed in Escherichia coli BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the well-characterized NAGases. The recombinant NAGaseA had a molecular mass of 92 kDa. Biochemical characterization of the purified NAGaseA revealed that the optimal reaction condition was at 40°C and pH 6.5, and exhibited great pH stability in the range of pH 6.5–9.5. The Vmax, Km, Kcat, and Kcat/Km of NAGaseA toward pNP-GlcNAc were 3333.33 µmol min−1 L−1, 39.99 µM, 4667.07 s−1, and 116.71 mL µmol −1 s−1, respectively. Analysis of the hydrolysis products of N-acetyl chitinoligosaccharides (N-Acetyl COSs) indicated that NAGaseA capable of converting N-Acetyl COSs((GlcNAc)2–(GlcNAc)6) into GlcNAc with hydrolysis ability order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6. Moreover, NAGaseA could generate (GlcNAc)3–(GlcNAc)6 from (GlcNAc)2–(GlcNAc)5, respectively. These results showed that NAGaseA is a multi-functional NAGase with transglycosylation activity. In addition, significantly synergistic action was observed between NAGaseA and other sources of chitinases during hydrolysis of colloid chitin. Finally, 0.759 g/L, 0.481 g/L and 0.986 g/L of GlcNAc with purity of 96% were obtained using different chitinase combinations, resulting in 1.6-2.69 fold increase, respectively. Conclusions The hydrolytic properties and good environmental adaptions indicate that NAGaseA has great potential in the bioconversion of chitin waste and behaved as an excellent candidate in GlcNAc producing.

Section: Effects Of Metal Ions On Activity Of Recombinant Nagaseasupporting

confidence: 59%

Section: Synergistic Action Between Nagasea and Chitinases On Chitin Degradationmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of New Multifunctional GH20 β-N-Acetylglucosaminidase From Chitinibacter sp. GC72 and Its Application in Converting Chitin Into N-Acetyl Glucosamine

Zhou

et al. 2022

Preprint

“… 26) On the other hand, a β- N -acetylhexosaminidase from S. alfalfae showed the highest activity against (GlcNAc) 4 , followed by (GlcNAc) 5 , (GlcNAc) 6 , (GlcNAc) 3 , and (GlcNAc) 2 . 27) The amino acid sequences of these enzymes were found to share more than 70 % identity with that of Sa HEX. NagC, a β- N -acetylglucosaminidase from S. thermoviolaceus OPC-520, which has low sequence identity with Sa HEX (22 %), hydrolyzed (GlcNAc) 2 faster than the other oligosaccharides, but did not hydrolyze (GlcNAc) 6 .…”

Section: Resultsmentioning

confidence: 98%

Characterization of a GH Family 20 Exo-β-<i>N</i>-acetylhexosaminidase with Antifungal Activity from <i>Streptomyces avermitilis</i>

et al. 2019

We characterized SaHEX, which is a glycoside hydrolase (GH) family 20 exo-β-Nacetylhexosaminidase found in Streptomyces avermitilis. SaHEX exolytically hydrolyzed chitin oligosaccharides from their non-reducing ends, and yielded N-acetylglucosamine (GlcNAc) as the end product. According to the initial rate of substrate hydrolysis, the rates of (GlcNAc) 3 and (GlcNAc) 5 hydrolysis were greater than the rates for the other oligosaccharides. The enzyme exhibited antifungal activity against Aspergillus niger, which was probably due to hydrolytic activity with regard to chitin in the hyphal tips. Therefore, SaHEX has potential for use in GlcNAc production and food preservation.

“…DL-6 is an endochitinase, and its products on crystalline α-chitin are a mixture of chitin COSs (DP 2–6), with (GlcNAc) 2 as the major product [ 27 ]. The mixture of purified chitinases SaChiB and SaHEX of strain Streptomyces alfalfa ACCC40021 can enhance the conversion of crystalline α-chitin to GlcNAc [ 62 ]. The chitinase of strain Chitinibacter sp.…”

Section: Resultsmentioning

confidence: 99%

Identification and Characterization of Three Chitinases with Potential in Direct Conversion of Crystalline Chitin into N,N′-diacetylchitobiose

Ren

Dang

et al. 2022

Marine Drugs

Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401T, with an ability to degrade crystalline chitin into (GlcNAc)2 (N,N’-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline α-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45–50 °C and pH optima of 7.0–7.5. They are all stable at 40 °C and in the pH range of 5.0–11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 °C. When acting on crystalline α-chitin, the main products of the three chitinases are all (GlcNAc)2, which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)2.