Secretory production and characterization of a highly effective chitosanase from <i>Streptomyces coelicolor</i> A3(2) M145 in <i>Pichia pastoris</i>

Liang, Jiayu; He, Shengbin; Sun, Jian; Bao, Haodong; Cui, Lanyu

doi:10.1002/biot.202300402

Biotechnology Journal

2024

DOI: 10.1002/biot.202300402

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Secretory production and characterization of a highly effective chitosanase from Streptomyces coelicolor A3(2) M145 in Pichia pastoris

Jiayu Liang,

Shengbin He,

Jian Sun

et al.

Abstract: In this study, a glycoside hydrolase family 46 chitosanase from Streptomyces coelicolor A3(2) M145 was firstly cloned and expressed in Pichia pastoris GS115 (P. pastoris GS115). The recombinant enzyme (CsnA) showed maximal activity at pH 6.0 and 65°C. Both thermal stability and pH stability of CsnA expressed in P. pastoris GS115 were significantly increased compared with homologous expression in Streptomyces coelicolor A3(2). A stable chitosanase activity of 725.7 ± 9.58 U mL−1 was obtained in fed‐batch fermen… Show more

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Cited by 3 publications

References 63 publications

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Depolymerization of the polyester–polyurethane by amidase GatA250 and enhancing the production of 4,4′‐methylenedianiline with cutinase LCC

Xin,

Lu,

Zeng

et al. 2024

Biotechnology Journal

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Polyurethane (PU) is a complex polymer synthesized from polyols and isocyanates. It contains urethane bonds that resist hydrolysis, which decreases the efficiency of biodegradation. In this study, we first expressed the amidase GatA250, and then, assessed the enzymatic characterization of GatA250 and its efficiency in degrading the polyester‐PU. GatA250 degraded self‐synthesized thermoplastic PU film and postconsumption foam with degradation efficiency of 8.17% and 4.29%, respectively. During the degradation, the film released 14.8 µm 4,4′‐methylenedianiline (MDA), but 1,4‐butanediol (BDO) and adipic acid (AA) were not released. Our findings indicated that GatA250 only cleaved urethane bonds in PU, and the degradation efficiency was extremely low. Hence, we introduced the cutinase LCC, which possesses hydrolytic activity on the ester bonds in PU, and then used both enzymes simultaneously to degrade the polyester‐PU. The combined system (LCC‐GatA250) had higher degradation efficiency for the degradation of PU film (42.2%) and foam (13.94%). The combined system also showed a 1.80 time increase in the production of the monomer MDA, and a 1.23 and 3.62 times increase in the production of AA and BDO, respectively, compared to their production recorded after treatment with only GatA250 or LCC. This study provides valuable insights into PU pollution control and also proposes applicable solutions to manage PU wastes through bio‐recycling.

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Depolymerization of the polyester–polyurethane by amidase GatA250 and enhancing the production of 4,4′‐methylenedianiline with cutinase LCC

Xin,

Lu,

Zeng

et al. 2024

Biotechnology Journal

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show abstract

High‐activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane

Li,

Shao,

Xiang

et al. 2024

Biotechnology Journal

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Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis‐sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty‐two single‐mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer‐aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1‐P6 showed a remarkable 114% increase in the catalytic rate constant (kcat), a 137% decrease in the Michaelis constant (Km), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1‐P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1‐P6 was produced by the secretion‐enhanced P. pastoris chassis to 199.6 ± 20 mg L−1 with a specific activity of 96 ± 0.32 U mg−1, resulting in a total enzyme activity of 19137 ± 1131 U L−1, demonstrating significant potential for industrial applications.

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Characterization of a Novel Acid-Stable Chitosanase from Lentinula edodes Suitable for Chitooligosaccharide Preparation

Wang,

Zhao,

et al. 2024

Foods

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As high-value chitosan derivatives, chitooligosaccharides (COSs) with biodegradable, biocompatible, nontoxic, antimicrobial, and antioxidant activities have been widely applied in food-related fields. Chitosanases can hydrolyze chitosan to produce COSs. Herein, a chitosanase (LeCho1) from Lentinula edodes was successfully expressed in Escherichia coli and was then purified and characterized. LeCho1 had a low sequence identity with other chitosanases reported from the GH75 family. The recombinant protein showed a molecular mass of 27 kDa on SDS-PAGE. LeCho1 preferentially hydrolyzed chitosan with a high degree of deacetylation (DDA) and exhibited maximal activity (71.88 U/mg) towards 95% DDA chitosan at pH 3.0 and 50 °C. It possessed good stability at pH 2.0–6.0 and temperatures below 45 °C. Its hydrolytic activity was remarkably enhanced by the metal ion Mn2+ at 1 mM, while it was totally inhibited by 1 mM Fe3+ or 10 mM EDTA. Its Km and Vmax values were 0.04 μM and 76.81 μmol·min−1·mg−1, respectively, indicating good substrate affinity. LeCho1 degraded chitosan into COSs with degrees of polymerization (DPs) of 2–5, while it had no action on COSs with DPs of less than 5, revealing its endo-chitosanase activity. This study proved that chitosanase LeCho1 is a promising candidate in the industrial preparation of COSs due to its excellent properties.

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Secretory production and characterization of a highly effective chitosanase from Streptomyces coelicolor A3(2) M145 in Pichia pastoris

Cited by 3 publications

References 63 publications

Depolymerization of the polyester–polyurethane by amidase GatA250 and enhancing the production of 4,4′‐methylenedianiline with cutinase LCC

Depolymerization of the polyester–polyurethane by amidase GatA250 and enhancing the production of 4,4′‐methylenedianiline with cutinase LCC

High‐activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane

Characterization of a Novel Acid-Stable Chitosanase from Lentinula edodes Suitable for Chitooligosaccharide Preparation

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