Engineering diacetylchitobiose deacetylase from Pyrococcus horikoshii towards an efficient glucosamine production

Huang, Ziyang; Mao, Xinzhu; Sun, Guoyun; Zhang, Hongzhi; Lü, Wei; Liu, Yanfeng; Li, Jianghua; Du, Guocheng; Liu, Long

doi:10.1016/j.biortech.2021.125241

Cited by 24 publications

(26 citation statements)

References 32 publications

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“…However, the optimal pH and temperature of PhDac was not suitable for the stability of GlcN. To solve this problem, Huang et al [38] predicted the amino acid sites related to the surface charge of PhDac by Rosetta software, and selected a mutant M20 with a high catalytic activity and a low optimal pH value by a high-throughput screening method.…”

Section: Semi-rational Evolution Strategiesmentioning

confidence: 99%

“…The design strategies mainly include random evolution and semi rational design. However, the catalytic activity and physicochemical properties of modified CDAs still barely meet the needs of industrial production [38,40]. With the continuous development of computer technology, Rosetta, alphafold 2, and other computer tools, the recognition of protein structure and function can be studies more rapidly.…”

Section: Rational Evolution Strategiesmentioning

confidence: 99%

“…The biochemical characterization of CDAs and CODs enable them to produce chitosan and COS in different application fields. Some researchers began to promote the industrial application of CDAs by constructing heterologous expression system with protein engineering technologies [37,38].…”

Section: Introductionmentioning

confidence: 99%

“…Many studies have tried to improve the heterogeneous expression of CDAs by Escherichia coli, Bacillus subtilis, Pichia pastoris or other hosts [39][40][41]. On the other hand, considering the cost and environmental problems, softer reacting conditions are required in industrial production, which has distance with the reaction characteristics of CDAs [38]. Therefore, CDAs are necessary to be modified by protein engineering to meet the industrial requirements.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, CDAs are necessary to be modified by protein engineering to meet the industrial requirements. Currently, researches on constructing expression systems and protein engineering of CDAs are still at the beginning stage and develop rapidly [37,38,40]. However, the work about these two fields has not been covered in the previous reviews.…”

Section: Introductionmentioning

confidence: 99%

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Chitin deacetylase: from molecular structure to practical applications

Huang

Sun

Mao

et al. 2022

Syst Microbiol and Biomanuf

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Chitosan and chitooligosaccharides (COS), as derivatives of chitin through deacetylation reaction, have broad applications due to their good biodegradability, biocompatibility, and solubility. In addition, chitosan and COS are involved in cell wall morphogenesis and host-pathogen interactions in vivo. Chitin deacetylases (CDAs) are enzymes that can catalyze the de-N-acetylation of chitin. They are widely distributed in protozoa, algae, bacteria, fungi, and insects with important physiological functions. Compared with the traditional chemical method, enzymatic catalysis by CDAs provides an enzymatic catalysis method to produce chitosan and COS with controllable deacetylation site and environmental friendliness. These characteristics attract researchers to produce CDAs by fungicides or pesticides. However, researches on heterologous expression and directed evolution of CDAs are still lacking. In this review, we summarize the latest knowledge of CDAs, especially for heterologous expression systems and directed evolution strategies, which may contribute to the industrial production and future application of CDAs.

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Section: Semi-rational Evolution Strategiesmentioning

confidence: 99%

Section: Rational Evolution Strategiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chitin deacetylase: from molecular structure to practical applications

Huang

Sun

Mao

et al. 2022

Syst Microbiol and Biomanuf

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A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

Sun

Lou

et al. 2022

Biotech & Bioengineering

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The pyrimidine metabolic pathway is tightly regulated in microorganisms, allowing limited success in metabolic engineering for the production of pathway‐related substances. Here, we constructed a four‐enzyme coupled system for the in vitro production of uridine triphosphate (UTP). The enzymes used include nucleoside kinase, uridylate kinase, nucleoside diphosphate kinase, and polyphosphate kinase for energy regeneration. All these enzymes are derived from extremophiles. To increase the total and unit time yield of the product, three enzymes other than polyphosphate kinase were modified separately by multiple protein engineering strategies. A nucleoside kinase variant with increased specific activity from 2.7 to 36.5 U/mg, a uridylate kinase variant (specific activity of 37.1 U/mg) with a 5.2‐fold increase in thermostability, and a nucleoside diphosphate kinase variant with a 2‐fold increase in a specific activity to over 900 U/mg were obtained, respectively. The reaction conditions of the coupled system were further optimized, and a two‐stage method was taken to avoid the problem of enzymatic pH adaptation mismatch. Under optimal conditions, this system can produce more than 65 mM UTP (31.5 g/L) in 3.0 h. The substrate conversion rate exceeded 98% and the maximum UTP productivity reached 40 mM/h.

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