几丁质脱乙酰酶的特点及应用
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摘要
几丁质是渔业生产过程中废弃虾蟹壳的主要组成成分。传统生产几丁质、壳聚糖和壳寡糖的方法是化学消化法,此方法的主要问题是环境污染严重,随着人们环保意识的提高,该方法已难以持续。此外由于化学法生产的壳聚糖和壳寡糖难以控制其聚合度、脱乙酰度和脱乙酰模式,因此很难研究壳寡糖的结构与功能之间的关系。几丁质脱乙酰酶(Chitin deacetylase EC 3.5.1.41)是一种可在温和条件下将几丁寡糖转化为壳寡糖的酶,利用此酶规模化生产具有特定乙酰化模式的壳寡糖已成为一个很重要的研究方向。鉴于该酶对于产业发展的重要性,本文对目前已经研究的几丁质脱乙酰酶的来源、结构特征、催化机制、脱乙酰化模式及几丁质脱乙酰化酶的应用等方面进行了综述。
Chitin is a major component of shrimp and crab shells in fishery production.There are about 6 million to 8 million tons of discarded crab and shrimp shells produced each year.These discarded shrimp shells are usually dumped in landfills or discharged into the ocean,causing serious environmental pollution and waste of resources.Useful chemicals such as protein,calcium carbonate and chitin contained in shrimp and crab shells are ignored.Therefore,finding sustainable methods to make rational use of the crustacean shell has attracted widespread attention.The traditional method for obtaining chitin,chitosan and chitooligosaccharides is through the strong acid chemical digestion method.With the improvement of people's environmental awareness,this method has been difficult to sustain.In addition,chitosan and chitosan oligosaccharides produced by chemical methods are difficult to control their degree of polymerization,degree of acetylation and acetylation mode,so it is difficult to study the relationship between the structure and function of chitooligosaccharides.Chitin deacetylase(E.C.3.5.1.41)is an enzyme that can convert chitin oligosaccharides into chitosan oligosaccharides under mild conditions.Chitosan oligosaccharides with specific acetylation patterns have become an important research direction.Chitosan,a chitin deacetylated product,is a linear polymer composed of β-1,4-N-glucosamine,which is the only basic polysaccharide in nature.The partially acetylated chitooligosaccharide(COS)consists of N-acetylglucosamine(GlcNAc,A)and glucosamine(GlcN,D)and is a complex biopolymer with a variety of biological activities and potential applications.Chitosan has a broad spectrum of antibacterial properties,water retention,reproducibility,good biocompatibility,degradability,cell membrane permeability,high drug loading,non-toxic side effects,etc.Through glutaraldehyde after cross-linking,grafting,acetylation and other chemical and physical modification,it is widely used in medicine,environmental protection,food,agriculture,paper,cosmetics and other fields.Partially acetylated chitooligosaccharides are potent biological agents with anti-inflammatory,anti-tumor,immunomodulatory,neuroprotective and other important pharmaceutical activities.Their biological activities are thought to depend on their structure,ie their degree of polymerization and degree of acetylation,and their mode of acetylation.Chitin deacetylase(CDA)can directly deacetylate from chitin oligosaccharides to form chitosan oligosaccharides,which are widely present in eukaryotes and prokaryotes.The reaction conditions for the bio-enzymatic production of chitosan are mild,non-polluting,and the degree of polymerization,deacetylation and deacetylation of the product chitosan oligosaccharides are single,so chitin deacetylase has attracted wide attention.It has been reported that chitin deacetylase was successfully constructed into Pichia pastoris or Escherichia coli for heterologous expression.Chitin deacetylase(CDA)can directly deacetylate from chitosan oligosaccharides to form chitosan oligosaccharides,which are widely present in eukaryotes and prokaryotes.Several chitin deacetylases have been expressed in prokaryotic and eukaryotic hosts.The reaction conditions for the bio-enzymatic production of chitosan are mild,non-polluting,and the structure of chitosan oligosaccharides are single,so chitin deacetylase has attracted wide attention.In view of the importance of this enzyme for industrial development,this paper reviews the sources of chitin deacetylase,deacetylation mode,catalytic mechanism and application of chitosan oligosaccharides.
Chitin is a major component of shrimp and crab shells in fishery production.There are about 6 million to 8 million tons of discarded crab and shrimp shells produced each year.These discarded shrimp shells are usually dumped in landfills or discharged into the ocean,causing serious environmental pollution and waste of resources.Useful chemicals such as protein,calcium carbonate and chitin contained in shrimp and crab shells are ignored.Therefore,finding sustainable methods to make rational use of the crustacean shell has attracted widespread attention.The traditional method for obtaining chitin,chitosan and chitooligosaccharides is through the strong acid chemical digestion method.With the improvement of people's environmental awareness,this method has been difficult to sustain.In addition,chitosan and chitosan oligosaccharides produced by chemical methods are difficult to control their degree of polymerization,degree of acetylation and acetylation mode,so it is difficult to study the relationship between the structure and function of chitooligosaccharides.Chitin deacetylase(E.C.3.5.1.41)is an enzyme that can convert chitin oligosaccharides into chitosan oligosaccharides under mild conditions.Chitosan oligosaccharides with specific acetylation patterns have become an important research direction.Chitosan,a chitin deacetylated product,is a linear polymer composed of β-1,4-N-glucosamine,which is the only basic polysaccharide in nature.The partially acetylated chitooligosaccharide(COS)consists of N-acetylglucosamine(GlcNAc,A)and glucosamine(GlcN,D)and is a complex biopolymer with a variety of biological activities and potential applications.Chitosan has a broad spectrum of antibacterial properties,water retention,reproducibility,good biocompatibility,degradability,cell membrane permeability,high drug loading,non-toxic side effects,etc.Through glutaraldehyde after cross-linking,grafting,acetylation and other chemical and physical modification,it is widely used in medicine,environmental protection,food,agriculture,paper,cosmetics and other fields.Partially acetylated chitooligosaccharides are potent biological agents with anti-inflammatory,anti-tumor,immunomodulatory,neuroprotective and other important pharmaceutical activities.Their biological activities are thought to depend on their structure,ie their degree of polymerization and degree of acetylation,and their mode of acetylation.Chitin deacetylase(CDA)can directly deacetylate from chitin oligosaccharides to form chitosan oligosaccharides,which are widely present in eukaryotes and prokaryotes.The reaction conditions for the bio-enzymatic production of chitosan are mild,non-polluting,and the degree of polymerization,deacetylation and deacetylation of the product chitosan oligosaccharides are single,so chitin deacetylase has attracted wide attention.It has been reported that chitin deacetylase was successfully constructed into Pichia pastoris or Escherichia coli for heterologous expression.Chitin deacetylase(CDA)can directly deacetylate from chitosan oligosaccharides to form chitosan oligosaccharides,which are widely present in eukaryotes and prokaryotes.Several chitin deacetylases have been expressed in prokaryotic and eukaryotic hosts.The reaction conditions for the bio-enzymatic production of chitosan are mild,non-polluting,and the structure of chitosan oligosaccharides are single,so chitin deacetylase has attracted wide attention.In view of the importance of this enzyme for industrial development,this paper reviews the sources of chitin deacetylase,deacetylation mode,catalytic mechanism and application of chitosan oligosaccharides.
引文
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[15]CANTAREL B L,COUTINHO P M,RANCUREL C,et al.The Carbohydrate-Active En Zymes database(CAZy):an expert resource for Glycogenomics[J].Nucleic Acids Research,2009,37(Database issue):233-238.
[16]BLAIR D E,SCHUTTELKOPF A W,MACRAE J I,et al.Structure and metal-dependent mechanism of peptidoglycan deacetylase,a streptococcal virulence factor[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(43):15429-15434.
[17]TUVENG T R,ROTHWEILER U,UDATHA G,et al.Structure and function of a CE4 deacetylase isolated from a marine environment[J].Plos One,2017,12(11):e0187544.
[18]TOKUYASU K,MITSUTOMI M,YAMAGUCHI I,et al.Recognition of chitooligosaccharides and their N-acetyl groups by putative subsites of chitin deacetylase from a deuteromycete,Colletotrichum lindemuthianum[J].Biochemistry,2000,39(30):8837-8843.
[19]LI X,WANG L X,WANG X,et al.The chitin catabolic cascade in the marine bacterium Vibrio cholerae:Characterization of a unique chitin oligosaccharide deacetylase[J].Glycobiology,2007,17(12):1377-1387.
[20]HEKMAT O,TOKUYASU K,WITHERS S G.Subsite structure of the endo-type chitin deacetylase from a Deuteromycete,Colletotrichum lindemuthianum:an investigation using steady-state kinetic analysis and MS[J].Biochemical Journal,2003,374(2):369-380.
[21]BARTLETT D H,AZAM F.Microbiology.Chitin,cholera,and competence[J].Science,2005,310(5755):1775-1777.
[22]GIASTAS P,ANDREOU A,PAPAKYRIAKOU A,et al.Structures of the peptidoglycan N-acetylglucosamine deacetylase Bc1974 and its complexes with zinc metalloenzyme inhibitors[J].Biochemistry,2018,57(5):753-763.
[23]YANG W J,XU K K,YAN X,et al.Functional characterization of chitin deacetylase 1 gene disrupting larval-pupal transition in the drugstore beetle using RNA interference[J].Comparative biochemistry and physiology Part B:Biochemistry&molecular biology,2018,219-220:10-16.
[24]YAN X P,ZHAO D,ZHANG Y K,et al.Identification and characterization of chitin deacetylase2 from the American white moth,Hyphantria cunea(Drury)[J].Gene,2018,670:98-105.
[25]ARAGUNDE H,BIARNES X,PLANAS A.Substrate Recognition and Specificity of Chitin Deacetylases and Related Family 4 Carbohydrate Esterases[J].International Journal of Molecular Sciences,2018,19(2):30.
[26]YIN H,DU Y G,DONG Z M.Chitin oligosaccharide and chitosan oligosaccharide:Two similar but different plant elicitors[J].Frontiers in Plant Science,2016,7:522.
[27]DING K G,WANG Y Y,WANG H W,et al.6-O-sulfated chitosan promoting the neural differentiation of mouse embryonic stem cells[J].ACS Applied Materials&Interfaces,2014,6(22):20043-20050.
[2]MUANPRASAT C,CHATSUDTHIPONG V.Chitosan oligosaccharide:Biological activities and potential therapeutic applications[J].Pharmacol Ther,2017,170:80-97.
[3]NAQVI S,CORD-LANDWEHR S,SINGH R,et al.Arecombinant fungal chitin deacetylase produces fully defined chitosan oligomers with novel patterns of acetylation[J].Applied and Environmental Microbiology,2016,82(22):6645-6655.
[4]ANDRES E,ALBESA-JOVE D,BIARNES X,et al.Structural basis of chitin oligosaccharide deacetylation[J].Angewandta Chemie Internation Edition,2014,53(27):6882-6887.
[5]ZHAO Y,PARK R D,MUZZARELLI R A.Chitin deacetylases:properties and applications[J].Marine Drugs,2010,8(1):24-46.
[6]WANG Y,SONG J Z,YANG Q,et al.Cloning of a heat-stable chitin deacetylase gene from Aspergillus nidulans and its functional expression in Escherichia coli[J].Applied Biochemistry Biotechnology,2010,162(3):843-854.
[7]LU Y M,LABORDA P,HUANG K,et al.Highly efficient and selective biocatalytic production of glucosamine from chitin[J].Green Chemistry,2017,19(2):527-535.
[8]MARTINOU A,KOUTSIOULIS D,BOURIOTIS V.Expression,purification,and characterization of a cobalt-activated chitin deacetylase(Cda2p)from Saccharomyces cerevisiae[J].Protein Expression and Purification,2002,24(1):111-116.
[9]JOHN M,ROHRIG H,SCHMIDT J,et al.Rhizobium nodb protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase[J].Proceedings of the National Academy of Sciences of the United States of America,1993,90(2):625-629.
[10]BAKER L G,SPECHT C A,DONLIN M J,et al.Chitosan,the deacetylated form of chitin,is necessary for cell wall integrity in Cryptococcus neoformans[J].Eukaryotic Cell,2007,6(5):855-867.
[11]TOPRAK U,BALDWIN D,ERLANDSON M,et al.A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata(Lepidoptera:Noctuidae)peritrophic matrix[J].Insect Molecular Biology,2008,17(5):573-585.
[12]BLAIR D E,HEKMAT O,SCHUTTELKOPF A W,et al.Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum[J].Biochemistry,2006,45(31):9416-9426.
[13]URCH J E,HURTADO-GUERRERO R,BROSSOND,et al.Structural and functional characterization of a putative polysaccharide deacetylase of the human parasite Encephalitozoon cuniculi[J].Protein Science,2009,18(6):1197-1209.
[14]DENG D M,URCH J E,TEN CATE J M,et al.Streptococcus mutans SMU.623c codes for a functional,metal-dependent polysaccharide deacetylase that modulates interactions with salivary agglutinin[J].Journal of Bacteriology,2009,191(1):394-402.
[15]CANTAREL B L,COUTINHO P M,RANCUREL C,et al.The Carbohydrate-Active En Zymes database(CAZy):an expert resource for Glycogenomics[J].Nucleic Acids Research,2009,37(Database issue):233-238.
[16]BLAIR D E,SCHUTTELKOPF A W,MACRAE J I,et al.Structure and metal-dependent mechanism of peptidoglycan deacetylase,a streptococcal virulence factor[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(43):15429-15434.
[17]TUVENG T R,ROTHWEILER U,UDATHA G,et al.Structure and function of a CE4 deacetylase isolated from a marine environment[J].Plos One,2017,12(11):e0187544.
[18]TOKUYASU K,MITSUTOMI M,YAMAGUCHI I,et al.Recognition of chitooligosaccharides and their N-acetyl groups by putative subsites of chitin deacetylase from a deuteromycete,Colletotrichum lindemuthianum[J].Biochemistry,2000,39(30):8837-8843.
[19]LI X,WANG L X,WANG X,et al.The chitin catabolic cascade in the marine bacterium Vibrio cholerae:Characterization of a unique chitin oligosaccharide deacetylase[J].Glycobiology,2007,17(12):1377-1387.
[20]HEKMAT O,TOKUYASU K,WITHERS S G.Subsite structure of the endo-type chitin deacetylase from a Deuteromycete,Colletotrichum lindemuthianum:an investigation using steady-state kinetic analysis and MS[J].Biochemical Journal,2003,374(2):369-380.
[21]BARTLETT D H,AZAM F.Microbiology.Chitin,cholera,and competence[J].Science,2005,310(5755):1775-1777.
[22]GIASTAS P,ANDREOU A,PAPAKYRIAKOU A,et al.Structures of the peptidoglycan N-acetylglucosamine deacetylase Bc1974 and its complexes with zinc metalloenzyme inhibitors[J].Biochemistry,2018,57(5):753-763.
[23]YANG W J,XU K K,YAN X,et al.Functional characterization of chitin deacetylase 1 gene disrupting larval-pupal transition in the drugstore beetle using RNA interference[J].Comparative biochemistry and physiology Part B:Biochemistry&molecular biology,2018,219-220:10-16.
[24]YAN X P,ZHAO D,ZHANG Y K,et al.Identification and characterization of chitin deacetylase2 from the American white moth,Hyphantria cunea(Drury)[J].Gene,2018,670:98-105.
[25]ARAGUNDE H,BIARNES X,PLANAS A.Substrate Recognition and Specificity of Chitin Deacetylases and Related Family 4 Carbohydrate Esterases[J].International Journal of Molecular Sciences,2018,19(2):30.
[26]YIN H,DU Y G,DONG Z M.Chitin oligosaccharide and chitosan oligosaccharide:Two similar but different plant elicitors[J].Frontiers in Plant Science,2016,7:522.
[27]DING K G,WANG Y Y,WANG H W,et al.6-O-sulfated chitosan promoting the neural differentiation of mouse embryonic stem cells[J].ACS Applied Materials&Interfaces,2014,6(22):20043-20050.