几丁质脱乙酰酶(chitin deacetylase, CDA)是几丁质生物转化为壳聚糖的关键酶。然而,目前报道的高活性CDAs非常少。为了获得高效的CDA,并提高其对几丁质的生物转化效率,该研究对来源于Rhizopus stolonifer中的CDA进行基因克隆、表达及纯化,获得几丁质脱乙酰酶RsCDA1,研究其酶学性质并探究其与来源于Vibrio harveyi的几丁质酶VhChit2对几丁质的协同水解作用。结果表明,RsCDA1的比酶活力为5.2 U/mg,在45 ℃、pH 6.5条件下活性最高,在温度低于40 ℃、pH 6.5～8.0保持较好的稳定性。1 mmol/L的Ca2+、Mn2+、Mg2+、Zn2+、Ba2+和Co2+对RsCDA1的活力有促进作用;1 mmol/L的Ni2+和Cu2+对该酶有抑制作用。此外,RsCDA1与VhChit2在几丁质水解中表现出良好的协同降解和脱乙酰作用。RsCDA1与VhChit2(摩尔浓度比为10∶1)在50 ℃、pH 6.5条件下水解20 mg/mL几丁质2 h时,对几丁质酶和CDA的协同度分别达到120.3%和175.6%。与单独使用RsCDA1相比(27.5%),两者协同水解时几丁质的脱乙酰度增加到35.3%。因此,RsCDA1在几丁质的生物转化中具有潜在的应用前景。
Chitin deacetylase (CDA) is the key enzyme in converting chitin to chitosan. However, the reported CDAs with high activity are very limited nowadays. To obtain a high-efficiency CDA and improve its biotransformation efficiency on chitin, a CDA from R. stolonifer was cloned, expressed, and purified, and RsCDA1 was obtained. Characterization of RsCDA1 and its synergistic action with a chitinase from V. harveyi (VhChit2) on the hydrolysis of chitin were investigated. Results indicated that RsCDA1 had a specific activity of 5.2 U/mg, and exhibited the maximum activity at 45 °C and pH 6.5. RsCDA1 was relatively stable when the temperature was lower than 40 °C and the pH was in the range of 6.5-8.0. The activity of RsCDA1 was promoted by 1 mmol/L of Ca2+, Mn2+, Mg2+, Zn2+, Ba2+ and Co2+, and it was inhibited by 1 mmol/L of Ni2+ and Cu2+. Additionally, RsCDA1 exhibited a good synergistic degradation and deacetylation of chitin with VhChit2. When the mixture of RsCDA1 and VhChit2 (molar concentration ratio of 10: 1) was used to hydrolyze chitin (20 mg/mL) at 50 ℃ and pH 6.5 for 2 h, the degrees of synergy on chitinase and CDA reached 120.3% and 175.6%, respectively. The degree of deacetylation of chitin synergistically treated with RsCDA1 and VhChit2 increased to 35.3% from 27.5% that alone treated with RsCDA1. Thus, RsCDA1 has potential application in the biotransformation of chitin.
[1] MINCEA M, NEGRULESCU A, OSTAFE V.Preparation, modification, and applications of chitin nanowhiskers:A review[J].Reviews on Advanced Materials Science, 2012, 30:225-242.
[2] TOKATLI K, DEMIRDÖVEN A.Effects of chitosan edible film coatings on the physicochemical and microbiological qualities of sweet cherry (Prunus avium L.)[J].Scientia Horticulturae, 2020, 259:108656.
[3] TSIGOS I, MARTINOU A, KAFETZOPOULOS D, et al.Chitin deacetylases:New, versatile tools in biotechnology[J].Trends in Biotechnology, 2000, 18(7):305-312.
[4] 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.
[5] BLAIR D E, HEKMAT O, SCHÜTTELKOPF A W, et al.Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum[J].Biochemistry, 2006, 45(31):9416-9426.
[6] HOßBACH J, BUßWINKEL F, KRANZ A, et al.A chitin deacetylase of Podospora anserina has two functional chitin binding domains and a unique mode of action[J].Carbohydrate Polymers, 2018, 183:1-10.
[7] WANG Y X, NIU X, GUO X L, et al.Heterologous expression, characterization and possible functions of the chitin deacetylases, Cda1 and Cda2, from mushroom Coprinopsis cinerea[J].Glycobiology, 2018, 28(5):318-332.
[8] ALFONSO C, NUERO O M, SANTAMARÍA F, et al.Purification of a heat-stable chitin deacetylase from Aspergillus nidulans and its role in cell wall degradation[J].Current Microbiology, 1995, 30(1):49-54.
[9] KAFETZOPOULOS D, MARTINOU A, BOURIOTIS V.Bioconversion of chitin to chitosan:Purification and characterization of chitin deacetylase from Mucor rouxii[J].Proceedings of the National Academy of Sciences of the United States of America, 1993, 90(7):2564-2568.
[10] CORD-LANDWEHR S, MELCHER R L J, KOLKENBROCK S, et al.A chitin deacetylase from the endophytic fungus Pestalotiopsis sp.efficiently inactivates the elicitor activity of chitin oligomers in rice cells[J].Scientific Reports, 2016, 6:38018.
[11] AYE K N, KARUPPUSWAMY R, AHAMED T, et al.Peripheral enzymatic deacetylation of chitin and reprecipitated chitin particles[J].Bioresource Technology, 2006, 97(4):577-582.
[12] ZHANG Q, ZHANG X Y, HE Y C, et al.The synergistic action of two chitinases from Vibrio harveyi on chitin degradation[J].Carbohydrate Polymers, 2023, 307:120640.
[13] MA L, LIU Z Y, KONG Z J, et al.Functional characterization of a novel copper-dependent lytic polysaccharide monooxygenase TgAA11 from Trichoderma guizhouense NJAU 4742 in the oxidative degradation of chitin[J].Carbohydrate Polymers, 2021, 258:117708.
[14] NGO T H D, NGO D N.Effects of low-frequency ultrasound on heterogenous deacetylation of chitin[J].International Journal of Biological Macromolecules, 2017, 104:1604-1610.
[15] LIU Z L, GAY L M, TUVENG T R, et al.Structure and function of a broad-specificity chitin deacetylase from Aspergillus nidulans FGSC A4[J].Scientific Reports, 2017, 7:1746.
[16] QIN Z, CHEN Q M, LIN S, et al.Expression and characterization of a novel cold-adapted chitosanase suitable for chitooligosaccharides controllable preparation[J].Food Chemistry, 2018, 253:139-147.
[17] YANG S Q, FU X, YAN Q J, et al.Cloning, expression, purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii[J].Food Chemistry, 2016, 192:1041-1048.
[18] VÁZQUEZ J A, RAMOS P, MIRÓN J, et al.Production of chitin from Penaeus vannamei by-products to pilot plant scale using a combination of enzymatic and chemical processes and subsequent optimization of the chemical production of chitosan by response surface methodology[J].Marine Drugs, 2017, 15(6):180.
[19] CHAI J L, HANG J H, ZHANG C G, et al.Purification and characterization of chitin deacetylase active on insoluble chitin from Nitratireductor aquimarinus MCDA3-3[J].International Journal of Biological Macromolecules, 2020, 152:922-929.
[20] BHAT P, PAWASKAR G M, RAVAL R, et al.Expression of Bacillus licheniformis chitin deacetylase in E.coli pLysS:Sustainable production, purification and characterisation[J].International Journal of Biological Macromolecules, 2019, 131:1008-1013.
[21] ZHAO Y, PARK R, MUZZARELLI R.Chitin deacetylases:Properties and applications[J].Marine Drugs, 2010, 8:24-46.
[22] TOKUYASU K, OHNISHI-KAMEYAMA M, HAYASHI K.Purification and characterization of extracellular chitin deacetylase from Colletotrichum lindemuthianum[J].Bioscience Biotechnology and Biochemistry, 1996, 60(10):1598-1603.
[23] 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].The Biochemical Journal, 2003, 374:369-380.
[24] BAI Y, WANG Y X, LIU X, et al.Heterologous expression and characterization of a novel chitin deacetylase, CDA3, from the mushroom Coprinopsis cinerea[J].International Journal of Biological Macromolecules, 2020, 150:536-545.
