Gene cloning, expression and characterization of an exo-chitinase with high β-glucanase activity from Aeromonas veronii B565
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    Abstract:

    [Objective] We aimed to express and characterize biochemical properties of Chi92, a chitinase from Aeromonas veronii B565, and study its potential application as aquafeed supplement. [Methods] The chitinase gene chi92 was cloned from A. veronii strain B565 and expressed in Pichia pastoris GS115. The recombinant chitinase (Chi92) was purified and characterized. Chi92 was supplemented in diets containing P. pastoris powder and fed to zebrafish for 14 days. By comparing with the control group, effect of Chi92 supplementation on growth, feed utilization, microvilli morphology, and disease resistance was investigated. [Results] The complete gene sequence encoded a polypeptide with 864 amino acids. Chi92 exhibited optimal activity at pH 6.0 and 40℃, and was resistant to proteases and not substantially inhibited by metal ions. Chi92 had high chitinase activity (69.4 U/mL). The specific activity was 809.2 U/mg and 235.6 U/mg on colloidal chitin and β-1,3-1,4-glucan, respectively. Thin-layer chromatography and electrospray ionization-coupled mass spectrometry revealed that N-diacetylglucosamine was the dominant product of Chi92 when colloidal chitin was used as substrate. Moreover, Chi92 showed advantages over other chitinases for degradation of yeast cell wall. Supplementation of Chi92 in diet containing yeast product significantly improved the intestine microvilli length and density of zebrafish after two weeks of feeding. Marginally improved growth performance, feed utilization, as well as disease resistance were also observed in the Chi92 supplement group. [Conclusion] The pH stability, resistance against metal ions/chemical reagents/proteases, and high yeast cell wall degradation activity of Chi92 suggest its potential use as feed additive enzyme for warm water aquaculture.

    Reference
    [1] Tsujibo H, Yoshida Y, Miyamoto K, Imada C, Okami Y, Inamori Y. Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7. Canadian Journal of Microbiology, 1992, 38(9):891-897.
    [2] Tsujibo H, Orikoshi H, Shiotani K, Hayashi M, Umeda J, Miyamoto K, Imada C, Okami Y, Inamori Y. Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure. Applied and Environmental Microbiology, 1998, 64(2):472-478.
    [3] Hou J, Han J, Cai L, Zhou J, Lü Y, Jin C, Liu JF, Xiang H. Characterization of genes for chitin catabolism in Haloferax mediterranei. Applied Microbiology and Biotechnology, 2014, 98(3):1185-1194.
    [4] Blaak H, Schnellmann J, Walter S, Henrissat B, Schrempf H. Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, putative protein domains and relationship to other chitinases. European Journal of Biochemistry, 1993, 214(3):659-669.
    [5] Khoushab F, Yamabhai M. Chitin research revisited. Marine Drugs, 2010, 8(7):1988-2012.
    [6] Ratledge C. Biochemistry of microbial degradation. 2nd ed. Dordrecht, Netherlands:Kluwer Academic Publishers, 1994, 279-331.
    [7] Tanaka T, Fukui T, Imanaka T. Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Journal of Biological Chemistry, 2001, 276(38):35629-35635.
    [8] Zhang YT, Zhou ZG, Liu YC, Cao YN, He SX, Huo FM, Qin CB, Yao B, Ringø E. High-yield production of a chitinase from Aeromonas veronii B565 as a potential feed supplement for warm-water aquaculture. Applied Microbiology and Biotechnolgy, 2014, 98(4):1651-1662.
    [9] Xia GQ, Jin CS, Zhou J, Yang SJ, Zhang SZ, Jin C. A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. European Journal of Biochemisty, 2001, 268(14):4079-4085.
    [10] Kirubakaran SI, Sakthivel N. Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli. Protein Expression and Purification, 2007, 52(1):159-166.
    [11] Nevoigt E. Progress in Metabolic engineering of Saccharomyces cerevisiae. Microbiol and Molecular Biology Reviews, 2008, 72(3):379-412.
    [12] Sanderson GW, Jolly SO. The value of PhafJia yeast as a feed ingredient for salmonid fish. Aquaculture, 1994, 124(1/4):193-200.
    [13] Valentín E, Herrero E, Rico H, Miragall F, Sentandreu R. Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae. Archives of Microbiology, 1987, 148(2):88-94.
    [14] Klis FM. Review:Cell wall assembly in yeast. Yeast, 1994, 10(7):851-869.
    [15] Adams DJ. Fungal cell wall chitinases and glucanases. Microbiology, 2004, 150(7):2029-2035.
    [16] Li YX, Liu YC, Zhou ZM, Huang HQ, Ren Y, Zhang YT, Li GN, Zhou ZG, Wang L. Complete genome sequence of Aeromonas veronii Strain B565. Journal of Bacteriology, 2011, 193(13):3389-3390.
    [17] Feng XJ, Jiang WQ, Hong W. The prepartion of chitosan and the it's application in wastewater treatment. Guizhou Chemical Industry, 2006, 31(3):36-38. (in Chinese)冯晓静, 蒋文强, 洪卫. 壳聚糖的制备及其在废水处理中的应用. 贵州化工, 2006, 31(3):36-38.
    [18] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 1959, 31(3):426-428.
    [19] Lee YS, Park IH, Yoo JS, Chung SY, Cho YS, Ahn SC, Kim CM, Choi YL. Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101. Bioresource Technology, 2007, 98(14):2734-2741.
    [20] Qin CB, Zhang YT, Liu WS, Xu L, Yang YL, Zhou ZG. Effects of chito-oligosaccharides supplementation on growth performance, intestinal cytokine expression, autochthonous gut bacteria and disease resistance in hybrid tilapia Oreohromis niloticus♀×Oreochromis aureus♂. Fish & Shellfish Immunology, 2014, 40(1):267-274.
    [21] Malik A, Wenuganen S, Suwanto A, Tjahjono B. Cloning, DNA sequence, and expression of Aeromonas caviae WS7b chitinase gene. Molecular Biotechnology, 2003, 23(1):1-10.
    [22] Wu ML, Chuang YC, Chen JP, Chen CS, Chang MC. Identification and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101. Applied and Environmental Microbiology, 2001, 67(11):5100-5106.
    [23] Orikoshi H, Baba N, Nakayama S, Kashu H, Miyamoto K, Yasuda M, Inamori Y, Tsujibo H. Molecular analysis of the gene encoding a novel cold-adapted chitinase (ChiB) from a marine bacterium, Alteromonas sp. Strain O-7. Journal of Bacteriology, 2003, 185(4):1153-1160.
    [24] Kim YS, Ho SB. Intestinal goblet cells and mucins in health and disease:recent insights and progress. Current Gastroenterology Reports, 2010, 12(5):319-330.
    [25] Schulz BL, Packer NH, Karlsson NG. Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis. Analytical Chemisty, 2002, 74(23):6088-6097.
    [26] Oliva-Teles A, Gonçalves P. Partial replacement of fishmeal by brewers yeast (Saccaromyces cerevisae) in diets for sea bass (Dicentrarchus labrax) juveniles. Aquaculture, 2001, 202(3/4):269-278.
    [27] Anderson DP, Siwicki AK, Rumsey GL. Injection or immersion delivery of selected immunostimulants to trout demonstrate enhancement of nonspecific defense mechanisms and protective immunity//Shariff M, Arthur JR, Subasinghe RP. (eds.). Diseases in Asian Aquaculture:II. Manila, Phillipines:Fish Health Section, Asian Fisheries Society, 1995, 413-426.
    [28] Lin SM, Mao SH, Guan Y, Lin X, Luo L. Dietary administration of chitooligosaccharides to enhance growth, innate immune response and disease resistance of Trachinotus ovatus. Fish & Shellfish Immunology, 2012, 32(5):909-913.
    [29] Liu HL, Sun MM, Wang HW, Wan WJ, Wang JT. Effects of chito-oligosaccharides on growth performance, serum biochemical in-dices and non-specific immunity function of rainbow trout (Oncorhynchus mykiss). Chinese Journal of Animal Nutrition, 2012, 24(3):479-486. (in Chinese)刘含亮, 孙敏敏, 王红卫, 万文菊, 王纪亭. 壳寡糖对虹鳟生长性能、血清生化指标及非特异性免疫功能的影响. 动物营养学报, 2012, 24(3):479-486.
    [30] Luo L, Cai XF, He C, Xue M, Wu XF, Cao HN. Immune response, stress resistance and bacterial challenge in juvenile rainbow trouts Oncorhynchus mykiss fed diets containing chitosan-oligosaccharides. Current Zoology, 2009, 55(6):416-422.
    [31] Gao J, Zhang HJ, Yu SH, Wu SG, Yoon I, Quigley J, Gao YP, Qi GH. Effects of yeast culture in broiler diets on performance and immunomodulatory functions. Poultry Science, 2008, 87(7):1377-1384.
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Fengmin Huo, Chao Ran, Yalin Yang, Jun Hu, Zhigang Zhou. Gene cloning, expression and characterization of an exo-chitinase with high β-glucanase activity from Aeromonas veronii B565. [J]. Acta Microbiologica Sinica, 2016, 56(5): 787-803

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  • Received:July 13,2015
  • Revised:September 30,2015
  • Online: May 06,2016
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