一种具有高β-葡聚糖酶活性的几丁质外切酶基因的克隆、表达和性质鉴定

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

摘要:

[目的] 表达并鉴定来源于维氏气单胞菌的几丁质酶Chi92并研究其作为水产饲用酶的有效性。[方法] 自A. veronii B565中克隆chi92基因并在Pichia pastoris GS115中进行表达,对表达成功的Chi92进行分离纯化和生化鉴定。最后将Chi92添加到含有毕赤酵母粉的饲料中饲喂斑马鱼2周,研究Chi92添加对斑马鱼生长、饲料利用率、肠道微绒毛形态和抗病性能的影响。[结果] chi92基因编码具有864个氨基酸残基的多肽。Chi92在pH 6.0和40℃时表现最佳酶活。Chi92对蛋白酶有抗性,同时酶活不受金属离子显著影响。Chi92具备高几丁质酶活(69.4 U/mL)。以胶体几丁质和β-1,3-1,4-葡聚糖作为底物时,比活力分别为809.2 U/mg和235.6 U/mg。薄层层析和电喷雾电离质谱联用技术均表明N-乙酰葡糖胺二聚体是Chi92酶解胶体几丁质的主要产物。Chi92在对酵母细胞壁的降解方面比其他几丁质酶性能更加优良。经过2周饲喂,添加有Chi92的饲料显著提高了斑马鱼肠道微绒毛的高度和密度,同时斑马鱼的生长,饲料利用率,以及抗病性能均得到了一定提高。[结论] Chi92具有pH稳定性、抗逆性和高酵母细胞壁降解功能,能较好地作为饲用酶用于温水水产养殖。

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.