Cloning and expression of Asperguillus endo-chitosanase gene in Escherichia coli

doi:10.3969/j.issn.2095-4344.2014.34.014

Abstract

Abstract:

BACKGROUND: Chitosanase is an enzyme for efficient and special hydrolysis of chitoan, and hence its effective and stable expression with enzymatic activity will contribute to improving gene therapeutic effect.

OBJECTIVE: To construct a chitosanase gene for the efficient and specifical hydrolysis of chitosan, and to investigate its expression in Escherichia coli and the main influencing factors of enzymatic activity.

METHODS: According to the sequences of endo-chitosanase of Aspergillus sp. CJ22-326 provided in Genbank (EU302818), primers were designed and synthesized. The Asperguillus endo-chitosanase gene was amplified by successive extension PCR. And then the recombinant pET28a-His6-CSN was constructed and expressed in Escherichia coli BL21. Finally the recombinant His6-CSN fusion protein was analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), the western blot and dinitrosalicylic acid assay for detecting the enzyme activity of eluted His6-CSN fusion protein. The influence of different pH value and temperature on the enzyme activity of the recombinant chitosanase was investigated.

RESULTS AND CONCLUSION: SDS-PAGE showed that 29 kDa proteins were expressed and the western blot assay showed that His6-CSN expressed successfully in the host. Dinitrosalicylic acid assay determined the enzymatic activity of His6-CSN was significantly higher than that of lysozyme, but lower than that of chitosanase from Streptomyces griseus (P < 0.05). The recombinant chitosanase displayed the maximal activity at temperature of 50 ℃ and pH value of 6.0. There were a higher enzymatic activity remaining at pH value of 4.0-7.0 and temperature of 30-50 ℃.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: biocompatible materials, chitosan, genes

CLC Number:

R318

Lu Hua-ding, Lian Li-yi, Chen Ming-wei, Dai Yu-hu. Cloning and expression of Asperguillus endo-chitosanase gene in Escherichia coli[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(34): 5490-5496.

Figures/Tables 1

2.1 壳聚糖酶基因片段的PCR扩增根据设计的壳聚糖酶基因序列所合成的23条引物，互为模板通过PCR拼接法扩增出目的片段，大小为717 bp，与曲霉菌CJ22-326内切型壳聚糖酶基因全长大小相符。 2.2 壳聚糖酶基因的克隆及双酶切鉴定质粒pET-28a(+)多克隆位点的两端有限制性内切酶BamHI/Xho I的识别位点，重组表达质粒进行BamHI/Xho I双酶切，进行琼脂糖凝胶电泳鉴定，获得约为5 300 bp和717 bp两个条带(图1)，证实构建重组原核表达质粒pET28a-His6-CSN成功。 2.3 重组原核表达质粒pET28a-His6-CSN表达壳聚糖酶的SDS-PAGE和Western-blot检测在IPTG诱导下，原核表达质粒pET28a-His6-CSN在E.coli BL21表达His6-CSN融合蛋白，使用Ni2+-NTA亲和层析得到纯化的融合蛋白。在SDS-PAGE中，经马考斯亮蓝染色后只有一个条带在相对分子质量29 000处出现，且在诱导表达1-4 h过程中，表达的融合蛋白的量逐渐增多，对照组不表达融合蛋白(图2A)。 Western-blot检测在相对分子质量25 000与35 000之间出现一蛋白条带(图2B)，该条带为纯化的融合蛋白His6-CSN。 2.4 原核表达融合蛋白His6-CSN的酶活性检测根据所得的氨基葡萄糖NAG作标准曲线，确定壳聚糖酶水解壳聚糖所产生的还原糖的量。在37 ℃、pH=6.0条件下，壳聚糖酶的活性为(14.055±0.375) U/μg，显著高于溶菌酶的活性(4.046±0.177) U/μg，两者均低于灰色链霉菌壳聚糖酶的活性(21.442±0.463) U/μg，差异有显著性意义(F=1 773.18，P < 0.05)，见图3。 2.5 pH和温度对壳聚糖酶活性的影响壳聚糖酶的最适pH值为6.0，且酶活性在pH为4.0-7.0范围内仍具有较高的活性；当pH=7.0时仍可达最高活性的73%；当pH=8时，酶活性下降为最高活性的46%(图4A)。壳聚糖酶的最适温度为 50 ℃，且在37 ℃时酶活性仍可达最大活性的89%；当温度为60 ℃时，酶活性急剧降低，仅为最高活性的30%；当温度为70 ℃时，酶完全失活(图4B)。"

References

[1] Kasaai MR.Various methods for determination of the degree of N-acetylation of chitin and chitosan: a review.J Agric Food Chem.2009;57(5):1667-1676.
[2] 张伟,林红,陈宇岳.甲壳素和壳聚糖的应用及发展前景[J].南通大学学报:自然科学版,2006,5(1):29-33.
[3] 李婷,胡小喜,周幸芝,等.从虾蟹壳中提取甲壳素的研究进展[J].食品工业,2014,35(6):209-212.
[4] 杨久林,谢红国,于炜婷,等.组织工程用壳聚糖研究进展[J].功能材料,2013,44(11):1521-1525.
[5] Raftery R,O'Brien FJ,Cryan SA.Chitosan for gene delivery and orthopedic tissue engineering applications. Molecules. 2013;18(5):5611-5647.
[6] Thirugnanasambandham K,Sivakumar V,Prakash MJ.Application of chitosan as an adsorbent to treat rice mill wastewater--mechanism, modelling and optimization. Carbohydr Polym.2013;97(2):451-457.
[7] Kouzai Y,Mochizuki S,Saito A,et al.Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus Magnaporthe oryzae. Plant Cell Rep. 2012;31(4):629-636.
[8] 单晓雪,郑人源,张涛,等.壳聚糖酶的研究进展[J].成都医学院学报,2007,2(3): 192-198.
[9] Jaiswal M,Chauhan D,Sankararamakrishnan N.Copper chitosan nanocomposite: synthesis, characterization, and application in removal of organophosphorous pesticide from agricultural runoff.Environ Sci Pollut Res Int.2012;19(6):2055-2062.
[10] 李广峰,杨建东.壳聚糖纳米粒子基因载体的研究现状[J].中国组织工程研究与临床康复,2011,15(47):8879-8882.
[11] Dai H,Jiang X,Tan GC,et al.Chitosan-DNA nanoparticles delivered by intrabiliary infusion enhance liver-targeted gene delivery.Int J Nanomedicine.2006;1(4): 507-522
[12] Jean M,Alameh M,De Jesus D,et al.Chitosan-based therapeutic nanoparticles for combination gene therapy and gene silencing of in vitro cell lines relevant to type 2 diabetes. Eur J Pharm Sci.2012;45(1-2):138-149.
[13] Plapied L,Vandermeulen G,Vroman B,et al.Bioadhesive nanoparticles of fungal chitosan for oral DNA delivery.Int J Pharm.2010;398(1-2):210-218.
[14] Gao S,Chen J,Dong L,et al.Targeting delivery of oligonucleotide and plasmid DNA to hepatocyte via galactosylated chitosan vector.Eur J Pharm Biopharm. 2005; 60(3):327-334.
[15] Smith JK,Moshref AR,Jennings JA,et al.Chitosan sponges for local synergistic infection therapy: a pilot study.Clin Orthop Relat Res.2013;471(10):3158-3164.
[16] Liang DC,Liu WG,Zuo AJ,et al.Pre-deliver chitosanase to cells: a novel strategy to improve gene expression by endocellular degradation-induced vector unpacking. Int J Pharm. 2006; 314(1):63-71.
[17] Jarmila V,Vavrikova E.Chitosan derivatives with antimicrobial, antitumour and antioxidant activities--a review.Curr Pharm Des.2011;17(32):3596-3607.
[18] Liu YL,Jiang S,Ke ZM,et al.Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production.Carbohydr Res.2009;344(6): 815-819.
[19] 金春阳,赵天勤,方良,等.壳寡糖作为基因载体的体外评价[J].医药导报,2013,32(9):1115-1119.
[20] Goo BG,Park JK.Characterization of an alkalophilic extracellular chitosanase from Bacillus cereus GU-02.J Biosci Bioeng.2014;117(6):684-689.
[21] Sugita A,Sugii A,Sato K,et al.Cloning and characterization of a gene coding for a major extracellular chitosanase from the koji mold Aspergillus oryzae.Biosci Biotechnol Biochem. 2012;76(1):193-195.
[22] Trombotto S,Ladaviere C,Delolme F,et al.Chemical preparation and structural characterization of a homogeneous series of chitin/chitosan oligomers. Biomacromolecules. 2008; 9(7):1731-1738.
[23] Sinha S,Tripathi P,Chand S.A new bifunctional chitosanase enzyme from Streptomyces sp. and its application in production of antioxidant chitooligosaccharides.Appl Biochem Biotechnol.2012;167(5):1029-1039.
[24] 张文清,夏玮,徐欢,等.非专一性酶催化壳聚糖水解反应的特性[J].功能高分子学报, 2003,16(1):44-48.
[25] 黄惠莉,朱利平.壳聚糖酶的研究进展[J].食品工业科技, 2012, 33(6):439-443.
[26] Xia W,Liu P,Liu J. Advance in chitosan hydrolysis by non-specific cellulases. Bioresour Technol. 2008;99(15): 6751-6762.
[27] Li S,Chen L,Wang C,et al.Expression, purification and characterization of endo-type chitosanase of Aspergillus sp. CJ22-326 from Escherichia coli. Carbohydr Res. 2008; 343 (17): 3001-3004.
[28] 涂绍勇,杨爱华,梅双喜,等. 3,5-二硝基水杨酸法(DNS)测定壳聚糖酶活力的探讨[J].食品科技,2012,37(1):240-242.
[29] Chen X,Zhai C,Kang L,et al.High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris. Biotechnol Lett. 2012; 34(4):689-694.
[30] Wang J,Zhou W,Yuan H,et al.Characterization of a novel fungal chitosanase Csn2 from Gongronella sp.JG.Carbohydr Res.2008;343(15):2583-2588.
[31] Choi YJ, Kim EJ,Piao Z,et al.Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides. Appl Environ Microbiol.2004;70(8): 4522-4531.
[32] 龚香艺,吴静,邬敏辰.真菌壳聚糖酶研究进展[J].食品科学, 2012, 33(17):308-311.
[33] Rodriguez-Martin A,Acosta R,Liddell S,et al.Characterization of the novel antifungal chitosanase PgChP and the encoding gene from Penicillium chrysogenum. Appl Microbiol Biotechnol. 2010;88(2):519-528.
[34] Cheng CY,Chang CH,Wu YJ,et al. Exploration of glycosyl hydrolase family 75, a chitosanase from Aspergillus fumigatus. J Biol Chem.2006;281(6):3137-3144.
[35] Heggset EB,Dybvik AI,Hoell IA,et al.Degradation of chitosans with a family 46 chitosanase from Streptomyces coelicolor A3(2).Biomacromolecules.2010;11(9):2487-2497.
[36] Katsumi T,Lacombe-Harvey ME,Tremblay H,et al.Role of acidic amino acid residues in chitooligosaccharide-binding to Streptomyces sp. N174 chitosanase.Biochem Biophys Res Commun.2005;338(4):1839-1844.
[37] 申杰,叶希韵,沈菊,等.壳寡糖对高脂血症小鼠降血脂及肝脏保护的作用[J].西北农林科技大学学报:自然科学版, 2007,35(9): 35-38.
[38] 张沛,韩宝芹,陈列欢,等.用酶解法制备壳寡糖及其对机体免疫功能的调节作用[J].中国免疫学杂志,2013,29(2):191-196.
[39] 金黎明,魏长征,田文杰,等.壳寡糖及其衍生物对体外培养成骨细胞增殖的作用特点[J].中国组织工程研究与临床康复, 2008, 12(19):3637-3640.
[40] 康立新,周玉玲,马立新.壳聚糖酶的克隆表达与壳寡糖的制备分析[J].生物技术,2012,22(2):20-23.
[41] 程仕伟,孙爱友.酶法制备甲壳低聚糖研究的进展[J].生物加工过程,2011,9(5):65-70.
[42] 吴爱祥.固态发酵黑曲霉产壳聚糖酶的纯化及其性质研究[J].华西药学杂志,2010,25(1):106-108.
[43] 杨俐,余蓉.贵州绿僵菌产壳聚糖酶的纯化鉴定及高产菌株的诱变选育[D]. 四川大学:微生物与生化药学,2007.
[44] 孙玉英,张继泉,王淑军.芽孢杆菌Bacillus sp.S-1壳聚糖酶基因的克隆与序列分析[J].中国生物工程杂志,2009,29(5):72-77.
[45] 徐瑞,郭占云,戚正武.壳聚糖酶的纯化、基因克隆、及其酶学特性的鉴定[J].药物生物技术,2008,15(2):94-99.
[46] 孙玉英,张继泉,王淑军,等.酶促反应制备壳寡糖条件的研究[J].中国生物工程杂志,2008,28(11):67-71.
[47] 杨俐,张涛,李晓红,等.固态发酵贵州绿僵菌产壳聚糖酶的亲和层析纯化及其性质研究[J].四川大学学报:医学版,2007,38(4): 713-716.
[48] 方文建,隋斯光,郑连英.青霉菌产壳聚糖酶制备低分子量壳聚糖的研究[J].中国医药工业杂志,2006,37(2):85-88.