微泡菌ALW1几丁质酶Chi18A的克隆及信息学分析
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摘要
以微泡菌(Microbulbifer sp.)ALW1的基因组为模板,利用几丁质酶基因的特异性引物进行PCR扩增,然后将产物插入到pMD18-T载体后进行DNA序列测定,并对目的基因编码的蛋白质序列进行信息学分析。结果显示,克隆的目的基因大小为1644 bp,预测编码含有547个氨基酸残基的蛋白质。该蛋白质序列与其他菌株来源的几丁质酶序列具有70%左右的相似性,表明预测目的基因编码几丁质酶。该蛋白质具有2个几丁质结合结构域和1个GH18家族酶的催化结构域,属于糖苷水解酶(glycoside hydrolase,GH)家族18,命名为几丁质酶Chi18A。模拟的三维结构显示,Chi18A含有(βα)_8桶状结构。
The genomic DNA of Microbulbifer sp.ALW1 was used as the template for the amplification of a chitinase gene by PCR with a pair of specific primers.The amplified products were cloned into pMD18-T vector and then sequenced.The deduced protein sequence of this gene was further analyzed by bioinformatics.The results showed that the cloned gene was 1644 bp,encoding 547 amino acid residues.The target protein sequence shared about 70% identity with the chitinase sequences from other strains.So the cloned target gene was predicted to encode an chitinase.The target protein had two chitin binding domains and one GH18 catalytic domain.It belonged to GH18 family and was named Chi18 A.The simulated 3 D structure showed that Chi18 A contained(βα)_8 barrel structures.
The genomic DNA of Microbulbifer sp.ALW1 was used as the template for the amplification of a chitinase gene by PCR with a pair of specific primers.The amplified products were cloned into pMD18-T vector and then sequenced.The deduced protein sequence of this gene was further analyzed by bioinformatics.The results showed that the cloned gene was 1644 bp,encoding 547 amino acid residues.The target protein sequence shared about 70% identity with the chitinase sequences from other strains.So the cloned target gene was predicted to encode an chitinase.The target protein had two chitin binding domains and one GH18 catalytic domain.It belonged to GH18 family and was named Chi18 A.The simulated 3 D structure showed that Chi18 A contained(βα)_8 barrel structures.
引文
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[4]AZUMA K,OSAKI T,WAKUDA T,et al.Suppressive effects of N-acetyl-D-glucosamine on rheumatoid arthritis mouse models[J].Inflammation,2012,35(4):1462-1465.DOI:10.1007/s10753-012-9459-0.
[5]SHI S Y,WANG W,LIU L Q,et al.Effect of chitosan/nano-silica coating on the physicochemical characteristics of longan fruit under ambient temperature[J].Journal of Food Engineering,2013,118(1):125-131.DOI:10.1016/j.jfoodeng.2013.03.029.
[6]CHEN J K,SHEN G R,LIU G L.N-acetyl glucosamine:production and applications[J].Marine Drugs,2010,8(9):2493-2516.DOI:10.3390/md8092493.
[7]RATHORE A S,GUPTA R D.Chitinases from bacteria to human:properties,applications,and future perspectives[J].Enzyme Research,2015,2015(2):1-8.DOI:10.1155/2015/791907.
[8]PURUSHOTHAM P,POIDLE A R.Synthesis of long-chain chitooligosaccharides by a hypertransglycosylating processive endochitinase of Serratia proteamaculans 568[J].Journal of Bacteriology,2012,194(16):4260- 4271.DOI:10.1128/JB.06473-11.
[9]GAO L,SUN J,SECUNDO F,et al.Cloning,characterization and substrate degradation mode of a novel chitinase from Streptomyces albolongus ATCC 27414[J].Food Chemistry,2018,261:329-336.DOI:10.1016/j.foodchem.2018.04.068.
[10]YANG S,FU X,YAN Q.Cloning,expression,purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii[J].Food Chemistry,2016,192(9/10):1041-1048.DOI:10.1016/j.foodchem.2015.07.092.
[11]WANG S L,LIN T Y,YEN Y H,et al.Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase[J].Carbohydrate Research,2006,341(15):2507-2515.DOI:10.1016/j.carres.2006.06.027.
[12]KANG S C,PARK S,LEE D G.Purification and characterization of a novel chitinase from the entomopathogenic fungus,Metarhizium anisopliae[J].Journal of Invertebrate Pathology,1999,73(3):276-281.
[13]KIM Y H,PARK S K,HUR J Y,et al.Purification and characterization of a major extracellular chitinase from a biocontrol bacterium,Paenibacillus elgii HOA73[J].Plant Pathology Journal,2017,33(3):318-328.DOI:10.5423/PPJ.FT.01.2017.0022.
[14]ZHU Y B,WU L Y,CHEN Y H,et al.Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp.ALW1 and antioxidant activity of enzymatic hydrolysates[J].Microbiological Research,2016,182:49-58.DOI:10.1016/j.micres.2015.09.004.
[15]MILLER G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical Chemistry,1959,31(3):426- 428.DOI:10.1021/ac60147a030.
[16]WATANABE T,ARIGA Y,SATO U,et al.Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin[J].Biochemical Journal,2003,376(1):237-244.DOI:10.1042/BJ20030419.