壳聚糖酶的性质及基因的克隆和表达研究
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摘要
本研究从自然界中分离筛选出一株高产壳聚糖酶的微生物菌株命名为OU01,经初步鉴定属于微杆菌属(Microbacterium),是目前所见报道中唯一产壳聚糖酶的微杆菌。
通过对菌株Microbacterium sp. OU01发酵产酶条件进行统计研究确定其最适产酶培养基组分为(%,w/v):粉末壳聚糖1.0,葡萄糖0.1,(NH4)2SO41.9,酵母提取物0.3,K2HPO4 0.14,KH2PO4 0.03,NaCl 0.5, MgSO4·7H2O 0.13;最适产酶培养条件是:500mL锥形瓶培养基装量为100mL,4.0%(v/v)的接种量,30℃,150 r/min培养96 h。在最适产酶培养条件下,96h时菌株OU01发酵液中壳聚糖酶活力可达到118U/mL,是迄今所见报道中发酵液壳聚糖酶活力最高的微生物菌株。Microbacterium sp. OU01所产壳聚糖酶是诱导酶,采用40-90% (NH4)2SO4分级盐析、Q-Sepharose Fast Flow阴离子交换层析、Sephadex G-75凝胶过滤相结合的方法对菌株OU01发酵液中的壳聚糖酶进行分离提纯,获得了SDS-PAGE呈单条带的两种壳聚糖酶ChiN和ChiX,分子量分别为30 kD和81kD,Km和Vmax分别为5 mg chitosan/mL和1260 U/mg protein(ChiN),1.9 mg chitosan/mL和154 U/mg protein(ChiX)。酶学性质研究表明,ChiN和ChiX的最适作用温度分别为50℃和60℃,最适反应pH分别为6.2和6.6,金属离子Mn2+对酶活力有明显的促进作用,Hg2+和Ag+对酶活力有明显的抑制作用,Zn2+对ChiX有明显的抑制作用;ChiN的最适作用底物为95%-100%脱乙酰度的壳聚糖,ChiX的最适作用底物为86%-100%脱乙酰度的壳聚糖;可降解壳聚糖及羧甲基壳聚糖,但不能降解胶体甲壳质和羧甲基纤维素。采用LC-ESI-MS对纯化的两种壳聚糖酶进行了鉴定,ChiN和ChiX与假单胞菌(Pseudomonas sp. A-01)壳聚糖酶的同源性最高,经胰蛋白酶酶解,肽段VYFDPAVAQGK和DAFGGIR,与假单胞菌壳聚糖酶的肽断完全匹配, ChiN降解壳聚糖制备壳寡糖的最适工艺条件为: 50℃,pH 6.2,最适底物浓度为5%,最适酶量30U/g壳聚糖,酶解的经济反应时间为12h。在此工艺条件下,酶解得到产物的数均分子量为1200。利用先前测定的部分氨基酸序列信息,设计简并引物,结合染色体步移的方法克隆了壳聚糖酶基因的全长序列;利用生物信息学的方法,对克隆的基因及其推导的氨基酸序列进行一系列分析;同时利用该基因中编码成熟肽部分的序列构建了重组表达载体,并实现了其在大肠杆菌体内的重组表达,经分离纯化及蛋白变性复性等步骤最终获得了具有生物活性的重组蛋白。
A strain with high chitosanase activity was isolated from soil samples and identified as Microbacterium sp. It is the only Microbacterium strain that can degrade chitosan reported so far. The optimal compositions of the medium producing chitosanase were as follows (g/L): chitosan powder 10, (NH4)2SO4 19.0, glucose 1, MgSO4·7H2O 1.3, K2HPO4·3H2O 1.4, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0. After sterilization at 121 oC for 20min, main culture medium was inoculated with 4% inoculum (V/V). The main culture was carried out in 500 mL Erlenmeyer flasks containing 100 mL of medium. The flasks were kept at 30°C at 150 r/min for 96h.The chitosanase activity of fermentation liquid could reach 118U/mL when OU01 was incubated in flask under the optimal cultivition conditions. The chitosanase from OU01 strain was an inducible and secreting extracellar enzyme. The chitosanase of the strain was extracted by ammonium sulfate precipitation(40-90% saturation), and purified by ion-exchange chromatography (Q-Sepharose Fast Flow column) and gel filtration (Sephadex G-75 column), SDS-PAGE showed single band for the purified chitosanases and the molecular weight was estimated to be 30 kD(ChiN),81kD(ChiX),respectively. Apparent Michaelis contents Km and Vmax were 5 mg chitosan/mL and 1260 U/mg protein(ChiN), 1.9 mg chitosan/mL and 154 U/mg protein(ChiX), respectively. Studies on characterization of ChiN and ChiX showed the optimal temperature and pH for hydrolysis of the chitosanase were 50℃and 6.2(ChiN),60℃and 6.6 (ChiX), respectively. The chitosanase activity was stable in the pH range of 6.6-7.4(ChiN)and 5.2-7.8 (ChiX), respectively under 50℃; Mn2+ could enhance the enzyme activity of ChiN and ChiX significantly, whereas Ag+ and Hg2+ could inhibite the enzyme activity; ChiN and ChiX showed activity for hydrolysis of chitosan and carboxymethyl chitosan. ChiN degraded 95%-100% deacetylated chitosan, and ChiX degraded 86%-100% deacetylated chitosan most effectively. Through in-gel digestion and identification using LC-ESI-MS, two peptide fragments (VYFDPAVAQGK and DAFGGIR) of ChiN and ChiX were matched with Pseudomonas sp. A-01. The enzymatic hydrolysis process for preparing chitooligosaccharides was studied. Experiment results demonstrated the optimal temperature, pH value, concentration of substrate, enzyme quantity were 50℃, 6.2, 5%, 30 U/g chitosanase respectively, 12 hours later the reaction reached balance. The components of the hydrolysis product were analyzed by acetylacetone method, and the average molecular weight of the chitosan hydrolyates was estimated as about 1200. A gene (mschito) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of MSCHITO was similar to those of the classical chitosanase belonging to glycoside hydrolase family 46. Chitosanase was successfully expressed in E. coli and purified with Ni-NTA affinity chromatography. After refolded and cleaved the fusion tag, the recombinant MSCHITO with chitoanase activity was obtained.
通过对菌株Microbacterium sp. OU01发酵产酶条件进行统计研究确定其最适产酶培养基组分为(%,w/v):粉末壳聚糖1.0,葡萄糖0.1,(NH4)2SO41.9,酵母提取物0.3,K2HPO4 0.14,KH2PO4 0.03,NaCl 0.5, MgSO4·7H2O 0.13;最适产酶培养条件是:500mL锥形瓶培养基装量为100mL,4.0%(v/v)的接种量,30℃,150 r/min培养96 h。在最适产酶培养条件下,96h时菌株OU01发酵液中壳聚糖酶活力可达到118U/mL,是迄今所见报道中发酵液壳聚糖酶活力最高的微生物菌株。Microbacterium sp. OU01所产壳聚糖酶是诱导酶,采用40-90% (NH4)2SO4分级盐析、Q-Sepharose Fast Flow阴离子交换层析、Sephadex G-75凝胶过滤相结合的方法对菌株OU01发酵液中的壳聚糖酶进行分离提纯,获得了SDS-PAGE呈单条带的两种壳聚糖酶ChiN和ChiX,分子量分别为30 kD和81kD,Km和Vmax分别为5 mg chitosan/mL和1260 U/mg protein(ChiN),1.9 mg chitosan/mL和154 U/mg protein(ChiX)。酶学性质研究表明,ChiN和ChiX的最适作用温度分别为50℃和60℃,最适反应pH分别为6.2和6.6,金属离子Mn2+对酶活力有明显的促进作用,Hg2+和Ag+对酶活力有明显的抑制作用,Zn2+对ChiX有明显的抑制作用;ChiN的最适作用底物为95%-100%脱乙酰度的壳聚糖,ChiX的最适作用底物为86%-100%脱乙酰度的壳聚糖;可降解壳聚糖及羧甲基壳聚糖,但不能降解胶体甲壳质和羧甲基纤维素。采用LC-ESI-MS对纯化的两种壳聚糖酶进行了鉴定,ChiN和ChiX与假单胞菌(Pseudomonas sp. A-01)壳聚糖酶的同源性最高,经胰蛋白酶酶解,肽段VYFDPAVAQGK和DAFGGIR,与假单胞菌壳聚糖酶的肽断完全匹配, ChiN降解壳聚糖制备壳寡糖的最适工艺条件为: 50℃,pH 6.2,最适底物浓度为5%,最适酶量30U/g壳聚糖,酶解的经济反应时间为12h。在此工艺条件下,酶解得到产物的数均分子量为1200。利用先前测定的部分氨基酸序列信息,设计简并引物,结合染色体步移的方法克隆了壳聚糖酶基因的全长序列;利用生物信息学的方法,对克隆的基因及其推导的氨基酸序列进行一系列分析;同时利用该基因中编码成熟肽部分的序列构建了重组表达载体,并实现了其在大肠杆菌体内的重组表达,经分离纯化及蛋白变性复性等步骤最终获得了具有生物活性的重组蛋白。
A strain with high chitosanase activity was isolated from soil samples and identified as Microbacterium sp. It is the only Microbacterium strain that can degrade chitosan reported so far. The optimal compositions of the medium producing chitosanase were as follows (g/L): chitosan powder 10, (NH4)2SO4 19.0, glucose 1, MgSO4·7H2O 1.3, K2HPO4·3H2O 1.4, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0. After sterilization at 121 oC for 20min, main culture medium was inoculated with 4% inoculum (V/V). The main culture was carried out in 500 mL Erlenmeyer flasks containing 100 mL of medium. The flasks were kept at 30°C at 150 r/min for 96h.The chitosanase activity of fermentation liquid could reach 118U/mL when OU01 was incubated in flask under the optimal cultivition conditions. The chitosanase from OU01 strain was an inducible and secreting extracellar enzyme. The chitosanase of the strain was extracted by ammonium sulfate precipitation(40-90% saturation), and purified by ion-exchange chromatography (Q-Sepharose Fast Flow column) and gel filtration (Sephadex G-75 column), SDS-PAGE showed single band for the purified chitosanases and the molecular weight was estimated to be 30 kD(ChiN),81kD(ChiX),respectively. Apparent Michaelis contents Km and Vmax were 5 mg chitosan/mL and 1260 U/mg protein(ChiN), 1.9 mg chitosan/mL and 154 U/mg protein(ChiX), respectively. Studies on characterization of ChiN and ChiX showed the optimal temperature and pH for hydrolysis of the chitosanase were 50℃and 6.2(ChiN),60℃and 6.6 (ChiX), respectively. The chitosanase activity was stable in the pH range of 6.6-7.4(ChiN)and 5.2-7.8 (ChiX), respectively under 50℃; Mn2+ could enhance the enzyme activity of ChiN and ChiX significantly, whereas Ag+ and Hg2+ could inhibite the enzyme activity; ChiN and ChiX showed activity for hydrolysis of chitosan and carboxymethyl chitosan. ChiN degraded 95%-100% deacetylated chitosan, and ChiX degraded 86%-100% deacetylated chitosan most effectively. Through in-gel digestion and identification using LC-ESI-MS, two peptide fragments (VYFDPAVAQGK and DAFGGIR) of ChiN and ChiX were matched with Pseudomonas sp. A-01. The enzymatic hydrolysis process for preparing chitooligosaccharides was studied. Experiment results demonstrated the optimal temperature, pH value, concentration of substrate, enzyme quantity were 50℃, 6.2, 5%, 30 U/g chitosanase respectively, 12 hours later the reaction reached balance. The components of the hydrolysis product were analyzed by acetylacetone method, and the average molecular weight of the chitosan hydrolyates was estimated as about 1200. A gene (mschito) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of MSCHITO was similar to those of the classical chitosanase belonging to glycoside hydrolase family 46. Chitosanase was successfully expressed in E. coli and purified with Ni-NTA affinity chromatography. After refolded and cleaved the fusion tag, the recombinant MSCHITO with chitoanase activity was obtained.
引文
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