苏云金芽胞杆菌几丁质酶活性研究
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摘要
几丁质是自然界储量第二的天然高聚物,几丁质酶是降解其的主要酶类。Bt是应用最成功的杀虫微生物,几丁质酶对Bt杀虫有增效作用。本研究以筛选到的具有较高几丁质水解能力的Bt菌株BRC-Bt1为试验材料,克隆表达该菌株的2个几丁质酶基因,对这2种几丁质酶的结构域组成、催化反应机制、表达调控以及水解产物的生物活性进行了系统研究。
BRC-Bt1菌株的chi74基因和目前已发现的Bt 70kDa几丁质酶在核酸序列上高度同源,该酶属18家族几丁质酶,由信号肽、催化区、FN3和ChBD等4个结构域组成,理论分子量74kDa,pI5.77。chi74基因和GST融合在E. coli表达产物为胞内可溶性蛋白,含chi74基因的E. coli细胞能在胶体几丁质平板产生水解圈。以胶体几丁质为底物,Chi74蛋白最适反应温度60℃、pH5,在20-80℃和pH4-8都有活性。Na~~+、K~~+、NO_3~-、Cl~-不影响该酶活性;Mg~(2+)和SO_4~(2-)对该酶有激活作用,其中6mmol/L浓度Mg~(2+)提高酶活力约40%,而SO_4~(2-)在0-3mmol/L浓度范围能提高酶活性,3mmol/L时,提高酶活力约20%,之后再增加浓度激活作用反而下降;Cu~(2+)、Ag~+都抑制酶活性。HPLC分析水解产物表明该酶能水解胶体几丁质形成GlcNAc、几丁二糖以及聚合度更高的几丁寡糖,随着反应时间的延长,GlcNAc占产物的比例不断提高。该酶为内切几丁质酶。
构建chi74基因不同截短酶基因chiA、chiAF、chiAC,三个截短基因编码的蛋白产物理论分子量和pI分别为:54kDa、6.04,63kDa、6.07和66kDa、5.74。三个截短基因GST融合在E. coli细胞表达产物ChiA、ChiAF、ChiAC水解胶体几丁质的活性分别是野生酶的81%、72%、39%。3种截短酶最适反应温度都是60℃,且温度变化对酶活性的影响和Chi74类似。Mg~(2+)能激活3种截短酶,且激活效率比Chi74高,6.0mmol/L的Mg~(2+)对ChiA、ChiAF、ChiAC分别提高酶活力85%、62%、60%。低浓度的Ag~+对3种截短酶活性抑制也比野生酶显著,500μmol/L的Ag~+对ChiAC、ChiAF、ChiA等3种截短酶活力分别为70.0%、63.2%、55.0%。截短酶的水解产物HPLC结果表明,Chi74的C端不同片段缺失,胶体几丁质水解不彻底,产物中GlcNAc含量不高,而几丁寡糖含量高。截短酶与Chi74催化活性的差异,表明FN3和ChBD对Chi74水解胶体几丁质很重要。
克隆chi36基因,推导蛋白分子量40kDa,pI6.52。该基因与Bt、Bc、Ba上发现的编码36kDa大小的几丁质酶基因同源性很高,而与chi74没有同源性。该酶也属于18家族几丁质酶,只有信号肽和催化区。chi36基因和GST融合表达产物,以胶体几丁质为底物,最适合反应温度55℃、pH5,在30-80℃、pH4-8都有活性。Na~+、K~+、Mg~(2+)、NO_3~-、Cl-对该酶活性基本没有影响;低浓度的SO_4~(2-)对该酶有激活作用;Cu~(2+)、Fe~(3+)、Ag~+则抑制该酶活性。Chi36水解胶体几丁质产物HPLC结果表明:该酶是内切几丁质酶,产物中GlcNAc含量低,多种几丁寡糖含量较高。从水解产物可以看出,Chi36水解胶体几丁质的机制和Chi74不同,而且Chi36水解的效率也不如Chi74。等体积混合Chi36和Chi74,催化活性协同提高。
用来自chi74的3个C端片段(ChBD、FN3、ChBD~+FN3)添加到chi36基因的C端构建3种加长酶基因chi36F、chi36C、chi36FC,预测分子量分别为49kDa、52kDa、60kDa。加长酶基因和GST融合表达产物Chi36F、Chi36C、Chi36FC几丁质酶活性分别比野生Chi36提高7%、21%、33%,最适合反应温度仍是55℃。Ag~+对3种加长酶活性抑制率比Chi36低,500μmol/L时,Chi36F、Chi36C、ChiFC加长酶活力分别保留为64.0%、66.0%、70.5%。加长酶水解胶体几丁质产物组成与Chi36类似,且产物中各个组分的浓度都提高,表明加长酶对底物的亲和力增强,进一步说明ChBD、FN3在催化反应中发挥作用,且ChBD比FN3更重要。
分别克隆6株Bt菌株chi74和chi36基因上游序列,序列功能分析结果表明,这两个基因都是独立表达的,且两个基因在Bt基因组距离很远。两个基因上游的启动子没有同源性,表明Bt的两个几丁质酶基因的表达调控方式不同。chi74和chi36基因启动子分析结果表明:chi74基因上游有多个潜在启动子,且不同菌株这些启动子的同源性较高,而在chi36上游发现的启动子较少,且菌株间的差异较大。6个菌株都在基因上游发现CAP/CRP位点、NIT-2等多种转录因子结合位点。产几丁质酶培养基和LB培养的BRC-Bt1菌株发酵液上清对香蕉炭疽菌生长有抑制作用,且产酶培养基上清的抑制率更高,抑制率分别为42.8%、27.4%。产酶培养基发酵的上清还能抑制稻瘟病菌丝生长和孢子萌发。E. coli表达的Chi74和Chi36蛋白混合,分别以浓度10mg/mL和5mg/mL作用于松材线虫,24h死亡率分别为87.5%、54.5%。以胶体几丁质为底物,混合酶解反应不同时间的产物对松材线虫的致死情况差异较大,其中酶解8h的产物对松材线虫的毒杀作用最好,平均死亡率为100%。1h、3h的水解产物处理松材线虫的死亡率分别为23.8%、69.3%,而处理时间长达24h的产物对线虫几乎没有毒杀作用,致死率仅为8.4%。此外,酶解产物对肝癌细胞SMMC-7721的生长有抑制作用,IC50为69μg/ml,而对正常肝细胞活性无影响。
Chitin is the second most abundant polysaccharides in nature. Chitinases are important and major enzymes which can degrade chitin. Bacillus thuringiensis is the most successfully used biological pesticide. Chitinase can increase the insecticidal activity of Bt. Herein, we present the cloning of two chitinase genes(chi74 and chi36) from a Bt strain(named BRC-Bt1) with high chitinase activity, as well as the expression in vitro, the domains of chitinases, the catalytic mechanisms, the regulation of chitinase genes expression and the bioactivity of chitinase hydrolysis products.
The nucleotide sequence of chi74 gene from BRC-Bt1 consists of 2025 bases and encodes a putative protein of 674 amino acids residus with a predicted molecular weight of 74kDa and pI 5.77. Homology comparison revealed that the chi74 gene had high homology in nucleotide sequence with the genes encoded 70kDa chitinases of Bt reported. The predicted protein of Chi74 was a modular enzyme consisting of a signal peptide, a FN3 domain, a ChBD domain and a catalytic domain belonging to chitinase family 18. The chi74 gene was expressed with GST fusion in E. coli and the characteristic of Chi74 was investigated. When using the colloid chitin as substrate, the optimum pH, optimum temperature and thermal stability of Chi74 were pH 5, 60℃and 40℃, respectively. The enzyme exhibited activity in broad temperature range, from 20 to 80℃, and pH between 4.0 and 8.0. The chitinase activity of Chi74 was not influenced by Na~+, K~+, NO_3~- and Cl-; however, it was improved by Mg~(2+)and SO_4~(2-), and inhibited by Cu~(2+) and Ag~+. HPLC analysis of the hydrolysis products indicated that it contained GlcNAc, (GlcNAc)2 and oligomers with higher degree of polymerization, and GlcNAc was predominant product increased gradually with the reaction time extension. The Chi74 maybe an endo-chitinase.
Three different C-terminal truncation chitinase genes of chiA, chiAF and chiAC from chi74 were constructed, which encoded the putative proteins with the predicted molecular weights and the pI values of 54kDa and 6.04, 63kDa and 6.07, 66kDa and 5.74, respectively. The 3 truncation genes were expressed with GST fusion in E.coli. With colloidal chitin as substrate, the relative activities of ChiA, ChiAF and ChiAC were 81%, 72% and 39% compared with the native chitinase Chi74. The optimum temperature of three truncation enzymes were all 60℃and the curves of enzyme activity on different temperature were similar to Chi74. The activities of three truncation enzymes were activated by Mg~(2+) and the activation efficiency was higher than that of native enzyme, and were inhibited by Ag~+ and the inhibition efficiency was higher than that of Chi74. HPLC analysis of the hydrolysates of three truncation enzymes showed that the contents of GlcNAc and chito-oligomers were at lower and higher level respectively, compared to Chi74. FN3 domain and ChBD domain may play important roles in insoluble chitin hydrolysis.
The nucleotide sequence of chi36 gene from BRC-Bt1 consists of 1083 bases and encodes a putative protein of 360 amino acids residus with a predicted molecular weight of 40kDa and a pI value of 6.52. The chi36 gene showed high homology with the genes encoded 36kDa chitinases from Bt, Bacillus cereus and Bacillus anthracis. Analysis of the predicted protein structure of Chi36 revealed that it also belongs to chitinase family 18, and contains a signal peptide and a single catalytic domain which has little homology with Chi74. The chi36 gene was expressed with GST fusion in E. coli and the characteristic of Chi36 was also investigated. When using the colloid chitin as substrate, the optimum pH, optimum temperature and thermal stability of Chi36 were pH 5, 55℃and 40℃, respectively. The enzyme exhibited activity in broad temperature range, from 30 to 80℃, and pH between 4.0 and 8.0. The chitinase activity of Chi36 was not influenced by Na~+, K~+, Mg~(2+), NO_3~- and Cl-; however, it was improved by SO_4~(2-), and inhibited by Fe~(3+), Cu~(2+) and Ag~+. HPLC analysis of hydrolysates showed that it contained GlcNAc, (GlcNAc)2 and oligomers with higher degree of polymerization, and the content of GlcNAc was lower than chito-oligomers. The Chi36 maybe an endo-chitinase and its catalytic mechanism different from that of Chi74. The enzyme activity of the mixture of Chi36 and Chi74 with equal volume was the highest.
Three different C-terminal prolonged chitinase genes of chi36F, chi36C and chi36FC were constructed by adding the different C-terminal fragments of Chi74 into the C-terminal of Chi36, which encoded the putative proteins with the predicted molecular weights of 49kDa, 52kDa and 60kDa, respectively. The three prolonged genes were expressed with GST in E. coli. With colloidal chitin as substrate, the relative activities of Chi36F, Chi36C and Chi36FC were 7%, 21% and 33% higher than native Chi36, respectively. The optimum temperature of three prolonged enzymes were all 55℃. The enzyme activities of three prolonged enzymes were inhibited by Ag+ and the inhibition efficiency was lower than Chi36. The components of colloid chitin hydrolysis products of three prolonged enzymes were similar with Chi36, but the concentrations of each component were higher than native enzyme. It supported that FN3 domain and ChBD domain may play important roles in insoluble chitin hydrolysis.
The upstream sequences of chi74 and chi36 from 6 Bt strains were cloned and sequenced. Analysis of the sequence revealed that the chi74 and chi36 were expressed independently and spaced long distance. The promoters of chi36 were not similar to the promoter of chi74. The result of promoter prediction showed that the amounts of promoters from chi74 were more than from chi36. The transcriptional factor of CAP/CRP and NIT-2 were found in both upstream sequences of chi74 and chi36.
The supernatants of BRC-Bt1 cultured in chitinase-produced medium and LB medium both inhibited spore germination of Colletotrichum musae, and the inhibition efficiency of the former was higher than the later. The supernatant of BRC-Bt1 cultured in chitinase-produced medium also inhibited spore germination and hyphal extension of Magnaporthe grisea. The mixture of Chi74 and Chi36 with the final concentration of 10mg/mL and 5mg/mL exhibited 87.5% and 54.5% mortality against Pinus massoniana, respectively. The mortality of hydrolysis products produced by mixture enzyme at 1h, 3h, 8h and 24h were 23.8%, 69.3%, 100% and 8.4%, respectively. Furthermore, the hydrolysis products exhibited in vitro anti-liver cancer cell (SMMC-7721) with IC_(50)69μg/ml, but had no influence on normal liver cell.
BRC-Bt1菌株的chi74基因和目前已发现的Bt 70kDa几丁质酶在核酸序列上高度同源,该酶属18家族几丁质酶,由信号肽、催化区、FN3和ChBD等4个结构域组成,理论分子量74kDa,pI5.77。chi74基因和GST融合在E. coli表达产物为胞内可溶性蛋白,含chi74基因的E. coli细胞能在胶体几丁质平板产生水解圈。以胶体几丁质为底物,Chi74蛋白最适反应温度60℃、pH5,在20-80℃和pH4-8都有活性。Na~~+、K~~+、NO_3~-、Cl~-不影响该酶活性;Mg~(2+)和SO_4~(2-)对该酶有激活作用,其中6mmol/L浓度Mg~(2+)提高酶活力约40%,而SO_4~(2-)在0-3mmol/L浓度范围能提高酶活性,3mmol/L时,提高酶活力约20%,之后再增加浓度激活作用反而下降;Cu~(2+)、Ag~+都抑制酶活性。HPLC分析水解产物表明该酶能水解胶体几丁质形成GlcNAc、几丁二糖以及聚合度更高的几丁寡糖,随着反应时间的延长,GlcNAc占产物的比例不断提高。该酶为内切几丁质酶。
构建chi74基因不同截短酶基因chiA、chiAF、chiAC,三个截短基因编码的蛋白产物理论分子量和pI分别为:54kDa、6.04,63kDa、6.07和66kDa、5.74。三个截短基因GST融合在E. coli细胞表达产物ChiA、ChiAF、ChiAC水解胶体几丁质的活性分别是野生酶的81%、72%、39%。3种截短酶最适反应温度都是60℃,且温度变化对酶活性的影响和Chi74类似。Mg~(2+)能激活3种截短酶,且激活效率比Chi74高,6.0mmol/L的Mg~(2+)对ChiA、ChiAF、ChiAC分别提高酶活力85%、62%、60%。低浓度的Ag~+对3种截短酶活性抑制也比野生酶显著,500μmol/L的Ag~+对ChiAC、ChiAF、ChiA等3种截短酶活力分别为70.0%、63.2%、55.0%。截短酶的水解产物HPLC结果表明,Chi74的C端不同片段缺失,胶体几丁质水解不彻底,产物中GlcNAc含量不高,而几丁寡糖含量高。截短酶与Chi74催化活性的差异,表明FN3和ChBD对Chi74水解胶体几丁质很重要。
克隆chi36基因,推导蛋白分子量40kDa,pI6.52。该基因与Bt、Bc、Ba上发现的编码36kDa大小的几丁质酶基因同源性很高,而与chi74没有同源性。该酶也属于18家族几丁质酶,只有信号肽和催化区。chi36基因和GST融合表达产物,以胶体几丁质为底物,最适合反应温度55℃、pH5,在30-80℃、pH4-8都有活性。Na~+、K~+、Mg~(2+)、NO_3~-、Cl-对该酶活性基本没有影响;低浓度的SO_4~(2-)对该酶有激活作用;Cu~(2+)、Fe~(3+)、Ag~+则抑制该酶活性。Chi36水解胶体几丁质产物HPLC结果表明:该酶是内切几丁质酶,产物中GlcNAc含量低,多种几丁寡糖含量较高。从水解产物可以看出,Chi36水解胶体几丁质的机制和Chi74不同,而且Chi36水解的效率也不如Chi74。等体积混合Chi36和Chi74,催化活性协同提高。
用来自chi74的3个C端片段(ChBD、FN3、ChBD~+FN3)添加到chi36基因的C端构建3种加长酶基因chi36F、chi36C、chi36FC,预测分子量分别为49kDa、52kDa、60kDa。加长酶基因和GST融合表达产物Chi36F、Chi36C、Chi36FC几丁质酶活性分别比野生Chi36提高7%、21%、33%,最适合反应温度仍是55℃。Ag~+对3种加长酶活性抑制率比Chi36低,500μmol/L时,Chi36F、Chi36C、ChiFC加长酶活力分别保留为64.0%、66.0%、70.5%。加长酶水解胶体几丁质产物组成与Chi36类似,且产物中各个组分的浓度都提高,表明加长酶对底物的亲和力增强,进一步说明ChBD、FN3在催化反应中发挥作用,且ChBD比FN3更重要。
分别克隆6株Bt菌株chi74和chi36基因上游序列,序列功能分析结果表明,这两个基因都是独立表达的,且两个基因在Bt基因组距离很远。两个基因上游的启动子没有同源性,表明Bt的两个几丁质酶基因的表达调控方式不同。chi74和chi36基因启动子分析结果表明:chi74基因上游有多个潜在启动子,且不同菌株这些启动子的同源性较高,而在chi36上游发现的启动子较少,且菌株间的差异较大。6个菌株都在基因上游发现CAP/CRP位点、NIT-2等多种转录因子结合位点。产几丁质酶培养基和LB培养的BRC-Bt1菌株发酵液上清对香蕉炭疽菌生长有抑制作用,且产酶培养基上清的抑制率更高,抑制率分别为42.8%、27.4%。产酶培养基发酵的上清还能抑制稻瘟病菌丝生长和孢子萌发。E. coli表达的Chi74和Chi36蛋白混合,分别以浓度10mg/mL和5mg/mL作用于松材线虫,24h死亡率分别为87.5%、54.5%。以胶体几丁质为底物,混合酶解反应不同时间的产物对松材线虫的致死情况差异较大,其中酶解8h的产物对松材线虫的毒杀作用最好,平均死亡率为100%。1h、3h的水解产物处理松材线虫的死亡率分别为23.8%、69.3%,而处理时间长达24h的产物对线虫几乎没有毒杀作用,致死率仅为8.4%。此外,酶解产物对肝癌细胞SMMC-7721的生长有抑制作用,IC50为69μg/ml,而对正常肝细胞活性无影响。
Chitin is the second most abundant polysaccharides in nature. Chitinases are important and major enzymes which can degrade chitin. Bacillus thuringiensis is the most successfully used biological pesticide. Chitinase can increase the insecticidal activity of Bt. Herein, we present the cloning of two chitinase genes(chi74 and chi36) from a Bt strain(named BRC-Bt1) with high chitinase activity, as well as the expression in vitro, the domains of chitinases, the catalytic mechanisms, the regulation of chitinase genes expression and the bioactivity of chitinase hydrolysis products.
The nucleotide sequence of chi74 gene from BRC-Bt1 consists of 2025 bases and encodes a putative protein of 674 amino acids residus with a predicted molecular weight of 74kDa and pI 5.77. Homology comparison revealed that the chi74 gene had high homology in nucleotide sequence with the genes encoded 70kDa chitinases of Bt reported. The predicted protein of Chi74 was a modular enzyme consisting of a signal peptide, a FN3 domain, a ChBD domain and a catalytic domain belonging to chitinase family 18. The chi74 gene was expressed with GST fusion in E. coli and the characteristic of Chi74 was investigated. When using the colloid chitin as substrate, the optimum pH, optimum temperature and thermal stability of Chi74 were pH 5, 60℃and 40℃, respectively. The enzyme exhibited activity in broad temperature range, from 20 to 80℃, and pH between 4.0 and 8.0. The chitinase activity of Chi74 was not influenced by Na~+, K~+, NO_3~- and Cl-; however, it was improved by Mg~(2+)and SO_4~(2-), and inhibited by Cu~(2+) and Ag~+. HPLC analysis of the hydrolysis products indicated that it contained GlcNAc, (GlcNAc)2 and oligomers with higher degree of polymerization, and GlcNAc was predominant product increased gradually with the reaction time extension. The Chi74 maybe an endo-chitinase.
Three different C-terminal truncation chitinase genes of chiA, chiAF and chiAC from chi74 were constructed, which encoded the putative proteins with the predicted molecular weights and the pI values of 54kDa and 6.04, 63kDa and 6.07, 66kDa and 5.74, respectively. The 3 truncation genes were expressed with GST fusion in E.coli. With colloidal chitin as substrate, the relative activities of ChiA, ChiAF and ChiAC were 81%, 72% and 39% compared with the native chitinase Chi74. The optimum temperature of three truncation enzymes were all 60℃and the curves of enzyme activity on different temperature were similar to Chi74. The activities of three truncation enzymes were activated by Mg~(2+) and the activation efficiency was higher than that of native enzyme, and were inhibited by Ag~+ and the inhibition efficiency was higher than that of Chi74. HPLC analysis of the hydrolysates of three truncation enzymes showed that the contents of GlcNAc and chito-oligomers were at lower and higher level respectively, compared to Chi74. FN3 domain and ChBD domain may play important roles in insoluble chitin hydrolysis.
The nucleotide sequence of chi36 gene from BRC-Bt1 consists of 1083 bases and encodes a putative protein of 360 amino acids residus with a predicted molecular weight of 40kDa and a pI value of 6.52. The chi36 gene showed high homology with the genes encoded 36kDa chitinases from Bt, Bacillus cereus and Bacillus anthracis. Analysis of the predicted protein structure of Chi36 revealed that it also belongs to chitinase family 18, and contains a signal peptide and a single catalytic domain which has little homology with Chi74. The chi36 gene was expressed with GST fusion in E. coli and the characteristic of Chi36 was also investigated. When using the colloid chitin as substrate, the optimum pH, optimum temperature and thermal stability of Chi36 were pH 5, 55℃and 40℃, respectively. The enzyme exhibited activity in broad temperature range, from 30 to 80℃, and pH between 4.0 and 8.0. The chitinase activity of Chi36 was not influenced by Na~+, K~+, Mg~(2+), NO_3~- and Cl-; however, it was improved by SO_4~(2-), and inhibited by Fe~(3+), Cu~(2+) and Ag~+. HPLC analysis of hydrolysates showed that it contained GlcNAc, (GlcNAc)2 and oligomers with higher degree of polymerization, and the content of GlcNAc was lower than chito-oligomers. The Chi36 maybe an endo-chitinase and its catalytic mechanism different from that of Chi74. The enzyme activity of the mixture of Chi36 and Chi74 with equal volume was the highest.
Three different C-terminal prolonged chitinase genes of chi36F, chi36C and chi36FC were constructed by adding the different C-terminal fragments of Chi74 into the C-terminal of Chi36, which encoded the putative proteins with the predicted molecular weights of 49kDa, 52kDa and 60kDa, respectively. The three prolonged genes were expressed with GST in E. coli. With colloidal chitin as substrate, the relative activities of Chi36F, Chi36C and Chi36FC were 7%, 21% and 33% higher than native Chi36, respectively. The optimum temperature of three prolonged enzymes were all 55℃. The enzyme activities of three prolonged enzymes were inhibited by Ag+ and the inhibition efficiency was lower than Chi36. The components of colloid chitin hydrolysis products of three prolonged enzymes were similar with Chi36, but the concentrations of each component were higher than native enzyme. It supported that FN3 domain and ChBD domain may play important roles in insoluble chitin hydrolysis.
The upstream sequences of chi74 and chi36 from 6 Bt strains were cloned and sequenced. Analysis of the sequence revealed that the chi74 and chi36 were expressed independently and spaced long distance. The promoters of chi36 were not similar to the promoter of chi74. The result of promoter prediction showed that the amounts of promoters from chi74 were more than from chi36. The transcriptional factor of CAP/CRP and NIT-2 were found in both upstream sequences of chi74 and chi36.
The supernatants of BRC-Bt1 cultured in chitinase-produced medium and LB medium both inhibited spore germination of Colletotrichum musae, and the inhibition efficiency of the former was higher than the later. The supernatant of BRC-Bt1 cultured in chitinase-produced medium also inhibited spore germination and hyphal extension of Magnaporthe grisea. The mixture of Chi74 and Chi36 with the final concentration of 10mg/mL and 5mg/mL exhibited 87.5% and 54.5% mortality against Pinus massoniana, respectively. The mortality of hydrolysis products produced by mixture enzyme at 1h, 3h, 8h and 24h were 23.8%, 69.3%, 100% and 8.4%, respectively. Furthermore, the hydrolysis products exhibited in vitro anti-liver cancer cell (SMMC-7721) with IC_(50)69μg/ml, but had no influence on normal liver cell.
引文
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