右旋糖酐酶产生菌的筛选、鉴定及酶学性质研究
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摘要
本论文自土壤中分离筛选得到了两株右旋糖酐酶产生菌,对两菌株进行了形态学的和分子手段鉴定,并研究了该右旋糖酐酶粗酶性质和菌株产酶发酵条件。研究内容如下：
右旋糖酐酶产生菌株的筛选：采用以右旋糖酐作唯一碳源的筛选培养基,经过多次筛选和单孢子分离法获得产右旋糖酐酶的单一菌株；通过小室培养法对各菌丝形态进行观察,结合菌落形态和液态培养形态对各菌株进行初步鉴别；采用蓝色葡聚糖培养基鉴定各菌株的产酶特性；用DNS测还原糖法计算各菌株发酵液酶活。结果如下：分离筛选获得5株产酶菌株,分别编号D1～D5,形态学初步判断认为D1和D5为青霉菌,D2和D3为镰刀霉菌,D4为头孢霉菌。D4和D5菌在蓝色葡聚糖平板上有明显透明圈,酶活测定结果同样显示这两种菌产酶最高,因此选择D4和D5菌进行下一步研究。
右旋糖酐酶产生菌的分类鉴定：分别对D4和D5菌进行分生孢子、分生孢子梗、菌落形态,色素颜色等形态学研究,并对菌株的ITS rDNA序列进行克隆测序,与GenBank中已知菌的ITS rDNA比对,用Neighbor-joining方法构建聚类分析树状图,并用Bootstrap法对其评估。鉴定结果如下：D4菌的ITS rDNA序列与Verticillium nigrescens有99%的同源性,然而该分子鉴定结果不与其形态学鉴定结果相符,根据更为准确的形态学特征鉴定为头孢霉属。D5菌通过形态学分析其为黄绿青霉(Penicillium ci treoviride),其ITS序列的分子鉴定支持了基于形态特征的鉴定结果,该菌株的ITS与Penicillium daleae, Penicillium janthinellum菌株的ITS rDNA序列同源性最高,分别达到97%和98%。
右旋糖酐酶酶学性质研究：采用薄层层析法分析右旋糖酐酶降解右旋糖酐、普鲁蓝糖、淀粉和羧甲基纤维素的酶促产物；通过DNS测还原糖法测酶活,研究酶的最适作用温度、热稳定性、最适作用pH、pH稳定性,以及金属离子、离子强度对酶活的影响。结果如下：来自D4、D5的右旋糖酐酶均只能降解连续的α-1,6糖苷键产生异麦芽三糖,对其他糖苷键无作用,证明该酶为外切型异麦芽三糖水解酶。该酶具有独特的底物特异性和产物均一性,在理论及应用上具有一定意义。D4所产的右旋糖酐酶最适作用温度为40℃,在40℃半衰期为80 min,最适作用pH为7,在pH 5.6～pH 7范围内稳定,低浓度的Ba2+’和Mg2+可促进酶活,150～300mmol/L NaCl所产生的离子强度促进酶活。D5所产的右旋糖酐酶最适作用温度为55℃,在50℃半衰期为3 h,最适作用pH为6.5,在pH 4.0～pH 6.5范围内稳定；金属离子K+、Ba2+显著促进酶活,Ca2+、Fe2+强烈抑制酶活；150mmol/L NaCl所产生的离子强度促进酶活。
产酶发酵研究：通过摇瓶发酵实验和正交实验对两菌株的产酶条件进行了优化,结果如下：D4的最优发酵条件为：培养基中蔗糖、葡聚糖、酪蛋白的质量分数分别为1%、1%、0.2%,pH 5.0,接种量1.5 mL,装液量80mL/250mL,转速120r/min。25℃培养11d酶活达到138.11 U/mL。D5的最优发酵条件为：培养基中淀粉、葡聚糖、氯化铵、酵母膏的质量分数分别为1%、1%、0.1%、0.1%,pH 6.0,接种量1%,装液量50mL/250mL,转速120 r/min。25℃培养13 d酶活达到176.80 U/mL。
D5菌所产右旋糖酐酶的分离纯化研究：对发酵13d所得的发酵液进行超滤浓缩,硫酸铵沉淀,DEAE-Sepharose离子交换层析,HPLC分子筛层析一系列的纯化操作,活性电泳和变性电泳结果显示得到两种蛋白质,其中右旋糖酐酶分子量为66kDa。
Two dextranase-producing fungi were screened from soil and were identified by morphological and molecular method. The fermentation of those two strains for producing dextranase and the enzymatic properties were studied. The contents were as follows.
Screening of dextranase-producing microbes:screening medium using dextran as carbon source was adopted; the strains were anticipated to be obtained through series of screening and monospore isolation. The strains were primarily identified according to their colonial morphology, fermentation morphology and hyphae morphology which was observed after cellule cultivation. The enzymatic activity of dextranase from fermentation supernatant was tested using DNS reagent. The results were as follows:five microorganisms were obtained, and were numbered from D1 to D5. Through morphology identification, D1 and D5 were considered as Penicillium sp., D2 and D3 were considered as Fusarium sp., D4 was considered as Cephalosporium sp. D4 and D5 formed apparent blank area when they grew in blue dextran plates, and the enzymatic activity test also confirmed that they both had higher enzymatic activity, thus the two strain were selected for the next experiments.
Identification of dextranase-producing species:The morphology of colony, conidiospores and conidiophores of D4 and D5 were studied. The ITS rDNA sequences of the two strains were cloned and sequenced, and then were compared with those available in the GenBank databases. The phylogenetic trees were derived with neighbor joining and analyzed with Bootstrapping. The results showed that the ITS sequence of D4 has a 99% similarity with Verticillium nigrescens, however, the result disagree with the identification based on morphological characteristics. After all D4 was identified as Cephalosporieae sp. according to its decided morphological characters. D5 was identified as Penicillium citreoviride according to its morphological characters, its ITS sequence analysis supported this conclusion. D5 strain had close consanguinities to P. daleae and P. janthinellum, which were 97% and 98%.
Enzymatic properties studies:hydrolytic products of enzyme released from dextran, pullulan, starch and carboxymethyl cellulose were analyzed by TLC method. Enzymatic activity was measured by DNS reagent, in this way enzymatic properties, such as optimum temperature, temperature stability, optimum pH, pH stability and the effects of metal ion and ionic strength on dextranase were studied. The outcomes showed that the dextranase yielded by D4 and D5 was exo-Isomaltotriodextranase, which hydrolyzed serial a-1,6 glucosidic bond to release isomaltotriose from dextran while had no effects on other bonds. The enzyme has special substrate specificity and products homogeneity, therefore it is meaningful in theory and application. The enzyme yielded by D4 displayed maximum activity at 40℃; the half-life of the enzyme was 80 min at 40℃; the optimum pH value was 7, and the enzyme activity was stable from pH 5.6 to pH 7; the enzyme activity was enhanced by low concentration Mg2+ and Ba2+, and the ionic strength of NaCl at 150～300 mmol/L. The enzyme yielded by D5 displayed maximum activity at 55℃; the half-life of the enzyme was 3 h at 50℃; the optimum pH value was 6.5, and the enzyme activity was stable in the range of pH4.0 to pH6.5; the enzyme activity was enhanced by K+ and Ba2+, but was inhibited by Ca2+、Fe2+; the enzyme activity could be improved by the ionic strength of NaCl at 150 mmol/L.
Fermentation for producing dextranase: The optimum conditions for Isomaltotriodextranase production were studied by flask-shaking fermentation and orthogonal experiments. The optimal condition for D4 strain were as follows:1% sucrose,1% dextran,0.2% casein, initial pH value 5.0,1.5 mL inoculating quantity, rate of shaking flask with 80 mL liquid in a 250 mL flask was 120 r/min. After fermentation for 11 days at 25℃, the enzyme activity reached 138.11 U/mL. The optimal condition for D5 strain were as follows:1% starch,1% dextran,0.1% NH4Cl,0.1% yeast extract, initial pH value 6.0, 1% inoculating quantity, rate of shaking flask with 50 mL liquid in a 250 mL flask was 120 r/min. After fermentation for 13 days at 25℃, the enzyme activity was 176.80 U/mL.
Extraction and purification of dextranase from D5: The supernant of fermentation medium cultured for 13 days was collected and a series of purification processes were carried out: ultrafiltration concentration, ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and HPLC chromatography. The results of SDS-PAGE and Native-PAGE showed that two proteins including the enzyme was gained, the molecular weight of the enzyme was 66kDa.
右旋糖酐酶产生菌株的筛选：采用以右旋糖酐作唯一碳源的筛选培养基,经过多次筛选和单孢子分离法获得产右旋糖酐酶的单一菌株；通过小室培养法对各菌丝形态进行观察,结合菌落形态和液态培养形态对各菌株进行初步鉴别；采用蓝色葡聚糖培养基鉴定各菌株的产酶特性；用DNS测还原糖法计算各菌株发酵液酶活。结果如下：分离筛选获得5株产酶菌株,分别编号D1～D5,形态学初步判断认为D1和D5为青霉菌,D2和D3为镰刀霉菌,D4为头孢霉菌。D4和D5菌在蓝色葡聚糖平板上有明显透明圈,酶活测定结果同样显示这两种菌产酶最高,因此选择D4和D5菌进行下一步研究。
右旋糖酐酶产生菌的分类鉴定：分别对D4和D5菌进行分生孢子、分生孢子梗、菌落形态,色素颜色等形态学研究,并对菌株的ITS rDNA序列进行克隆测序,与GenBank中已知菌的ITS rDNA比对,用Neighbor-joining方法构建聚类分析树状图,并用Bootstrap法对其评估。鉴定结果如下：D4菌的ITS rDNA序列与Verticillium nigrescens有99%的同源性,然而该分子鉴定结果不与其形态学鉴定结果相符,根据更为准确的形态学特征鉴定为头孢霉属。D5菌通过形态学分析其为黄绿青霉(Penicillium ci treoviride),其ITS序列的分子鉴定支持了基于形态特征的鉴定结果,该菌株的ITS与Penicillium daleae, Penicillium janthinellum菌株的ITS rDNA序列同源性最高,分别达到97%和98%。
右旋糖酐酶酶学性质研究：采用薄层层析法分析右旋糖酐酶降解右旋糖酐、普鲁蓝糖、淀粉和羧甲基纤维素的酶促产物；通过DNS测还原糖法测酶活,研究酶的最适作用温度、热稳定性、最适作用pH、pH稳定性,以及金属离子、离子强度对酶活的影响。结果如下：来自D4、D5的右旋糖酐酶均只能降解连续的α-1,6糖苷键产生异麦芽三糖,对其他糖苷键无作用,证明该酶为外切型异麦芽三糖水解酶。该酶具有独特的底物特异性和产物均一性,在理论及应用上具有一定意义。D4所产的右旋糖酐酶最适作用温度为40℃,在40℃半衰期为80 min,最适作用pH为7,在pH 5.6～pH 7范围内稳定,低浓度的Ba2+’和Mg2+可促进酶活,150～300mmol/L NaCl所产生的离子强度促进酶活。D5所产的右旋糖酐酶最适作用温度为55℃,在50℃半衰期为3 h,最适作用pH为6.5,在pH 4.0～pH 6.5范围内稳定；金属离子K+、Ba2+显著促进酶活,Ca2+、Fe2+强烈抑制酶活；150mmol/L NaCl所产生的离子强度促进酶活。
产酶发酵研究：通过摇瓶发酵实验和正交实验对两菌株的产酶条件进行了优化,结果如下：D4的最优发酵条件为：培养基中蔗糖、葡聚糖、酪蛋白的质量分数分别为1%、1%、0.2%,pH 5.0,接种量1.5 mL,装液量80mL/250mL,转速120r/min。25℃培养11d酶活达到138.11 U/mL。D5的最优发酵条件为：培养基中淀粉、葡聚糖、氯化铵、酵母膏的质量分数分别为1%、1%、0.1%、0.1%,pH 6.0,接种量1%,装液量50mL/250mL,转速120 r/min。25℃培养13 d酶活达到176.80 U/mL。
D5菌所产右旋糖酐酶的分离纯化研究：对发酵13d所得的发酵液进行超滤浓缩,硫酸铵沉淀,DEAE-Sepharose离子交换层析,HPLC分子筛层析一系列的纯化操作,活性电泳和变性电泳结果显示得到两种蛋白质,其中右旋糖酐酶分子量为66kDa。
Two dextranase-producing fungi were screened from soil and were identified by morphological and molecular method. The fermentation of those two strains for producing dextranase and the enzymatic properties were studied. The contents were as follows.
Screening of dextranase-producing microbes:screening medium using dextran as carbon source was adopted; the strains were anticipated to be obtained through series of screening and monospore isolation. The strains were primarily identified according to their colonial morphology, fermentation morphology and hyphae morphology which was observed after cellule cultivation. The enzymatic activity of dextranase from fermentation supernatant was tested using DNS reagent. The results were as follows:five microorganisms were obtained, and were numbered from D1 to D5. Through morphology identification, D1 and D5 were considered as Penicillium sp., D2 and D3 were considered as Fusarium sp., D4 was considered as Cephalosporium sp. D4 and D5 formed apparent blank area when they grew in blue dextran plates, and the enzymatic activity test also confirmed that they both had higher enzymatic activity, thus the two strain were selected for the next experiments.
Identification of dextranase-producing species:The morphology of colony, conidiospores and conidiophores of D4 and D5 were studied. The ITS rDNA sequences of the two strains were cloned and sequenced, and then were compared with those available in the GenBank databases. The phylogenetic trees were derived with neighbor joining and analyzed with Bootstrapping. The results showed that the ITS sequence of D4 has a 99% similarity with Verticillium nigrescens, however, the result disagree with the identification based on morphological characteristics. After all D4 was identified as Cephalosporieae sp. according to its decided morphological characters. D5 was identified as Penicillium citreoviride according to its morphological characters, its ITS sequence analysis supported this conclusion. D5 strain had close consanguinities to P. daleae and P. janthinellum, which were 97% and 98%.
Enzymatic properties studies:hydrolytic products of enzyme released from dextran, pullulan, starch and carboxymethyl cellulose were analyzed by TLC method. Enzymatic activity was measured by DNS reagent, in this way enzymatic properties, such as optimum temperature, temperature stability, optimum pH, pH stability and the effects of metal ion and ionic strength on dextranase were studied. The outcomes showed that the dextranase yielded by D4 and D5 was exo-Isomaltotriodextranase, which hydrolyzed serial a-1,6 glucosidic bond to release isomaltotriose from dextran while had no effects on other bonds. The enzyme has special substrate specificity and products homogeneity, therefore it is meaningful in theory and application. The enzyme yielded by D4 displayed maximum activity at 40℃; the half-life of the enzyme was 80 min at 40℃; the optimum pH value was 7, and the enzyme activity was stable from pH 5.6 to pH 7; the enzyme activity was enhanced by low concentration Mg2+ and Ba2+, and the ionic strength of NaCl at 150～300 mmol/L. The enzyme yielded by D5 displayed maximum activity at 55℃; the half-life of the enzyme was 3 h at 50℃; the optimum pH value was 6.5, and the enzyme activity was stable in the range of pH4.0 to pH6.5; the enzyme activity was enhanced by K+ and Ba2+, but was inhibited by Ca2+、Fe2+; the enzyme activity could be improved by the ionic strength of NaCl at 150 mmol/L.
Fermentation for producing dextranase: The optimum conditions for Isomaltotriodextranase production were studied by flask-shaking fermentation and orthogonal experiments. The optimal condition for D4 strain were as follows:1% sucrose,1% dextran,0.2% casein, initial pH value 5.0,1.5 mL inoculating quantity, rate of shaking flask with 80 mL liquid in a 250 mL flask was 120 r/min. After fermentation for 11 days at 25℃, the enzyme activity reached 138.11 U/mL. The optimal condition for D5 strain were as follows:1% starch,1% dextran,0.1% NH4Cl,0.1% yeast extract, initial pH value 6.0, 1% inoculating quantity, rate of shaking flask with 50 mL liquid in a 250 mL flask was 120 r/min. After fermentation for 13 days at 25℃, the enzyme activity was 176.80 U/mL.
Extraction and purification of dextranase from D5: The supernant of fermentation medium cultured for 13 days was collected and a series of purification processes were carried out: ultrafiltration concentration, ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and HPLC chromatography. The results of SDS-PAGE and Native-PAGE showed that two proteins including the enzyme was gained, the molecular weight of the enzyme was 66kDa.
引文
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