溶菌酶高产菌株的筛选及其产酶研究
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摘要
本论文系统研究了产溶菌酶菌株的分离筛选及鉴定,发酵条件的优化和粗酶液酶学性质,研究结果如下:
从不同地区采集的156个土壤样品中进行筛选,最终,从河北农业大学标本园的果园土壤中筛选得到一高产菌株。通过产酶菌株的分离筛选,建立了一套快速、准确的溶菌酶高产菌株筛选方法:通过添加金黄色葡萄球菌的双层平板分离初筛,琼脂打孔法进行高抑菌活性菌株的初步筛选,验证抑菌物质的初筛方法,测定酶活力的复筛的方法;确定了合理、客观的综合考察酶活的方法。
对菌株S2-6b进行鉴定,分别在光学显微镜和扫描电镜下观察了菌体形态、大小,并对其在营养琼脂平板上的单菌落形态、大小、颜色等特征进行了描述;通过一系列生理生化试验及16SrDNA保守序列的分析,将此菌株初步鉴定为溶杆菌(LysobacterS2-6b)。
对溶杆菌S2-6b的发酵条件进行了研究,确定其最佳培养基成分及最佳培养条件。研究了不同碳源、氮源、无机盐、表面活性剂、不同培养温度、时间、pH值等主要因素对溶杆菌S2-6b产酶的影响,进行发酵条件优化,优化结果表明,菌株产酶优化的培养基为,可溶性淀粉1.25%,牛肉膏2.5%,玉米浆1.0%,Tween-80 0.1%;优化发酵条件为,接种量3%,发酵培养基的起始pH为7.0,装液量40/300 mL,摇床28℃,200 r/min,培养40 h。经过发酵条件优化,菌株产酶活力可达6 166.67 U/mL。
酶学性质研究发现,菌株溶菌酶的基本酶学性质为:最适作用温度75℃,75℃下的半衰期为17.3 min;最适作用pH值为7.0,酶的pH稳定范围在3.0～11.0之间;其中,Mg~(2+),Mn~(2+),Cu~(2+),Al~(3+),Ca~(2+)对溶菌酶水解底物有不同程度的抑制,酶活力分别下降了41.7%,40.8%,40.6%,16.7%,12.5%,其他金属离子对酶活力基本没有影响。SDS对溶菌酶的活性抑制明显,使酶活力下降了33.5%。EDTA使酶活力升高了17%。与鸡卵清溶菌酶相比较,S2-6b溶菌酶对受试菌株有不同程度的抑制和杀灭作用,抑菌谱比卵清溶菌酶广泛。
Systematic study on the screening of lysozyme-yielding strains,liquid fermentation of lactase high producing strain S2-6b and enzyme properties were carried out in this paper. The main results were as following:
Screening of high-yielding lysozyme strains,the method of the primary screening has three standards,lyric zone on the double layer plates,lyric zone of liquid fermentation, identifying the ferment product,respectively,the second screening was enzyme activity assay.Then we obtained a bacterium named S2-6b from the orchard soil sample from Agricultural University of Hebei.It included that we can find the high-yielding lysozyme strains by the optimal method.
The strain S2-6b was identified.Through optic microscope and scanning electron microscope,the S2-6b's modality,size wsa observed.Modality,size and colour of colony both on broth agar culture medium were described.Through the traditional physiological and biochemical test,together with the 16SrDNA sequence and phylogenetic analysis,the strain S2-6b was finally ascertained to be Lysobacter(Lysobacter S2-6b).
The fermentation conditions of Lysobacter S2-6b,including carbon source,nitrogen source,inorganic salt,surfactant,temperature,formation time,pH value and so on,were investigated in shake flask level.The orthogonal test of the three factors showed that the optimized culture medium was soluble starch 1.5%,meat-extract 2.5%,corn steep 0.5%, Tween-80 0.1%.Through shake flask experiments,the optimum fermentation conditions for S2-6 strain were 28℃incubation,inoculum's level 3.0%(V/V),volume ratio 40mL/300mL,shake speed 200rpm formation time 40 h and initial pH 7.5.The maximal activity was 6 167 U/mL under the optimum conditions,which was about 3 times of the initial strain.
The test of crude enzyme indicated that optimal temperature of Lysobacter S2-6b was 75℃,the half life of reaction temperature was 17.3 min.Optimal pH was 7.0 and it was stable at pH 3.0-11.0 at room temperature.The metal irons and reagent Mg~(2+),Mn~(2+),Cu~(2+), Al~(3+),Ca~(2+),SDS have inhibited on lysozyme.EDTA has activation on lysozyme.Compared with chicken lysozyme,the Lysobacter S2-6b lysozyme has more widely antimicrobial spectrum.Lysobacter S2-6b lysozyme has different effect on experimented bacterium.
从不同地区采集的156个土壤样品中进行筛选,最终,从河北农业大学标本园的果园土壤中筛选得到一高产菌株。通过产酶菌株的分离筛选,建立了一套快速、准确的溶菌酶高产菌株筛选方法:通过添加金黄色葡萄球菌的双层平板分离初筛,琼脂打孔法进行高抑菌活性菌株的初步筛选,验证抑菌物质的初筛方法,测定酶活力的复筛的方法;确定了合理、客观的综合考察酶活的方法。
对菌株S2-6b进行鉴定,分别在光学显微镜和扫描电镜下观察了菌体形态、大小,并对其在营养琼脂平板上的单菌落形态、大小、颜色等特征进行了描述;通过一系列生理生化试验及16SrDNA保守序列的分析,将此菌株初步鉴定为溶杆菌(LysobacterS2-6b)。
对溶杆菌S2-6b的发酵条件进行了研究,确定其最佳培养基成分及最佳培养条件。研究了不同碳源、氮源、无机盐、表面活性剂、不同培养温度、时间、pH值等主要因素对溶杆菌S2-6b产酶的影响,进行发酵条件优化,优化结果表明,菌株产酶优化的培养基为,可溶性淀粉1.25%,牛肉膏2.5%,玉米浆1.0%,Tween-80 0.1%;优化发酵条件为,接种量3%,发酵培养基的起始pH为7.0,装液量40/300 mL,摇床28℃,200 r/min,培养40 h。经过发酵条件优化,菌株产酶活力可达6 166.67 U/mL。
酶学性质研究发现,菌株溶菌酶的基本酶学性质为:最适作用温度75℃,75℃下的半衰期为17.3 min;最适作用pH值为7.0,酶的pH稳定范围在3.0～11.0之间;其中,Mg~(2+),Mn~(2+),Cu~(2+),Al~(3+),Ca~(2+)对溶菌酶水解底物有不同程度的抑制,酶活力分别下降了41.7%,40.8%,40.6%,16.7%,12.5%,其他金属离子对酶活力基本没有影响。SDS对溶菌酶的活性抑制明显,使酶活力下降了33.5%。EDTA使酶活力升高了17%。与鸡卵清溶菌酶相比较,S2-6b溶菌酶对受试菌株有不同程度的抑制和杀灭作用,抑菌谱比卵清溶菌酶广泛。
Systematic study on the screening of lysozyme-yielding strains,liquid fermentation of lactase high producing strain S2-6b and enzyme properties were carried out in this paper. The main results were as following:
Screening of high-yielding lysozyme strains,the method of the primary screening has three standards,lyric zone on the double layer plates,lyric zone of liquid fermentation, identifying the ferment product,respectively,the second screening was enzyme activity assay.Then we obtained a bacterium named S2-6b from the orchard soil sample from Agricultural University of Hebei.It included that we can find the high-yielding lysozyme strains by the optimal method.
The strain S2-6b was identified.Through optic microscope and scanning electron microscope,the S2-6b's modality,size wsa observed.Modality,size and colour of colony both on broth agar culture medium were described.Through the traditional physiological and biochemical test,together with the 16SrDNA sequence and phylogenetic analysis,the strain S2-6b was finally ascertained to be Lysobacter(Lysobacter S2-6b).
The fermentation conditions of Lysobacter S2-6b,including carbon source,nitrogen source,inorganic salt,surfactant,temperature,formation time,pH value and so on,were investigated in shake flask level.The orthogonal test of the three factors showed that the optimized culture medium was soluble starch 1.5%,meat-extract 2.5%,corn steep 0.5%, Tween-80 0.1%.Through shake flask experiments,the optimum fermentation conditions for S2-6 strain were 28℃incubation,inoculum's level 3.0%(V/V),volume ratio 40mL/300mL,shake speed 200rpm formation time 40 h and initial pH 7.5.The maximal activity was 6 167 U/mL under the optimum conditions,which was about 3 times of the initial strain.
The test of crude enzyme indicated that optimal temperature of Lysobacter S2-6b was 75℃,the half life of reaction temperature was 17.3 min.Optimal pH was 7.0 and it was stable at pH 3.0-11.0 at room temperature.The metal irons and reagent Mg~(2+),Mn~(2+),Cu~(2+), Al~(3+),Ca~(2+),SDS have inhibited on lysozyme.EDTA has activation on lysozyme.Compared with chicken lysozyme,the Lysobacter S2-6b lysozyme has more widely antimicrobial spectrum.Lysobacter S2-6b lysozyme has different effect on experimented bacterium.
引文
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