单宁酶产生菌的筛选及其发酵条件研究
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摘要
单宁酶全称单宁酯酰水解酶(Tannase,EC 3.11.20),可以水解单宁中的酯键和缩酚羧键,生成没食子酸和葡萄糖。微生物产单宁酶主要来源于真菌,目前研究最多的产酶微生物是青霉和黑曲霉。本文从产单宁酶菌株的筛选开始,对单宁酶发酵工艺条件以及部分酶学性质进行了研究,主要研究结果如下:
采用平板透明圈法和固体发酵筛选相结合的方法,从土壤中分离、筛选得到了一株产单宁酶的黑曲霉菌株A-6,初筛酶活力为8.1U/g。
在固体发酵的基础上,通过对培养基组分进行单因素实验和响应面实验分析,确定了菌株A-6的最佳发酵培养基组成为:在250mL三角瓶中装入5g麸皮和5mL营养液;营养液的组成为(g/L):蔗糖12、KNO_3 2.5、玉米浆22.4、五倍子65.1、MgSO_4 1.36、CoCl_2 0.2、柠檬酸钠3.0、NaCl 2.5;通过对接种量、发酵温度、初始pH值、固液比等培养条件的考察,获得了菌株A-6的较优培养条件:接种量1mL的孢子悬液(浓度为11×10~9个/mL),发酵温度30℃,初始pH7.0,固液比1:1.8,发酵时间108h左右。菌株A-6在最佳培养基和较优培养条件下,酶活可达15.68U/g,比优化前提高了约94%。
本文对菌株A-6粗酶性质进行了系统的研究,结果表明:菌株A-6所产单宁酶在4℃储存时稳定性好,贮存8周后依然保存有94%的酶活性;最适作用温度为60℃左右,在65℃以下稳定性较好,超过75℃易失活;最适作用pH 8.0,在pH 6.0-9.0范围内稳定性较好,酸性环境下非常易失活,碱性条件下相对稳定;该酶可耐受1.5mmol/L的离子强度;K~+、Mn~(2+)、Fe~(3+)对酶活力有比较明显的激活作用,Zn2+对酶活力有明显的抑制作用,Ca~(2+)和Fe~(2+)有轻微的抑制作用。
Tanin acyl hydrolase(E.C.3.11.20) commonly referred to as tannase, Tannase catalyzes the hydrolysis of ester and depside bonds in hydrolysable tannins such as tannic acid ,releasing glucose and gallic acid . It produced mainly by fungi., at present Penicillium and Aspergillus niger were studied more than others. In this paper, we carried out a series of experiments, including screening, identifying optimizing fermentation process, and studying characteristics of tannase. The main results are as follows:
A tannase producing strain Aspergillus niger A-6 was obtained from soil by assaying hydrolyzed circle on the selective medium and tannase activity under sol1id-state fermentation, the yield of tannase was 8.1U/g.
By single-factor experiments and response surface methodology, the culture medium and conditions of the strain A-6 outputing tannase were optimized. The optimum culture composition were as follows: wheat bran 5g and 5mL nutrition solution was taken in 250mL conical flasks,the nutrition solution containing(g/L):sucrose 12, KNO_3 2.5, corn steep liquor 22.4, gallnut 65.1 , MgSO_4·7H_2O 1.36, CaCl 2·2H_2O 0.2, Sodium Citrate 3.0, NaCl 2.5; the optimal culture conditions were as follows: inoculation 1mL spore suspension(11×10~9个/mL), temperature 30℃, initial pH value 7.0, solid-liquid ratio1:2 and culture time 108h. Under these optimal conditions, the yield of tannase was 15.81U/g, increased for about 0.94 times more than that of before.
Studies on characterization of tannase showed: The crude tannase from the strain A-6 showed well stability at 4℃, the enzyme retained 94% of maximal activity at 4℃after 3 mouths; tannase activity was optimal at 60℃, pH 8.0, and in 1.5mmol/L NaCl solution. The enzyme was stable below 65℃and pH6.0-9.0; The tannase activity was strongly activtied by K~+, Mn~(2+)、, Fe~(3+); and inhibited by Zn2+, Ca~(2+)and Fe~(2+).
采用平板透明圈法和固体发酵筛选相结合的方法,从土壤中分离、筛选得到了一株产单宁酶的黑曲霉菌株A-6,初筛酶活力为8.1U/g。
在固体发酵的基础上,通过对培养基组分进行单因素实验和响应面实验分析,确定了菌株A-6的最佳发酵培养基组成为:在250mL三角瓶中装入5g麸皮和5mL营养液;营养液的组成为(g/L):蔗糖12、KNO_3 2.5、玉米浆22.4、五倍子65.1、MgSO_4 1.36、CoCl_2 0.2、柠檬酸钠3.0、NaCl 2.5;通过对接种量、发酵温度、初始pH值、固液比等培养条件的考察,获得了菌株A-6的较优培养条件:接种量1mL的孢子悬液(浓度为11×10~9个/mL),发酵温度30℃,初始pH7.0,固液比1:1.8,发酵时间108h左右。菌株A-6在最佳培养基和较优培养条件下,酶活可达15.68U/g,比优化前提高了约94%。
本文对菌株A-6粗酶性质进行了系统的研究,结果表明:菌株A-6所产单宁酶在4℃储存时稳定性好,贮存8周后依然保存有94%的酶活性;最适作用温度为60℃左右,在65℃以下稳定性较好,超过75℃易失活;最适作用pH 8.0,在pH 6.0-9.0范围内稳定性较好,酸性环境下非常易失活,碱性条件下相对稳定;该酶可耐受1.5mmol/L的离子强度;K~+、Mn~(2+)、Fe~(3+)对酶活力有比较明显的激活作用,Zn2+对酶活力有明显的抑制作用,Ca~(2+)和Fe~(2+)有轻微的抑制作用。
Tanin acyl hydrolase(E.C.3.11.20) commonly referred to as tannase, Tannase catalyzes the hydrolysis of ester and depside bonds in hydrolysable tannins such as tannic acid ,releasing glucose and gallic acid . It produced mainly by fungi., at present Penicillium and Aspergillus niger were studied more than others. In this paper, we carried out a series of experiments, including screening, identifying optimizing fermentation process, and studying characteristics of tannase. The main results are as follows:
A tannase producing strain Aspergillus niger A-6 was obtained from soil by assaying hydrolyzed circle on the selective medium and tannase activity under sol1id-state fermentation, the yield of tannase was 8.1U/g.
By single-factor experiments and response surface methodology, the culture medium and conditions of the strain A-6 outputing tannase were optimized. The optimum culture composition were as follows: wheat bran 5g and 5mL nutrition solution was taken in 250mL conical flasks,the nutrition solution containing(g/L):sucrose 12, KNO_3 2.5, corn steep liquor 22.4, gallnut 65.1 , MgSO_4·7H_2O 1.36, CaCl 2·2H_2O 0.2, Sodium Citrate 3.0, NaCl 2.5; the optimal culture conditions were as follows: inoculation 1mL spore suspension(11×10~9个/mL), temperature 30℃, initial pH value 7.0, solid-liquid ratio1:2 and culture time 108h. Under these optimal conditions, the yield of tannase was 15.81U/g, increased for about 0.94 times more than that of before.
Studies on characterization of tannase showed: The crude tannase from the strain A-6 showed well stability at 4℃, the enzyme retained 94% of maximal activity at 4℃after 3 mouths; tannase activity was optimal at 60℃, pH 8.0, and in 1.5mmol/L NaCl solution. The enzyme was stable below 65℃and pH6.0-9.0; The tannase activity was strongly activtied by K~+, Mn~(2+)、, Fe~(3+); and inhibited by Zn2+, Ca~(2+)and Fe~(2+).
引文
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