微生物酯酶产生菌的选育、菌株Bacillus sp.EB-87产酶特性及酶学性质的研究
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摘要
微生物酯酶在生物转化和动力学拆分方面具有广阔的应用前
景，因而本文对产酯微生物酶菌种的选育、菌株的鉴定、Bacillus sp.Strain EB-87
产酶特性以及酶学性质做了系统的研究。
通过比较试验，建立起一个有效、简便、快速的筛选模型。采用富集法、
添加溴甲酚紫和酯酶染色液的快速平板显色法和发酵测酶活对与酯类长期接触
的30多份土样以及实验室保存的菌种进行大量的筛选。按照相对酶活值大于
40％为标准，初筛得到174株产酯酶菌株；复筛得到24株产酯酶的优良菌株。
经验证实验最后筛选到一株高产酯酶菌株EB-87，酶活达38.5U/ml。另外，还
以不同底物对复筛菌株进行酶活测定和对泛酰内酯、乳酸乙酯具有的立体选择
性菌株进行筛选。
结果发现产酯酶的菌株分布较为广泛；添加溴甲酚紫和酯酶染色液的两种
指示剂的平板检测法得到的初筛结果与复筛结果的有所不同，前者相关性不如
后者。
对菌株EB-87分离纯化，进行形态特征、生理生化特征、生长特性等项目
鉴定。依据《伯杰氏细菌手册》及相关书籍，该菌属于芽孢杆菌属（Bacillus），
并命名为Bacillus sp.Strain EB-87。
为了弄清楚Bacillus sp.Strain.EB-87的产酶机理和提高其产酶水平，采用单
因素试验，Plackett-Burman法和响应面回归分析相结合进行产酶特性研究。得
出最佳培养基组成为：玉米浆2.4％，葡萄糖1.0％，蛋白胨2.6％，MgSO_4.7H_2O
0.071％，（NH_4）_2SO_4 0.2％，K_2HPO_4 0.2％，NaH_2PO_4 0.05％。最佳发酵条件为：
温度32℃，时间26-28小时，pH为7.2，摇瓶转速150rpm。酶活达84.5U/ml，
提高2.2倍。不同结构、类型及数量的酯类、油脂及结构类似物对产酶效果有
不同的影响。
最后，对分离纯化后的酶做温度、pH、底物特异性以及酶的动力学研究。
结果表明：该酯酶的最适作用温度为40℃，最适作用pH为7.2，酶在pH6.0-8.0
之间稳定性较好：在40℃以下，酶很稳定，在50℃以上不稳定，为此构建酶热
失活动力学模型，得出该酯酶在50℃和60℃的半衰期t_（1/2）分别为56.4min和
21.7min，残留酶活力为85％时的存放时间t_0.85分别为11.2min和8.6min。金属
离子Fe~(2+)、Mg~(2+)、K~+、Na~+和Ca~(2+)对酶有激活作用，Co~(2+)、Zn~(2+)和Cu~(2+)对酶有抑
制作用，Mn~(2+)对酶活几乎没有影响。酶动力学参数K_m为0.331mmol/L，V_m为1.005
μmol/ml.min。
该酯酶对碳链长短不同单甘酯、二元酯、甘油酯等酯表现出不同的酶活。
In this paper, the screening and determinant of microbial esterase-producing, the
fermentation conditions and properties of the Bacillus sp. Strain EB-87 were studied
systematically, because the application of microbial esterase was increasing,
especially in the field of bioconversion, kinetic resolution.
Based on studies and comparisons among methods of screening microbial esterase,
a rapid, simple and efficient Screening Model for microbial esteraseroducing was
developed. More than thirty soil samples from all kinds of water ester or oil and
strains from myself laboratory. An efficient and rapid plating methods for determinant
of microbial esterase activity by observing the diameter of the strain and the
hydrolytic color circle around on the culture was adopted. The underlying principle
employed in this Model was based on color change of the indicator-Fast Blue B Salt
caused by a -NA, f3 -NA from a -NP and P -NP under the proper conditions. This
was preliminary screening and 174 strains obtained according to relative esterase
activity more than 40%. After culturing in a triangle bottle on the shake and color halo
is used to determine their esterase activity. This was secondary screening and 24 fine
strains were selected and the highest esterase activity of the strain EB-87 was
38.5UIml.
The mixed liquor of esterase results of the preliminary screening was a nearly linear
with results of the secondary screening, but the Bromocresol Purple later didn so.
Besides, studies and compared esterase activity of these strains with different
substance, including the esterase roducing strain for stereospecific hydrolysis of
(D,L)-Pantyl lactone and (D,L)-Ethyl lactate.
Isolation aiid purification the strain EB-87, and through detection of its appearance,
physiological and biochemical characteristics, growth characters, it was identified
Bacillus Genus according to Bergeysmanual of Deternmiutue Bacteriology(8) and
Microbiology Bacteriological Analytical Manual(FDA), and named Bacillus sp.
Strain EB-87.
In order to investigate the conditions and the principle of producing esterase by
Bacillus sp. Strain EB-87, the methods of combination Single Factor design
Plackett-Burman design and Response Surface Analysis had been employed for the
fermentation conditions and the composition of the fermentation medium.
The results showed that the optimum composition of the fermentation medium as
following: Corn steep liquor 2.4%, Glucose l.0%, Peptone 2.6%, MgSO.7H,O
0.07l%, peH.),SO. 0.2%, K,HPO, 0.2%, NaH,PO. 0.05%, the optimum fermentation
conditions as following: the optimum fermentation tempendre was 32oC, the
optimum fermentation shaking frequency was l50rpm, the optimum fermentation pH
was 7.2, the optimum fermentation time was 26-28h. In doing so Bacillus sp. strain
EB-87 esterase activity was 84.5U/ml, enlarging 2.2 times.
Finally the purification and properties including substance specificity, temperature,
pH, Bacillus sp. strain EB-87 esterase were studied in detail. The results showed that
the optimal temperature of BaciUus sp. strain EB-87 esterase reaction was 40C,
pH7.2, the esterase activity was stable in the range of pH6.0-8.0. and the enzyme was
stable less than 40C, but not stable above 50C. The parameters: tI/2 was 56.4min, k 85
was l l .2min at 50oC and tll2 was 2l .7min, to85 was 8.6min at 60C. The lions Fe2+.
Mg'+. K+. Na+ and Ca2+ were beneficial to esterase activity, but the lions Co2+. Zn2+
and Cu2+ were inhibited esterase activity. The kinetic parameters K. was
0.33 lmmoI/L, V. was l .005 ll mol/ml.min
The enzyme exhibits different esterase
景，因而本文对产酯微生物酶菌种的选育、菌株的鉴定、Bacillus sp.Strain EB-87
产酶特性以及酶学性质做了系统的研究。
通过比较试验，建立起一个有效、简便、快速的筛选模型。采用富集法、
添加溴甲酚紫和酯酶染色液的快速平板显色法和发酵测酶活对与酯类长期接触
的30多份土样以及实验室保存的菌种进行大量的筛选。按照相对酶活值大于
40％为标准，初筛得到174株产酯酶菌株；复筛得到24株产酯酶的优良菌株。
经验证实验最后筛选到一株高产酯酶菌株EB-87，酶活达38.5U/ml。另外，还
以不同底物对复筛菌株进行酶活测定和对泛酰内酯、乳酸乙酯具有的立体选择
性菌株进行筛选。
结果发现产酯酶的菌株分布较为广泛；添加溴甲酚紫和酯酶染色液的两种
指示剂的平板检测法得到的初筛结果与复筛结果的有所不同，前者相关性不如
后者。
对菌株EB-87分离纯化，进行形态特征、生理生化特征、生长特性等项目
鉴定。依据《伯杰氏细菌手册》及相关书籍，该菌属于芽孢杆菌属（Bacillus），
并命名为Bacillus sp.Strain EB-87。
为了弄清楚Bacillus sp.Strain.EB-87的产酶机理和提高其产酶水平，采用单
因素试验，Plackett-Burman法和响应面回归分析相结合进行产酶特性研究。得
出最佳培养基组成为：玉米浆2.4％，葡萄糖1.0％，蛋白胨2.6％，MgSO_4.7H_2O
0.071％，（NH_4）_2SO_4 0.2％，K_2HPO_4 0.2％，NaH_2PO_4 0.05％。最佳发酵条件为：
温度32℃，时间26-28小时，pH为7.2，摇瓶转速150rpm。酶活达84.5U/ml，
提高2.2倍。不同结构、类型及数量的酯类、油脂及结构类似物对产酶效果有
不同的影响。
最后，对分离纯化后的酶做温度、pH、底物特异性以及酶的动力学研究。
结果表明：该酯酶的最适作用温度为40℃，最适作用pH为7.2，酶在pH6.0-8.0
之间稳定性较好：在40℃以下，酶很稳定，在50℃以上不稳定，为此构建酶热
失活动力学模型，得出该酯酶在50℃和60℃的半衰期t_（1/2）分别为56.4min和
21.7min，残留酶活力为85％时的存放时间t_0.85分别为11.2min和8.6min。金属
离子Fe~(2+)、Mg~(2+)、K~+、Na~+和Ca~(2+)对酶有激活作用，Co~(2+)、Zn~(2+)和Cu~(2+)对酶有抑
制作用，Mn~(2+)对酶活几乎没有影响。酶动力学参数K_m为0.331mmol/L，V_m为1.005
μmol/ml.min。
该酯酶对碳链长短不同单甘酯、二元酯、甘油酯等酯表现出不同的酶活。
In this paper, the screening and determinant of microbial esterase-producing, the
fermentation conditions and properties of the Bacillus sp. Strain EB-87 were studied
systematically, because the application of microbial esterase was increasing,
especially in the field of bioconversion, kinetic resolution.
Based on studies and comparisons among methods of screening microbial esterase,
a rapid, simple and efficient Screening Model for microbial esteraseroducing was
developed. More than thirty soil samples from all kinds of water ester or oil and
strains from myself laboratory. An efficient and rapid plating methods for determinant
of microbial esterase activity by observing the diameter of the strain and the
hydrolytic color circle around on the culture was adopted. The underlying principle
employed in this Model was based on color change of the indicator-Fast Blue B Salt
caused by a -NA, f3 -NA from a -NP and P -NP under the proper conditions. This
was preliminary screening and 174 strains obtained according to relative esterase
activity more than 40%. After culturing in a triangle bottle on the shake and color halo
is used to determine their esterase activity. This was secondary screening and 24 fine
strains were selected and the highest esterase activity of the strain EB-87 was
38.5UIml.
The mixed liquor of esterase results of the preliminary screening was a nearly linear
with results of the secondary screening, but the Bromocresol Purple later didn so.
Besides, studies and compared esterase activity of these strains with different
substance, including the esterase roducing strain for stereospecific hydrolysis of
(D,L)-Pantyl lactone and (D,L)-Ethyl lactate.
Isolation aiid purification the strain EB-87, and through detection of its appearance,
physiological and biochemical characteristics, growth characters, it was identified
Bacillus Genus according to Bergeysmanual of Deternmiutue Bacteriology(8) and
Microbiology Bacteriological Analytical Manual(FDA), and named Bacillus sp.
Strain EB-87.
In order to investigate the conditions and the principle of producing esterase by
Bacillus sp. Strain EB-87, the methods of combination Single Factor design
Plackett-Burman design and Response Surface Analysis had been employed for the
fermentation conditions and the composition of the fermentation medium.
The results showed that the optimum composition of the fermentation medium as
following: Corn steep liquor 2.4%, Glucose l.0%, Peptone 2.6%, MgSO.7H,O
0.07l%, peH.),SO. 0.2%, K,HPO, 0.2%, NaH,PO. 0.05%, the optimum fermentation
conditions as following: the optimum fermentation tempendre was 32oC, the
optimum fermentation shaking frequency was l50rpm, the optimum fermentation pH
was 7.2, the optimum fermentation time was 26-28h. In doing so Bacillus sp. strain
EB-87 esterase activity was 84.5U/ml, enlarging 2.2 times.
Finally the purification and properties including substance specificity, temperature,
pH, Bacillus sp. strain EB-87 esterase were studied in detail. The results showed that
the optimal temperature of BaciUus sp. strain EB-87 esterase reaction was 40C,
pH7.2, the esterase activity was stable in the range of pH6.0-8.0. and the enzyme was
stable less than 40C, but not stable above 50C. The parameters: tI/2 was 56.4min, k 85
was l l .2min at 50oC and tll2 was 2l .7min, to85 was 8.6min at 60C. The lions Fe2+.
Mg'+. K+. Na+ and Ca2+ were beneficial to esterase activity, but the lions Co2+. Zn2+
and Cu2+ were inhibited esterase activity. The kinetic parameters K. was
0.33 lmmoI/L, V. was l .005 ll mol/ml.min
The enzyme exhibits different esterase
引文
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