一株产蛋白酶细菌的筛选、鉴定及发酵工艺优化

doi:10.13560/j.cnki.biotech.bull.1985.2024-0945

摘要/Abstract

摘要：

目的从酸菜汁中分离筛选产蛋白酶细菌，明确菌株种属分类地位，优化发酵产酶工艺，以丰富蛋白酶菌种资源，提高发酵产酶效能。方法以某酸菜作坊大白菜酸菜汁为分离源，采用10倍梯度稀释法通过酪蛋白平板初筛，福林酚法进一步复筛获得产蛋白酶菌株，结合形态学观察和16S rRNA基因系统发育分析完成种属鉴定；单因素试验优化发酵培养基成分及条件，响应面法进一步优化产酶工艺条件，提高产酶效能。结果初筛共得到15株蛋白酶产生菌，福林酚法复筛得到同时具备酸性、中性和碱性蛋白酶能力的菌株P-133，其酸性、中性和碱性蛋白酶活力分别为1.00、52.50和48.50 U/mL，经鉴定为假单胞菌（Pseudomonas sp.）；单因素及响应面试验结果表明，菌株P-133最佳产酶工艺条件为可溶性淀粉5.0 g/L、菜粕5.9 g/L、NaCl 3.3 g/L、K₂HPO₄ 5.0 g/L，pH 7.8，温度28℃，接种量5%及装液量75 mL/250 mL，该条件下酸性、中性和碱性蛋白酶活力分别为4.14、185.19和177.30 U/mL，是优化前的4.14、3.53和3.66倍。结论从酸菜汁分离筛选得到同时具备产酸性、中性和碱性蛋白酶的假单胞菌P-133，发酵产酶工艺优化后产酸性、中性和碱性蛋白酶活性分别是优化前的4.14、3.53和3.66倍。

关键词: 蛋白酶, 筛选, 鉴定, 假单胞菌, 发酵优化, 单因素法, 响应面法

Abstract:

Objective To isolate and screen protease-producing bacteria from sauerkraut juice, clarify the species classification status of the strains, optimize the fermentation enzyme production process, enrich the resources of protease strains, and improve the efficiency of fermentation enzyme production. Method Using the sauerkraut juice of Chinese cabbage in a sauerkraut workshop as the isolation source, the ten-fold layer dilution method was used to screen the casein plate, and the folinol method was further re-screened to obtain the protease-producing strains. Morphological observation and 16S rRNA gene phylogenetic analysis were combined to complete the species identification. Single factor test was to optimize the composition and conditions of the fermentation medium, and the response surface method was further to optimize the enzyme-producing process for improving the efficiency of enzyme production. Result A total of 15 protease-producing strains were obtained by initial screening, and strain P-133 with acidic, neutral and alkaline protease abilities was obtained by re-screening with forinol method. The acidic, neutral and alkaline protease activities were 1.00, 52.50 and 48.50 U/mL, respectively, and were identified as Pseudomonas sp. The results via single factor and response surface tests showed that the optimal conditions for enzyme production of strain P-133 were soluble starch 5.0 g/L, rapeseed meal 5.9 g/L, NaCl 3.3 g/L, K₂HPO₄ 5.0 g/L, pH 7.8, temperature 28℃, inoculation volume 5%. and liquid-loading capacity 75 mL/250 mL. Under these conditions, the acidic, neutral and alkaline protease activities were 4.14, 185.19 and 177.30 U/mL, respectively, which were 4.14, 3.53 and 3.66 times higher than those before optimization. Conclusion Pseudomonas sp. P-133 with acidic, neutral and alkaline proteases is isolated and screened from sauerkraut juice. The activities of acidic, neutral and alkaline proteases were 4.14, 3.53 and 3.66 times higher than those before optimization, respectively.

Key words: protease, screening, identification, Pseudomonas sp., fermentation optimization, single factor method, response surface method

刘爽, 江洲, 赵帅, 赵雷真, 黄峰, 周佳, 屈建航. 一株产蛋白酶细菌的筛选、鉴定及发酵工艺优化[J]. 生物技术通报, 2025, 41(4): 335-344.

LIU Shuang, JIANG Zhou, ZHAO Shuai, ZHAO Lei-zhen, HUANG Feng, ZHOU Jia, QU Jian-hang. Screening， Identification， and Fermentation Optimization of a Protease-producing Bacterial Strain[J]. Biotechnology Bulletin, 2025, 41(4): 335-344.

图/表 12

表1 发酵条件优化响应面试验设计

Table 1 Response surface design for the optimization of fermentation conditions

水平 Level
水平 Level	A pH	B菜粕浓度 Rapeseed meal concentration/（g·L^-1）	C NaCl浓度 NaCl concentration/（g·L^-1）
-1	5	1	0.5
0	7	5	1
1	9	10	5

表2 产蛋白酶菌株初筛结果

Table 2 Results of preliminary screening of proteinase-producing strains

编号 Number	水解圈直径Diameter of hydrolysis ring （D）/mm	菌落直径 Diameter of colony （d）/mm	圈径比Ring diameter ratio （D/d）	编号 Number	水解圈直径 Diameter of hydrolysis ring （D）/mm	菌落直径 Diameter of colony （d）/mm	圈径比Ring diameter ratio （D/d）
P-6	20	11	1.8	P-18	12	6	2.0
P-7	11	6	1.8	P-25	18	7	2.6
P-8	13	6	2.7	P-34	24	5	4.8
P-9	12	5	2.4	P-35	20	10	2.0
P-12	14	6	2.3	P-36	14	7	2.0
P-13	20	5	4.0	P-37	10	5	2.0
P-14	12	6	2.0	P-133	20	4	5.0
P-15	10	6	1.7

图1 产蛋白酶菌株复筛结果N.D. 代表未检出，下同

Fig. 1 Results of rescreening of proteinase-producing strainsN.D. stands for not detected, the same below

图2 菌株P-133的菌落和菌体形态A：菌落形态；B：菌体形态

Fig. 2 Morphology of colony and cell of strain P-133A: Morphology of colony. B: Morphology of cell

图3 菌株P-133基于16S rRNA基因序列的系统发育树括号内为菌株16S rRNA基因序列的GenBank 登录号；分支节点数字为 Bootstrap 值（低于50 不显示）；标尺的数据为进化距离

Fig. 3 Phylogenetic tree of strain P-133 based on the 16S rRNA gene sequencesCode in parentheses is GenBank accession number of the strain. The numbers of branch nodes are bootstrap values (<50 not displayed). The data of the scale is the evolutionary distance

图4 菌株P-133的生长曲线和产酶曲线

Fig. 4 Growth curve and enzyme production curve of strain P-133

图5 菌株P-133的发酵培养基优化不同小写字母、大写字母、数字，分别代表酸性蛋白酶活性、中性蛋白酶活性和碱性蛋白酶活性的显著差异（P＜0.05），下同

Fig. 5 Optimization of fermentation medium for strain P-133Different lowercase letters, uppercase letters and numbers, indicate significant differences in acid protease activity, neutral protease activity and alkaline protease activity (P<0.05). The same below

图6 菌株P-133的发酵条件优化

Fig. 6 Optimization of fermentation conditions for strain P-133

表3 IBM SPSS Statistics 方差分析

Table 3 Analysis of variance in IBM SPSS statistics

方差来源 Source of variance	平方和 Sum of squares	自由度 Freedom	均方 Mean	F值 F value	P值 P value	显著性 Significance
pH	64 659.717	4	16 164.929	1 349.359	1.30×10^-13	**
菜粕浓度	21 586.241	6	3 597.707	203.065	8.62×10^-13	**
NaCl浓度	20 247.864	6	3 374.644	126.434	2.24×10^-11	**
装液量	2 923.535	5	584.707	208.34	3.15×10^-11	**
温度	53 335.824	4	13 333.96	349.142	1.08×10^-10	**
接种量	1 205.128	4	301.282	64.771	4.12×10^-7	**
可溶性淀粉浓度	3 331.388	6	555.231	4.269	0.012	*

表4 Box-Behnken 试验设计及结果

Table 4 Box-Behnken design and test results

Test number	A	B	C	Y/（U·mL^-1）
1	1	-1	0	38.43
2	-1	1	0	129.26
3	0	0	0	144.55
4	1	0	1	145.14
5	0	0	0	170.45
6	1	1	0	115.7
7	0	0	0	159.06
8	-1	0	-1	89.69
9	0	0	0	149.34
10	-1	0	1	81.90
11	-1	-1	0	17.51
12	0	1	1	147.61
13	0	0	0	140.501
14	0	1	-1	152.94
15	1	0	-1	95.91
16	0	-1	-1	72.67
17	0	-1	1	75.88

表5 二次回归方程方差分析

Table 5 Analysis of variance via the quadratic regression equation

来源Source	平方和 Sum of square	自由度 Freedom	均方 Mean	F值 F value	P值 P value	显著性Significance
模型	31 171.60	9	3 463.51	26.81	0.000 1	**
A	1 462.82	1	1 462.82	11.32	0.012 0	*
B	10 789.03	1	10 789.03	83.50	<0.000 1	**
C	171.86	1	171.86	1.33	0.286 6
AB	308.14	1	308.14	2.38	0.166 4
AC	1 216.09	1	1 216.09	9.41	0.018 1	*
BC	122.90	1	122.90	0.951 2	0.361 9
A²	7 969.72	1	7 969.72	61.68	0.000 1	**
B²	6 347.18	1	6 347.18	49.12	0.000 2	**
C²	326.71	1	326.71	2.53	0.155 8
残差	904.44	7	129.21
失拟项	322.52	3	107.51	0.739 0	0.581 5	不显著
纯误差	581.91	4	145.48
总和	32 076.04	16

图7 各因素交互作用对中性蛋白酶活影响的响应面与等高线

Fig. 7 Response surfaces and contour lines of the interaction between various factors on neutral protease activity

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