高产银耳多糖酶菌株的分离、鉴定、发酵条件优化及其酶的特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0271

摘要/Abstract

摘要：

目的银耳多糖因具有高分子量、高黏度等特性限制了其生物活性的发挥与应用。筛选高产银耳多糖酶的菌株并优化其发酵条件，以实现酶的量产，从而通过酶降解法降低银耳多糖分子量。方法根据微生物产酶分解银耳多糖的特性，从腐烂银耳样本中分离出以银耳多糖为唯一碳源的菌株，结合银耳多糖降解率测定与3，5-二硝基水杨酸（DNS）酶活测定法筛选出高产银耳多糖菌株。采用单因素实验与响应面法优化高产银耳多糖酶菌株的发酵培养基及发酵条件，并对所产银耳多糖酶的催化特性进行分析。结果从腐烂银耳样本中分离出40株能够以银耳多糖为唯一碳源的菌株，其中10株具有较高降解银耳多糖能力，酶活最高的菌株为Y3522，经16S rRNA基因测序鉴定为属间芽胞杆菌（Mesobacillus）。Y3522优化后的最佳发酵培养基组分为：银耳多糖（分子量为1 250 kD）8.07 g/L，酪蛋白胨25.47 g/L，K₂HPO₄ 7.11 g/L，NaCl 2.0 g/L，MgSO₄·7H₂O 1.0 g/L；最佳发酵条件为温度35 ℃、pH 7.5、接种量3%、转速250 r/min、发酵时间18-24 h。优化后酶活力提升60.1%。Y3522来源的银耳多糖酶的最适催化条件为pH 7.5、温度35 ℃。此外，20%（V/V）粗酶液可将3 000 kD银耳多糖（0.5%，m/V）在30、60、90、120 min分别降解至922、308、85、18 kD。结论分离并鉴定了高产银耳多糖酶菌株为属间芽胞杆菌Y3522，并优化了其产酶发酵体系，所产酶展现出对银耳多糖分子量高效的降解能力。

关键词: 银耳多糖酶, 分离鉴定, 属间芽胞杆菌, 发酵条件优化, 响应面实验, 分子量

Abstract:

Objective Tremella fuciformis polysaccharides’ (TFPs) high molecular weight, high viscosity and other characteristics limit its biological activity and application. The purpose of this study is to screen strains with high yield of TFPs enzyme and optimize its fermentation conditions, so as to achieve the mass production of enzyme, thus effectively reduce the molecular weights of TFPs by enzymatic degradation method, and expand its application potential. Method Based on the characteristics of microbial enzymatic decomposition of TFPs, strains with TFPs as the sole carbon source were isolated from decomposed Tremella fuciformis samples. Combined the determination of TFPs degradation rate with 3,5-dinitrosalicylic acid (DNS) enzyme activity, strains with high yield of TFPs enzyme were screened. Single factor experiments and response surface methodology were used to optimize the fermentation medium (carbon source, nitrogen source, inorganic salts, etc.) and fermentation conditions (temperature, pH, rotation speed, etc.) of high-yield TFPs enzyme strains, and the catalytic characteristics (optimal pH, temperature, and degradation efficiency) of the TFPs enzyme were analyzed. Result The 40 strains isolated from the rotten T. fuciformis samples could use TFPs as sole carbon source, and 10 strains of them had high ability to degrade TFPs. The strain Y3522 with the highest enzyme activity was identified as Mesobacillus by 16S rRNA gene sequencing. The optimized medium components of Mesobacillus sp. Y3522 were as follows: TFPs (molecular weight of 1 250 kD) 8.07 g/L, casein peptone 25.47 g/L, K₂HPO₄ 7.11 g/LNaCl 2.0 g/L,MgSO₄·7H₂O 1.0 g/L. The optimal fermentation conditions were a temperature of 35 ℃, pH of 7.5, inoculation volume of 3%, rotation speed of 250 r/min, and fermentation time of 18-24 h. After optimization, the enzyme activity increased by 60.1%. The optimal catalytic conditions for TFPs enzyme derived from Mesobacillus sp. Y3522 were pH 7.5 and temperature 35 ℃. In addition, 20% (V/V) crude enzyme solution degraded TFPs with a molecular weight of 3 000 kD (0.5%, m/V) to 922, 308, 85, and 18 kD at 30, 60, 90, and 120 min, respectively, demonstrating efficient molecular weight regulation ability. Conclusion The strain Mesobacillus sp. Y3522 producing high yield of TFPS are isolated and identified, its enzyme production fermentation system is optimized. The produced enzyme demonstrates efficient degradation ability on the molecular weights of TFPs.

Key words: Tremella fuciformis polysaccharides enzyme, isolation and identification, Mesobacillus, optimization of fermentation conditions, response surface experiment, molecular weight

廉少杰, 唐胜硕, 康传利, 刘磊, 郑德强, 杜帅, 汤丽伟, 张美霞, 刘蔷. 高产银耳多糖酶菌株的分离、鉴定、发酵条件优化及其酶的特性分析[J]. 生物技术通报, 2025, 41(9): 302-313.

LIAN Shao-jie, TANG Sheng-shuo, KANG Chuan-li, LIU Lei, ZHENG De-qiang, DU Shuai, TANG Li-wei, ZHANG Mei-xia, LIU Qiang. Isolation， Identification， Optimization of Fermentation Conditions of High-yield Tremella fuciformis Polysaccharides Enzyme-producing Strain and Its Enzyme Characteristics Analysis[J]. Biotechnology Bulletin, 2025, 41(9): 302-313.

图/表 10

表1 发酵培养基优化响应面试验设计

Table 1 Response surface design for the optimization of fermentation medium

水平 Level	因素 Factor
水平 Level	A 银耳多糖浓度 TFPs concentration （g/L）	B 酪蛋白胨浓度 Casein tryptone concentration （g/L）	C K₂HPO₄浓度 K₂HPO₄ concentration （g/L）
-1	7.5	22.5	6.0
0	8.0	25.0	7.0
1	8.5	27.5	8.0

图1 高产银耳多糖酶菌株的筛选A：银耳多糖降解实验结果； B：葡萄糖醛酸标准曲线； C：酶活测定结果

Fig. 1 Screening of strains producing high-yield TFPs enzymeA: Experimental results of TFPs degradation. B: Standard curve of glucuronic acid. C: Enzyme activity assay results

图2 菌株Y3522的形态学与分子生物学鉴定A：菌落形态；B：菌体形态；C：基于16S rRNA基因的系统发育分析

Fig. 2 Morphological and molecular biology identification of strain Y3522A: Morphology of colony; B: morphology of cell; C: phylogenetic analysis based on the 16S rRNA gene

图3 属间芽胞杆菌Y3522产酶发酵培养基的优化

Fig. 3 Optimization of enzyme-producing fermentation medium for Mesobacillus sp.Y3522

表2 响应面试验设计及结果

Table 2 Response surface design and test results

序号 Test No.	A 银耳多糖 TFPs	B酪蛋白胨 Casein peptone	C K₂HPO₄	Y 银耳多糖酶活 Enzyme activity of TFPs（U/mL）
1	7.5	22.5	7.0	332.47
2	8.5	22.5	7.0	333.33
3	7.5	27.5	7.0	333.36
4	8.5	27.5	7.0	348.39
5	7.5	25.0	6.0	334.14
6	8.5	25.0	6.0	333.28
7	7.5	25.0	8.0	327.40
8	8.5	25.0	8.0	349.77
9	8.0	22.5	6.0	340.28
10	8.0	27.5	6.0	348.17
11	8.0	22.5	8.0	343.75
12	8.0	27.5	8.0	349.75
13	8.0	25.0	7.0	362.98
14	8.0	25.0	7.0	372.05
15	8.0	25.0	7.0	370.73
16	8.0	25.0	7.0	373.57
17	8.0	25.0	7.0	364.28

表3 回归模型方差分析结果

Table 3 Results of variance analysis by regression model

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 Model	3 727.49	9	414.17	29.42	< 0.001	***
A	174.85	1	174.85	12.42	0.010	**
B	111.30	1	111.30	7.91	0.026	*
C	27.38	1	27.38	1.94	0.206
AB	50.20	1	50.20	3.57	0.101
AC	134.91	1	134.91	9.58	0.017	*
BC	0.893 0	1	0.893 0	0.063 4	0.808
A²	1 784.57	1	1 784.57	126.75	< 0.001	***
B²	532.63	1	532.63	37.83	< 0.001	***
C²	605.03	1	605.03	42.97	< 0.001	***
残差 Residual	98.55	7	14.08
失拟向 Lack of fit	7.24	3	2.41	0.105 7	0.952	Not significant
纯误差 Pure error	91.31	4	22.83
总差 Total error	3 826.0	16

图4 三因素交互作用对属间芽胞杆菌Y3522银耳多糖酶活力影响的曲面图

Fig. 4 Surface plots for interaction effects of three factors on TFPs enzyme activity of Mesobacillus sp.Y3522

图5 属间芽胞杆菌Y3522产酶发酵条件的优化

Fig. 5 Optimization of enzyme-producing fermentation condition for Mesobacillus sp.Y3522

图6 不同反应条件对属间芽胞杆菌Y3522来源的银耳多糖酶活力的影响

Fig. 6 Effects of different reaction conditions on the activity of TFPs enzyme from Mesobacillus sp.Y3522

图7 不同条件下酶解液的主成分分子量的变化及酶解前后银耳多糖的GPC图谱

Fig. 7 Variations in the molecular weights of the main components of enzymatic hydrolysate under different conditions and GPC spectra of TFPs before and after enzymatic hydrolysis

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