丹参药渣木质素降解菌的分离及酶学特性

doi:10.13560/j.cnki.biotech.bull.1985.2024-0419

生物技术通报 ›› 2024, Vol. 40 ›› Issue (11): 269-276.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0419

丹参药渣木质素降解菌的分离及酶学特性

杨秉乾¹(), 恽辰珂¹, 常思源¹^,²(), 郭盛¹, 张森¹()

1.南京中医药大学江苏省中药资源产业化过程协同创新中心，南京 210023
2.南京科技职业学院江苏省生物基平台化合物催化工程技术研究开发中心，南京 210048

收稿日期:2024-05-06 出版日期:2024-11-26 发布日期:2024-12-19
通讯作者: 张森，男，博士，副教授，研究方向：中药资源化利用；E-mail: zhangsci@njucm.edu.cn；
常思源，男，博士，副教授，研究方向：中药资源化利用；E-mail: siyuanchang@njpi.edu.cn
作者简介:杨秉乾，男，硕士研究生，研究方向：中药资源化利用；E-mail: ybq20010329@163.com
基金资助:
国家自然科学基金项目(221006073);中国石油和化学工业联合会责任关怀专项研究课题(2022CRCB004);山东省重点研发计划项目(2021SFGC1203);江苏省高职院校教师专业带头人高端研修项目(2023GRFX038);江苏省研究生实践创新计划项目(SJCX23_0766);江苏省青蓝工程

Isolation and Enzymatic Characterization of Fungus Degrading Salvia miltiorrhiza Residue Lignin

YANG Bing-qian¹(), YUN Chen-ke¹, CHANG Si-yuan¹^,²(), GUO Sheng¹, ZHANG Sen¹()

1. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023
2. Bio-based Platform Chemicals Catalysis Engineering Technology Research and Development Center of Jiangsu Province, Nanjing Polytechnic Institute, Nanjing 210048

Received:2024-05-06 Published:2024-11-26 Online:2024-12-19

摘要/Abstract

摘要：

【目的】 筛选可降解丹参（Salvia miltiorrhiza）药渣木质素的菌株并探究其酶学特性，以筛得菌株降解丹参药渣木质素以提高纤维素的酶解效率。【方法】 以丹参药渣表面自然生长的菌株为筛菌来源，通过碱性木素培养基和愈创木酚培养基进行初筛和复筛，利用添加丹参药渣的培养基验证产酶能力，优选产漆酶菌株。通过ITS序列分析对菌株进行鉴定。对分离菌株漆酶酶学特性进行研究。采用NREL法测定木质纤维素含量，利用商业纤维素酶探究酶解效果。【结果】 筛选出一株产漆酶且耐受丹参药渣的真菌MY-5，经ITS序列鉴定为猬木霉（Trichoderma erinaceum）。对漆酶酶学性质进行考察，漆酶最适反应温度为50℃，60℃下保温24 h后酶活保持在85.3%，4℃低温贮藏25 d酶活保持在80.7%；最适pH为5，在pH 4-8时保温24 h活性保持在90%以上；添加Cu²⁺、Mg²⁺和香兰素提高了漆酶活性，其中5 mmol/L Cu²⁺提高1.68倍；添加4%丹参药渣保温24 h后漆酶仍能保持90.6%的酶活。液态发酵20 d后丹参药渣木质素的降解率达到了36.3%，葡萄糖产量和最大产率分别提高了62.6%和81.0%。【结论】 筛选得到一株产漆酶菌株猬木霉MY-5，其漆酶热稳定性、低温贮藏稳定性、pH稳定性及丹参药渣耐受性良好，可以有效降解丹参药渣木质素，提高纤维素的酶解效率。

关键词: 丹参药渣, 猬木霉, 漆酶, 酶学特性, 木质素降解

Abstract:

【Objective】 Selecting fungus that degrades Salvia miltiorrhiza residue（SMR）lignin and exploring its enzymatic characteristics is for having a strain to degrade SMR lignin for improving the enzymatic hydrolysis efficiency of cellulase. 【Method】 The strains naturally growing on the surface of SMR were selected as the screening source. Basic lignin medium and guaiacol medium were used for preliminary screening, and the medium added with SMR was used for re-screening to select the laccase-producing strains. The strains were identified by ITS sequence analysis. The enzymatic characteristics of laccase from isolated strains were studied. The content of lignocellulose was determined by NREL method, and the enzymolysis effect was investigated by commercial cellulase. 【Result】 A laccase-producing and SMR-tolerant fungus MY-5 was selected and identified as Trichoderma erinaceum by ITS sequence. The enzymatic properties of laccase were investigated. The optimal reaction temperature was 50℃, the enzyme activity was maintained at 85.3% after 24 h at 60℃, and 80.7% after 25 d of low temperature storage at 4℃. The optimal pH was 5, and the activity remained above 90% at pH 4-8 for 24 h. Adding Cu²⁺ and Mg²⁺ and vanillin increased the activity of laccase, and 5 mmol/L Cu²⁺ increased to 1.68 times. Adding 4% SMR and holding it for 24 h, 90.6% laccase activity still was maintained. After 20 d of liquid fermentation, SMR lignin degradation rate reached 36.3%, and the glucose yield and maximum yield increased by 62.6% and 81.0%, respectively. 【Conclusion】 A laccase-producing and SMR-tolerant T. erinaceum MY-5 had been isolated, which had good thermal stability, low temperature storage stability, pH stability and resistance to SMR, which may effectively degrade SMR lignin and improve the efficiency of cellulolytic enzyme hydrolysis, providing a basis for the bio-utilization of Chinese medicinal residue.

Key words: Salvia miltiorrhiza residue, Trichoderma erinaceum, laccase, enzymatic characterization, delignification

杨秉乾, 恽辰珂, 常思源, 郭盛, 张森. 丹参药渣木质素降解菌的分离及酶学特性[J]. 生物技术通报, 2024, 40(11): 269-276.

YANG Bing-qian, YUN Chen-ke, CHANG Si-yuan, GUO Sheng, ZHANG Sen. Isolation and Enzymatic Characterization of Fungus Degrading Salvia miltiorrhiza Residue Lignin[J]. Biotechnology Bulletin, 2024, 40(11): 269-276.

图/表 9

图1 4株真菌的显色反应

Fig. 1 Colorimetric reactions of 4 fungi A: YY3. B: YY3-A1. C: BQ-A3. D: MY-5

图2 4株真菌发酵玉米秸秆和丹参药渣漆酶酶活图中误差线表示标准偏差。小写字母表示不同组间差异达到（P<0.05）显著水平，下同

Fig. 2 Laccase activity in 4 fungi fermenting in the corn straw and SMR fermentation The error line in the figure refers to the standard deviation. The lowercase letters indicate that the difference among different groups reached a significant level（P < 0.05）. The same below

图3 菌株MY-5鉴定 A：MY-5菌落形态；B：MY-5系统发育树

Fig. 3 Identification of strain MY-5 A: MY-5 colony morphology. B: MY-5 phylogenetic tree

图4 温度对漆酶酶活的影响 A：漆酶最适温度；B：漆酶热稳定性

Fig. 4 Effect of temperature on laccase activity A: Optimal laccase temperature. B: Laccase thermal stability

图5 pH对漆酶酶活的影响 A：漆酶最适pH；B：漆酶pH稳定性

Fig. 5 Effect of pH on laccase activity A: Optimal laccase pH. B: Laccase pH stability

图6 金属离子及诱导剂对漆酶酶活的影响组1：金属离子浓度1 mmol/L，香兰素浓度0.01 g/L；组2：金属离子浓度5 mmol/L，香兰素浓度0.05 g/L

Fig. 6 Effects of metal ions and inducers on laccase activity Group 1: Metal ion concentration is 1 mmol/L and vanillin concentration is 0.01 g/L. Group 2: Metal ion concentration is 5 mmol/L and vanillin concentration is 0.05 g/L

图7 丹参药渣对漆酶酶活的影响

Fig. 7 Effect of SMR on laccase activity

表1 发酵前后丹参药渣木质纤维素含量变化

Table 1 Lignocellulose content changes in SMR before and after fermentation

组别Group	酸溶性木质素Acid soluble lignin/%	酸不溶性木质素Acid insoluble lignin/%	纤维素Cellulose/%	半纤维素Hemicellulose/%
未处理Untreated	4.53±1.06a	10.58±1.36a	17.46±1.27a	14.02±1.67a
处理后Treated	3.07±0.59b	6.56±0.47b	15.06±0.92b	7.32±0.71b

图8 处理前后丹参药渣葡萄糖产率及酶解效率

Fig. 8 Glucose yield and enzymatic hydrolysis efficiency of SMR before and after treatment

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丹参药渣木质素降解菌的分离及酶学特性

Isolation and Enzymatic Characterization of Fungus Degrading Salvia miltiorrhiza Residue Lignin

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