食品与发酵工业 >

2023 , Vol. 49 >Issue 1: 10 - 18

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.031522

研究报告

纤维微菌β-1,3-葡聚糖酶的克隆表达及对灵芝细胞壁的水解作用

  • 杨梦莲 ,
  • 单逸蓝 ,
  • 沈微 ,
  • 朱新文 ,
  • 杨海泉 ,
  • 陈献忠 ,
  • 陈磊 ,
  • 夏媛媛
展开
  • 1(江南大学 生物工程学院 工业生物技术教育部重点实验室,江苏 无锡,214122)
    2(山东省东阿县农业农村局,山东 聊城,252200)
第一作者：硕士,副教授(沈微副教授为通信作者,E-mail:2270723473@qq.com)

收稿日期: 2022-03-12

  修回日期: 2022-04-02

  网络出版日期: 2023-02-14

收起

Cloning and expression of Cellulosimicrobium β-1,3-glucanase and its hydrolysis on the cell wall of Ganoderma lucidum

  • YANG Menglian ,
  • SHAN Yilan ,
  • SHEN Wei ,
  • ZHU Xinwen ,
  • YANG Haiquan ,
  • CHEN Xianzhong ,
  • CHEN Lei ,
  • XIA Yuanyuan
Expand
  • 1(School of Biotechnology, Key Laboratory of Industrial Biotechnology, Jiangnan University, Wuxi 214122, China)
    2(Agricultural and Rural Bureau of Dong’e County, Shandong Province, Liaocheng 252200, China)

Received date: 2022-03-12

  Revised date: 2022-04-02

  Online published: 2023-02-14

Fold

摘要

该文旨在研究一种酶解灵芝(Ganoderma lucidum)菌丝体从而提高其胞内蛋白提取效率的方法。通过PCR方法扩增获得纤维微菌(Cellulosimicrobium sp.)CICIM6906的β-1,3-葡聚糖酶编码基因,该基因命名为bgl6906并与表达载体连接,构建重组质粒pET28a-bgl6906,重组质粒转化大肠杆菌BL21(DE3),获得重组大肠杆菌BL21(DE3)/pET28a-bgl6906。重组菌在TB培养基中进行诱导表达,表达产物在自身信号肽引导下大部分分泌到细胞外。以茯苓多糖为底物时,重组酶BGL6906纯酶比酶活力为567.8 U/mg,最适pH为5.5,最适温度为50 ℃。重组菌在15 L发酵罐中发酵72 h胞外酶活力达到67 U/mL。重组酶BGL6906与果胶酶交替水解灵芝菌丝体培养物,可以有效水解细胞壁,获得部分原生质体。进一步辅助短时超声波破碎可以大幅度提高灵芝胞内产物的释放。

关键词： 纤维微菌; β-1,3-葡聚糖酶; 分泌表达; 灵芝; 细胞破碎

本文引用格式

杨梦莲 , 单逸蓝 , 沈微 , 朱新文 , 杨海泉 , 陈献忠 , 陈磊 , 夏媛媛 . 纤维微菌β-1,3-葡聚糖酶的克隆表达及对灵芝细胞壁的水解作用[J]. 食品与发酵工业, 2023 , 49(1) : 10 -18 . DOI: 10.13995/j.cnki.11-1802/ts.031522

Abstract

This paper aims to study the enzymatic hydrolysis of Ganoderma lucidum mycelium to improve the protein extraction efficiency. The gene bgl6906 encoding β-1,3-glucanase was amplified from the genome DNA of Cellulosimicrobium sp. CICIM B6906. The recombinant plasmid pET28a-bgl6906 was constructed and transformed into E. coli BL21(DE3), the recombinant strain Escherichia coli BL21(DE3)/pET28a-bgl6906 was constructed to express bgl6906. The recombinant strain was cultured in TB medium and induced gene expression. Most of the expression products of the gene bgl6906 was secreted outside the cell under the guidance of its own signal peptide. The recombinant enzyme BGL6906 showed the highest activity when pachyman was used as a substrate, and the specific activity of the pure enzyme was 567.8 U/mg. The optimum temperature and pH of BGL6906 was 50 °C and 5.5, respectively. The recombinant strain was used in a fermentation experiment using a 15 L fermenter. The highest enzyme activity was 67 U/mL, which was reached after 72 h of fermentation. BGL6906 and pectinase were used alternatively for the cell wall hydrolysis of the G. lucidum mycelium. The cell wall was successfully degraded, and a small amount of protoplast was observed. The mycelium cells treated with BGL6906 and pectinase alternatively could be disrupted effectively by short-time ultrasonic treatment. About 76% of the total protein could be extracted to the alkaline solution.

Key words： Cellulosimicrobium; β-1,3-glucanase; secretory expression; Ganoderma lucidum; cell disruption

参考文献

[1] 贾蕾, 向极钎, 殷红清, 等.生物活性硒肽的研究进展[J].食品科学, 2021, 42(15):346-355.
JIA L, XIANG J Q, YIN H Q, et al.Progress in bioactive selenium-containing peptides[J].Food Science, 2021, 42(15):346-355.
[2] 张俊杰. 硒的生理功能及富硒强化食品的研究进展[J].微量元素与健康研究, 2006, 23(3):58-60.
ZHANG J J.The biological functions of selenium and research development of Se-enriched foodstuff[J].Studies of Trace Elements and Health, 2006, 23(3):58-60.
[3] 王俊, 黄明, 徐幸莲, 等.硒及富硒功能食品研究进展[J].江苏农业科学, 2003,31(2):53-56.
WANG J, HUANG M, XU X L, et al.Advance in selenium and selenium-enriched functional foods[J].Jiangsu Agricultural Science, 2003,31(2):53-56.
[4] 李海蓉, 杨林生, 谭见安, 等.我国地理环境硒缺乏与健康研究进展[J].生物技术进展, 2017, 7(5):381-386.
LI H R, YANG L S, TAN J A, et al.Progress on selenium deficiency in geographical environment and its health impacts in China[J].Current Biotechnology, 2017, 7(5):381-386.
[5] 胡婷, 惠改芳, 赵桂慎, 等.富硒食用菌研究进展[J].食用菌学报, 2019, 26(1):68-76.
HU T, HUI G F, ZHAO G S, et al.Advances in selenium-enriched edible fungi[J].Acta Edulis Fungi, 2019, 26(1):68-76.
[6] 赵镭, 胡小松.富硒灵芝的研究进展[J].中国食用菌, 2003, 22(3):35-36.
ZHAO L, HU X S.Researches on the Se-accumulating Ganoderma lucidum[J].Edible Fungi of China, 2003, 22(3):35-36.
[7] BATRA P, SHARMA A K, KHAJURIA R.Probing Lingzhi or Reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes):A bitter mushroom with amazing health benefits[J].International Journal of Medicinal Mushrooms, 2013, 15(2):127-143.
[8] RZYMSKI P, MLECZEK M, NIEDZIELSKI P, et al.Potential of cultivated Ganoderma lucidum mushrooms for the production of supplements enriched with essential elements[J].Journal of Food Science, 2016, 81(3):C587-C592.
[9] 郭莹莹, 汪桐, 徐峥辉, 等.富硒灵芝的研究进展[J].园艺与种苗, 2012, 32(9):55-58;61.
GUO Y Y, WANG T, XU Z H, et al.Research progress on the Se-accumulating Ganoderma lucidum[J].Horticulture & Seed, 2012, 32(9):55-58;61.
[10] 尚德静, 崔乔, 高林.灵芝硒多糖SeGLP-1抑制小鼠肝腹水癌作用的研究[J].食用菌学报, 2001, 8(3):34-38.
SHANG D J, CUI Q, GAO L.Study on anti-HCa-f tumor activity of polysaccharide containing selenium in Ganoderma lucidum (SeGLP 1)[J].Acta Edulis Fungi, 2001, 8(3):34-38.
[11] 尚德静, 李庆伟, 崔乔, 等.灵芝硒多糖Se-GLP-1抗氧化与抗肿瘤作用的研究[J].营养学报, 2002, 24(3):249-251.
SHANG D J, LI Q W, CUI Q, et al.Study on antioxidative and antitumor effect of selenium containing polysaccharide in Ganoderma lucidum in mice[J].Acta Nutrimenta Sinica, 2002, 24(3):249-251.
[12] SHANG D J, LI Y, WANG C, et al.A novel polysaccharide from Se-enriched Ganoderma lucidum induces apoptosis of human breast cancer cells[J].Oncology Reports, 2011, 25(1):267-272.
[13] 杜明, 赵镭, 李朝睿, 等.富硒灵芝中一种新含硒蛋白的纯化、性质及其自由基清除活性研究[J].高等学校化学学报, 2007, 28(1):75-78.
DU M, ZHAO L, LI C R, et al.Purification and properties of a novel Se-containing protein from Se-enriched Ganoderma lucidum and its antioxidant activity[J].Chemical Journal of Chinese Universities, 2007, 28(1):75-78.
[14] ZHAO L, ZHAO G H, ZHAO Z D, et al.Selenium distribution in a Se-enriched mushroom species of the genus Ganoderma[J].Journal of Agricultural and Food Chemistry, 2004, 52(12):3 954-3 959.
[15] 吴裕豪, 龙志标, 肖虹, 等.灵芝菌丝胞内多糖提取条件研究[J].轻工科技, 2012, 28(11):14-15.
WU Y H, LONG Z B, XIAO H, et al.Study on extraction conditions of intracellular polysaccharide from Ganoderma lucidum hyphae[J].Light Industry Science and Technology, 2012, 28(11):14-15.
[16] 漆倩涯, 贠建民, 黄玉琴, 等.超声破碎辅助蜗牛酶提取杏鲍菇蛋白工艺优化[J].食品科学, 2016, 37(22):85-91.
QI Q Y, YUN J M, HUANG Y Q, et al.Optimization of ultrasonic-assisted snailase hydrolysis for extraction of protein from Pleurotus eryngii[J].Food Science, 2016, 37(22):85-91.
[17] 肖亚朋, 沈微, 李婷霖, 等.嗜热脂肪土芽孢杆菌普鲁兰酶基因的异源表达及重组酶性质[J].食品与发酵工业, 2017, 43(5):30-36.
XIAO Y P, SHEN W, LI T L, et al.Heterologous expression of pullulanase gene from Geobacillus stearothermophilus and characterization of the recombinant enzyme[J].Food and Fermentation Industries, 2017, 43(5):30-36.
[18] 邓乾坤, 纪彦宇, 蒋霞, 等.酵母富硒蛋白提取条件优化[J].中国酿造, 2021, 40(9):206-210.
DENG Q K, JI Y Y, JIANG X, et al.Optimization of extraction conditions for yeast selenium-enriched protein[J].China Brewing, 2021, 40(9):206-210.
[19] 薛伟, 罗晖, 常雁红, 等.藤黄节杆菌β-1, 3-葡聚糖酶基因在大肠杆菌中的克隆及表达[J].生物技术通报, 2010(10):210-214.
XUE W, LUO H, CHANG Y H, et al.Cloning and expression of the β-1, 3-glucanase from Arthrobacter luteus in Escherichia coli[J].Biotechnology Bulletin, 2010(10):210-214.
[20] BAI L, KIM J, SON K H, et al.Novel anti-fungal D-laminaripentaose-releasing endo-β-1, 3-glucanase with a RICIN-like domain from Cellulosimicrobium funkei HY-13[J].Biomolecules, 2021, 11(8):1080.
[21] LI K K, CHEN W, WANG W X, et al.Effective degradation of curdlan powder by a novel endo-β-1→3-glucanase[J].Carbohydrate Polymers, 2018, 201:122-130.
[22] 颜梦秋, 张丹, 周帅, 等.紫芝细胞壁大分子量葡聚糖的纯化及结构鉴定[J].菌物学报, 2020, 39(8):1 502-1 509.
YAN M Q, ZHANG D, ZHOU S, et al.Purification and structure elucidation of high molecular weight polysaccharide from the cell wall of Ganoderma sinense[J].Mycosystema, 2020, 39(8):1 502-1 509.
[23] 黄敏, 廖春燕, 陆志鸿.酶法制备金针菇提取物的工艺研究[J].食品工业科技, 2014, 35(3):216-220.
HUANG M, LIAO C Y, LU Z H.Study of Flammulina velutipes extract by enzymatic preparation[J].Science and Technology of Food Industry, 2014, 35(3):216-220.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司