重组毕赤酵母产<i>β</i>-甘露聚糖酶的高密度发酵研究

杜少平; 胡海艳; 甘祥武; 黄秀敏; 叶俊豪

doi:10.12187/2020.04.001

CN 41-1437/TS ISSN 2096-1553

重组毕赤酵母产β-甘露聚糖酶的高密度发酵研究

杜少平 , 胡海艳 , 甘祥武 , 黄秀敏 , 叶俊豪

杜少平, 胡海艳, 甘祥武, 等. 重组毕赤酵母产β-甘露聚糖酶的高密度发酵研究[J]. 轻工学报, 2020, 35(4): 1-7. doi: 10.12187/2020.04.001

引用本文: 杜少平, 胡海艳, 甘祥武, 等. 重组毕赤酵母产β-甘露聚糖酶的高密度发酵研究[J]. 轻工学报, 2020, 35(4): 1-7. doi: 10.12187/2020.04.001

DU Shaoping, HU Haiyan, GAN Xiangwu, et al. Study on high-density fermentation of β-mannanase produced by constitutive Pichia pastoris[J]. Journal of Light Industry, 2020, 35(4): 1-7. doi: 10.12187/2020.04.001

Citation: DU Shaoping, HU Haiyan, GAN Xiangwu, et al. Study on high-density fermentation of β-mannanase produced by constitutive Pichia pastoris[J]. Journal of Light Industry, 2020, 35(4): 1-7. doi: 10.12187/2020.04.001

重组毕赤酵母产β-甘露聚糖酶的高密度发酵研究

广州市微生物研究所, 广东广州 510663

作者简介: 杜少平(1974-),男,广东省丰顺县人,广州市微生物研究所高级工程师,主要研究方向为微生物技术与相关检测.;

基金项目: 广州市科技计划项目（201710010154）
中图分类号: TS201.3

Study on high-density fermentation of β-mannanase produced by constitutive Pichia pastoris

Guangzhou Institute of Microbiology, Guangzhou 510663, China
Received Date: 2020-05-21

CLC number: TS201.3

摘要: 通过摇瓶实验，对重组毕赤酵母X-33/pGAPZαA-Man26A-39发酵培养基的碳源、氮源进行筛选，并在50 L发酵罐中采用间歇流加策略，对其发酵条件进行调控，以实现重组毕赤酵母高密度发酵产β-甘露聚糖酶.结果表明：葡萄糖为发酵培养基的最适碳源，其初始添加质量浓度为30 g/L；初始质量分数为6%的玉米浆作为发酵培养基的氮源较为适宜；在间歇补料发酵调控中，通过间歇流加体积分数为25%的氨水、质量分数为50%的葡萄糖溶液和质量分数为20%的玉米浆，可使发酵液pH值维持在5.0~5.5之间，且保证发酵培养基的碳源、氮源充足；发酵60 h时采取放罐措施，此时酶活力可达2 685.5 U/mL，菌体质量浓度达331.1 g/L，OD₆₀₀达302.8，实现了β-甘露聚糖酶的高效表达.
- β-甘露聚糖酶 /
- 毕赤酵母 /
- 高密度发酵 /
- 发酵调控
Abstract: The carbon and nitrogen sources of the constitutive Pichia pastoris X-33/pGAPZαA-Man26A-39 fermentation medium were optimized by shake flask experiment,and the fermentation conditions were regulated in a 50 L fermentor by using intermittent feeding strategy,in order to realize high-density fermentation of constitutive Pichia pastoris to produce β-mannanase.The results showed that glucose was the most suitable carbon source for fermentation medium,and its initial centration was 30 g/L;corn syrup was the most suitable nitrogen source,and its initial mass fraction was 6%;during the regulation of intermittent fed fermentation,the pH value of bacteria could be maintained between 5.0~5.5 by intermittent flow adding 25% ammonia hydroxide,50% glucose solution and 20% corn syrup,and make sure the carbon and nitrogen sources of the fermentation medium were sufficient;after 60 h fermentation,took measures to put the fermentater tank,at this time,the maximum enzyme activity was 2 685.5 U/mL,the mass concentration of bacteria reached 331.1 g/L,and the OD₆₀₀ reached 302.8,which achieved the high expression of β-mannanase.
- β-mannanase /
- Pichia pastoris /
- high-density fermentation /
- fermentation control
1. [1]
  SCHELLER H V,ULVSKOV P.Hemicelluloses[J].Annual Review of Plant Biology,2010,61:263.
2. [2]
  STÅLBRAND H,SIIKAAHO M,TENKANEN M,et al.Purification and characterization of two β-mannanases from Trichoderma reesei[J].Journal of Biotechnology,1993,29(3):229.
3. [3]
  KANSOH A L,NAGIEB Z A.Xylanase and mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp[J].Antonie Van Leeuwonhoek,2004,85(2):103.
4. [4]
  NUNES C S,MALMLÖF K.Effects of guar gum and cellulose on glucose absorption,hormonal release and hepatic metabolism in the pig[J].British Journal of Nutrition,1992,68(3):693.
5. [5]
  BHAT M K.Cellulases and related enzymes in biotechnology[J].Biotechnology Advances,2000,18(5):355.
6. [6]
  熊进,黄魁英,夏枫耿,等.耐高温β-甘露聚糖酶M27-8菌株发酵条件的优化[J].中国饲料,2017,19:13.
7. [7]
  吴秀秀,吕晓慧,胡亚冬,等.耐高温耐酸稳定假密环菌(Armillariella tabescens)MAN47β-甘露聚糖酶体外分子定向进化[J].中国生物工程杂志,2012,32(3):83.
8. [8]
  ZHAO W,ZHENG J,ZHOU H B,et al.A thermotolerant and cold-active mannan endo-1,4-β-mannosidase from Aspergillus niger CBS 513.88:Constitutive overexpression and high-density fermentation in Pichia pastoris[J].Bioresource Technology,2011,102:7538.
9. [9]
  ZHENG J,ZHAO W,GUO N,et al.Development of an industrial medium and a novel fed-batch strategyfor high-level expression of recombinant β-mananase by Pichia pastoris[J].Bioresource Technology,2012,118:257.
10. [10]
  赵凯,许鹏举,谷光烨.3,5-二硝基水杨酸比色法测定还原糖含量的研究[J].食品科学,2008,29(8):534.
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杜少平, 胡海艳, 甘祥武, 等. 重组毕赤酵母产β-甘露聚糖酶的高密度发酵研究[J]. 轻工学报, 2020, 35(4): 1-7. doi: 10.12187/2020.04.001

引用本文: 杜少平, 胡海艳, 甘祥武, 等. 重组毕赤酵母产β-甘露聚糖酶的高密度发酵研究[J]. 轻工学报, 2020, 35(4): 1-7. doi: 10.12187/2020.04.001

重组毕赤酵母产β-甘露聚糖酶的高密度发酵研究

作者简介:杜少平(1974-),男,广东省丰顺县人,广州市微生物研究所高级工程师,主要研究方向为微生物技术与相关检测.

广州市微生物研究所, 广东广州 510663

收稿日期: 2020-05-21

基金项目: 广州市科技计划项目（201710010154）

关键词:

摘要: 通过摇瓶实验，对重组毕赤酵母X-33/pGAPZαA-Man26A-39发酵培养基的碳源、氮源进行筛选，并在50 L发酵罐中采用间歇流加策略，对其发酵条件进行调控，以实现重组毕赤酵母高密度发酵产β-甘露聚糖酶.结果表明：葡萄糖为发酵培养基的最适碳源，其初始添加质量浓度为30 g/L；初始质量分数为6%的玉米浆作为发酵培养基的氮源较为适宜；在间歇补料发酵调控中，通过间歇流加体积分数为25%的氨水、质量分数为50%的葡萄糖溶液和质量分数为20%的玉米浆，可使发酵液pH值维持在5.0~5.5之间，且保证发酵培养基的碳源、氮源充足；发酵60 h时采取放罐措施，此时酶活力可达2 685.5 U/mL，菌体质量浓度达331.1 g/L，OD₆₀₀达302.8，实现了β-甘露聚糖酶的高效表达.