Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (11): 70-77    DOI: 10.13523/j.cb.20191108
技术与方法     
DA-F127水凝胶包埋固定化含腈水合酶细胞
张颖,王莹,杨立荣,吴坚平()
浙江大学化学工程与生物工程学院生物工程研究所 杭州 310027
DA-F127 Hydrogel Embedded Immobilized the Nitrile Hydratase-Containing Cells
ZHANG Ying,WANG Ying,YANG Li-rong,WU Jian-ping()
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
 全文: PDF(757 KB)   HTML
摘要：

腈水合酶是一类可催化腈类化合物转化生成相应酰胺类物质的酶。含腈水合酶的游离细胞催化水合反应存在酶容易失活、细胞无法重复利用、分离纯化困难等缺陷,细胞固定化技术可有效解决这些问题。为探索合适的固定化方法,以含腈水合酶的重组E.coli细胞为研究对象,以固定化酶活回收率和批次反应情况为评价指标,筛选比较了几种常用的包埋固定化方法。结果表明,DA-F127水凝胶包埋固定化细胞不仅具有较高的酶活回收率,而且稳定性也很好。对该方法进行了固定化条件和操作稳定性优化,当DA-F127浓度为15%、UV光源距离为20cm、光照时间为6min、菌体含量为20mg/g 固定化细胞时,酶活回收率为89.74%,并且可以催化9批次150g/L的3-氰基吡啶完成转化,第九批次转化率可达98.26%。与游离细胞催化过程相比,单位质量游离细胞的烟酰胺产量提高了12倍,具有良好的工业应用前景。
关键词： DA-F127;  腈水合酶;  细胞固定化;  烟酰胺    
Abstract:

Nitrile hydratase are enzymes that catalyze the conversion of nitriles to their corresponding amides. There are many problems in the catalytic process of free cells containing nitrile hydratases, such as low cell utilization rate and the high cost of later separation, which can be solved effectively by immobilization of cells. Several commonly embedding immobilization methods by evaluating the reaction batches and the activity recovery rate of immobilized enzymes with nitrile hydratase containing recombinant E.coli cells as the research object to find a suitable immobilization method were compared.The results showed that DA-F127 was the most suitable material for immobilization of recombinant E. coli cells. When the concentration of DA-F127 was 15%, the UV irradiation distance was 20cm, the irradiation time was 6min, and the cell content was 20mg/g carrier, the recovery rate of enzyme activity is 89.74%, and it could be reused up for 9 batches to covert 150g/L 3-cyanopyridine. The conversion rate of the ninth batch could reach to 98.26%. The nicotinamide yield was increased by 12 times compared with the same amount of free cell catalysis.
Key words: DA-F127    Nitrile hydratase    Immobilization cell    Nicotinamide
收稿日期: 2019-03-12 出版日期: 2019-12-17
ZTFLH:  Q814  
通讯作者: 吴坚平     E-mail: wjp@zju.edu.cn
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
张颖
王莹
杨立荣
吴坚平

引用本文:

张颖,王莹,杨立荣,吴坚平. DA-F127水凝胶包埋固定化含腈水合酶细胞[J]. 中国生物工程杂志, 2019, 39(11): 70-77.

ZHANG Ying,WANG Ying,YANG Li-rong,WU Jian-ping. DA-F127 Hydrogel Embedded Immobilized the Nitrile Hydratase-Containing Cells. China Biotechnology, 2019, 39(11): 70-77.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191108    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I11/70

Immobilization
method		 Shape Strength Enzyme recovery
(%)
Calcium alginate Bead ++++ 48.08±2.16
Polyacryamide Cube ++++ 54.93±1.83
DA-F127 Cube ++++ 61.28±2.64
Carrageenan Cube ++ 47.43±2.27
Agar Cube +++ 34.76±1.86
表1  不同固定化方法的比较
图1  不同固定化方法批次反应情况
DA-F127
concentration
(%)		 UV
irradiation time
(min)		 Cell embedding
amount(mg)①		 Enzyme
recovery
(%)
10 10 12 62.32 ± 3.71
15 10 12 57.86 ± 2.47
20 10 12 56.61 ± 3.34
25 10 12 44.67 ± 1.73
表2  DA-F127浓度对细胞固定化回收率的影响
图2  不同DA-F127浓度对反应批次的影响
图3  UV照射距离对固定化细胞酶活回收的影响
UV irradiation
time(min)		 DA-F127
concentration
(%)		 Cell embedding
amount (mg)①		 Enzyme
recovery(%)
4 15 12 80.14 ± 2.42
6 15 12 86.28 ± 3.57
8 15 12 66.68 ± 1.66
10 15 12 53.31 ± 3.13
12 15 12 20.56 ± 2.85
表3  UV照射时间对细胞固定化回收率的影响
图4  UV光照时间对反应批次的影响
Cell embedding
amount (mg)①		 DA-F127
concentration
(%)		 UV irradiation
time(min)		 Enzyme
recovery
(%)
4 15 6 74.13 ± 2.29
8 15 6 76.08 ± 3.57
12 15 6 83.16 ± 2.64
16 15 6 84.30 ± 1.76
20 15 6 89.74 ± 1.58
24 15 6 82.92 ± 1.47
28 15 6 80.57 ± 2.56
表4  细胞添加量对细胞固定化回收率的影响
图5  pH对固定化细胞反应的影响
  
图7  温度对固定化细胞反应的影响
  
图9  DA-F127固定化细胞催化3-氰基吡啶批次反应
[1] Chen Y Z, Jiao S, Wang M M , et al. A novel molecular chaperone GroEL2 from Rhodococcus ruber and its fusion chimera with nitrile hydratase for co-enhanced activity and stability. Chemical Engineering Science, 2018,192(1):235-243.
doi: 10.1016/j.ces.2018.07.045
[2] Shaw N M, Robins K T, Kiener A . Lonza: 20 years of biotransformations. Adv Synth Catal, 2003,345(4) : 425-435.
doi: 10.1002/adsc.200390049
[3] Singh R, Devi N, Chand D , et al. Bench scale production of butyramide using free and immobilized cells of Bacillus sp. APB-6. Bioprocess Biosyst Eng, 2018,41(8) : 1225-1232.
doi: 10.1007/s00449-018-1951-y pmid: 29748858
[4] 尹灵富 . 生物催化技术生产烟酰胺的研究. 杭州: 浙江工业大学, 2004.
Yin L F . Production of nicotinamide by biological catalyst. Hangzhou: Zhejiang University of Technology, 2004.
[5] Zigova J, Robins K, Bartek J. Polyacrylamide beads containing encapsulated cells: America, US20070822928. 2007-11-08[2019-4-10]. .
[6] 刘善和, 钱前, 梁锡臣 , 等. 一种利用固定化细胞将烟腈转化为烟酰胺的方法: 中国, CN201510115815.9. 2015-07-08[2019-4-10]. .
Liu S H, Qian Q, Liang X C. Method of converting nicotinonitrile into nicotinamide with immobilized cells: China, CN201510115815.9. 2015-07-08[2019-4-10]. .
[7] Singh R, Pandey D, Devi N , et al. Bench scale production of butyramide using free and immobilized cells of Bacillus sp. APB-6. Bioprocess Biosyst Eng, 2018,41(8):1225-1232.
doi: 10.1007/s00449-018-1951-y pmid: 29748858
[8] Kubac D, Cejkova A, Masak J , et al. Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats. Journal of Molecular Catalysis B Enzymatic, 2006,39(1-4) : 59-61.
doi: 10.1016/j.molcatb.2006.01.004
[9] Maksimova Y G, Nikulin S M, Osovetskii B M , et al. Heterogeneous biocatalyst for nitrile and amide transformation based on cells of nitrile-hydrolyzing bacteria and multiwalled carbon nanotubes. Appl Biochem Microbiol , 2017,53(5):506-512.
doi: 10.1134/S0003683817050118
[10] Lee S Y, Tae G . Formulation and in vitro characterization of an in situ gelable photo-polymerizable Pluronic hydrogel suitable for injection. J Control Release, 2007,119(3) : 313-319.
doi: 10.1016/j.jconrel.2007.03.007 pmid: 17490772
[11] Rodrigues R O, Baldi G, Doumett S , et al. Multifunctional graphene-based magnetic nanocarriers for combined hyperthermia and dual stimuli-responsive drug delivery. Materials Science and Engineering, 2018,93(1):206-217.
doi: 10.1016/j.msec.2018.07.060 pmid: 30274052
[12] Pei X L, Zhang H Y, Meng L J , et al. Effciernt cloning and expression of a thermostable nitrile hydratase in Eschericha coli using an auto-induction fed-batch strategy. Process Biochemistry, 2013,48(1):1921-1927.
doi: 10.1016/j.procbio.2013.09.004
[13] Yang Z F, Pei X L, Xu G , et al. N-terminal engineering of overlapping genes in the nitrile hydratase gene cluster improved its activity. Enzyme & Microbial Technology, 2018,117(1):9-14.
doi: 10.3760/cma.j.issn.0254-6450.2019.11.016 pmid: 31838816
[14] Duque S M M, Castro I J L, Flores D M . Evaluation of antioxidant and nutritional properties of sago and its utilization for direct lactic acid production using immobilized Enterococcus faecium DMF78. International Food Research Journal, 2018,25(1) : 83-91.
[15] Li Y M, Gao J Q, Pei X Z , et al. Production of l-alanyl-l-glutamine by immobilized Pichia pastoris GS115 expressing -amino acid ester acyltransferase. Microbial Cell Factories, 2019,18(1):18-27.
doi: 10.1186/s12934-019-1069-1 pmid: 30696431
[16] Lee S, Tae G, Kim Y H . Thermal gellation and photo-polymerization of di-acrylated Pluronic F 127. Journal of Biomaterials Science Polymer Edition, 2007,18(10):1335-1353.
doi: 10.1163/156856207782177855 pmid: 17939890
[17] Chun K W, Lee J B, Kim S H , et al. Controlled release of plasmid DNA from photo-cross-linked pluronic hydrogels. Biomaterials, 2005,26(16) : 3319-3326.
doi: 10.1016/j.biomaterials.2004.07.055
[18] Raj J, Sharma N N, Prasad S , et al. Acrylamide synjournal using agar entrapped cells of Rhodococcus rhodochrous PA-34 in a partitioned fed batch reactor. J Ind Microbiol Biotechnol, 2008,35(1):35-40.
doi: 10.1007/s10295-007-0263-z pmid: 17994258
[19] 邹树平, 颜海蔚, 胡忠策 , 等. 固定化重组大肠杆菌细胞催化合成(R)一环氧氯丙烷. 现代化工, 2013,33(7):55-59.
Zou S P, Yan H W, Hu Z C , et al. Synjournal of (R)-epichlorohydrin catalyzed by immobilized recombinant Escherichia coli cells. Modern Chemical Industry, 2013,33(7):55-59.
[20] 裴晓林 . 腈水合酶基因资源开发及其重组表达体系在制备烟酰胺中的应用. 杭州: 浙江大学, 2013.
Pei X L . Discovery of nitrile hydratase genes and their recombinant expression for the production of nicotinamide. Hangzhou: Zhejiang University, 2013.
[1] 王世伟, 王敏, 王卿惠. Rhodococcus ruber CGMCC3090腈水合酶纯化、酶学性质及结晶研究[J]. 中国生物工程杂志, 2017, 37(10): 42-52.
[2] 王丽燕, 王煜, 吴坚平, 徐刚, 杨立荣. 腈水合酶NHaseK在大肠杆菌中的功能表达[J]. 中国生物工程杂志, 2016, 36(12): 42-48.
[3] 张丽铃, 王筱兰. 耐底物腈水合酶融合子的产酶条件优化[J]. 中国生物工程杂志, 2012, 32(08): 75-80.
[4] 王世伟, 王敏. 腈类物降解菌多样性和产腈水合酶研究进展[J]. 中国生物工程杂志, 2011, 31(9): 117-123.
[5] 高慧慧, 陈晟, 吴敬, 陈坚. 用于腈纶表面改性的Corynebacterium nitrilophilus 腈水合酶的摇瓶发酵优化[J]. 中国生物工程杂志, 2011, 31(8): 54-60.
[6] 申渝, 白凤武. 串联填充管式反应系统中高浓度乙醇连续发酵[J]. 中国生物工程杂志, 2011, 31(03): 66-70.
[7] 史悦, 于慧敏, 田卓玲, 沈忠耀. 产腈水合酶重组大肠杆菌的质粒稳定性研究[J]. 中国生物工程杂志, 2005, 25(8): 70-75.
[8] 史悦, 于慧敏, 孙旭东, 田卓玲, 沈忠耀. 腈水合酶基因克隆与调控表达的研究进展[J]. 中国生物工程杂志, 2004, 24(7): 34-39.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会