壳聚糖-聚乙烯醇固定化细胞在生物转化合成烟酸中的应用
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摘要
传统的腈水解酶游离催化剂在腈类化合物生物转化过程中存在易失活、稳定性低、重复利用率不高等特点,本研究采用复合固定方式对基因工程腈水解酶进行固定化以期提升其应用性能。通过固定化壳聚糖和聚乙烯醇(PVA)作为包埋材料,获得了具有高机械强度和酶活性的固定化细胞。初步探究了固定化聚乙烯醇和壳聚糖的浓度及固定剂的优选条件,分别为80g/L的PVA、40g/L的壳聚糖及60g/L三聚磷酸钠的饱和硼酸溶液;与游离细胞相比,固定化细胞的热稳定性和贮藏稳定性均显著提高,可增加约2倍;在525min的转化时间内,采用底物流加模式可转化合成烟酸208g/L。本文为复合固定化催化剂在腈化合物生物转化及烟酸合成中的应用奠定了坚实基础。
The free nitrilase enzymes are usually easy to be inactivated in the biotransformation reaction of nitriles; and they showed poor stability and low reuse ratio. All of those would lead to high production costs. In this study, the combination immobilization of resting cells was employed to improve the application performance of nitrilase. Chitosan and polyvinyl alcohol were selected for encapsulation experiments, and the moderate residual activity and mechanical strength were observed. The immobilization conditions include the concentration of polyvinyl alcohol and chitosan, as well as immobilization reagents, which were preliminarily optimized. Results showed that 80 g/L of polyvinyl alcohol, 40 g/L of chitosan, and saturated boric acid solution containing 60 g/L of sodium tripolyphosphate were the optimal immobilization conditions. Compared with free cells, the thermal stability and storage stability of immobilized cells were improved significantly. The 3-cyanopyridine bioconversion was carried out by feeding batch reaction with immobilized cells. Finally, 208 g/L of nicotinic acid was obtained through 525 min of conversion, and the results laid the foundation for the practical application of nicotinic acid bioproduction.
The free nitrilase enzymes are usually easy to be inactivated in the biotransformation reaction of nitriles; and they showed poor stability and low reuse ratio. All of those would lead to high production costs. In this study, the combination immobilization of resting cells was employed to improve the application performance of nitrilase. Chitosan and polyvinyl alcohol were selected for encapsulation experiments, and the moderate residual activity and mechanical strength were observed. The immobilization conditions include the concentration of polyvinyl alcohol and chitosan, as well as immobilization reagents, which were preliminarily optimized. Results showed that 80 g/L of polyvinyl alcohol, 40 g/L of chitosan, and saturated boric acid solution containing 60 g/L of sodium tripolyphosphate were the optimal immobilization conditions. Compared with free cells, the thermal stability and storage stability of immobilized cells were improved significantly. The 3-cyanopyridine bioconversion was carried out by feeding batch reaction with immobilized cells. Finally, 208 g/L of nicotinic acid was obtained through 525 min of conversion, and the results laid the foundation for the practical application of nicotinic acid bioproduction.
引文
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[12]KAUL P,BANERJEE A,BANERJEE U C.Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst[J].Biomacromolecules,2006,7:1536-1541.
[13]KUMAR S,MOHAN U,KAMBLE A L,et al.Cross-linked enzyme aggregates of recombinant Pseudomonas putida nitrilase for enantioselective nitrile hydrolysis[J].Bioresource Technology,2010,101:6856-6858.
[14]LIU Z Q,LU M M,ZHANG X H,,et al.Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid[J].International Journal of Biological Macromolecules,2018,116:563-571.
[15]ZHANG X H,LIU Z Q,XUE Y P,et al.Nitrilase-catalyzed conversion of(R,S)-mandelonitrile by immobilized recombinant Escherichia coli cells harboring nitrilase[J].Biotechnology and Applied Biochemistry,2016,63:479-489.
[16]ZHANG Z J,PAN J,LI C X,et al.Efficient production of(R)-(-)-mandelic acid using glutaraldehyde cross-linked Escherichia coli cells expressing Alcaligenes sp.nitrilase[J].Bioprocess and Biosystems Engineering,2014,37:1241-1248.
[17]ZHANG J F,LIU Z Q,ZHANG X H,et al.Biotransformation of iminodiacetonitrile to iminodiacetic acid by Alcaligenes faecalis cells immobilized in ACA-membrane liquid-core capsules[J].Chemical Papers,2014,68:53-64.
[2]BHALLA T C,KUMAR V,THAKUR N,et al.Nitrile metabolizing enzymes in biocatalysis and biotransformation[J].Applied Biochemistry and Biotechnology,2018,4:1-22.
[3]GONG J S,SHI J S,LU Z M,et al.Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis:recent insights and promises[J].Critical Reviews in Biotechnology,2017,37(1):69-81.
[4]SCHMID A,DORDICK J,S,HAUER B,et al.Industrial biocatalysis today and tomorrow[J].Nature,2001,409:258-266.
[5]MARTINKOVA L,KREN V.Biocatalytic production of mandelic acid and analogues:a review and comparison with chemical processes[J].Applied Microbiology and Biotechnology,2018,102:3893-3900.
[6]LIU Z Q,LU M M,ZHANG X H,et al.Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid[J].International Journal of Biological Macromolecules,2018,116:563-571.
[7]DIAS J,REZENDE R,LINARD V.Bioconversion of nitriles by Candida guilliermondii CCT 7207 cells immobilized in barium alginate[J].Applied Microbiology and Biotechnology,2001,56:757-761.
[8]LIU Z Q,ZHOU M,ZHANG X H,et al.Biosynthesis of iminodiacetic acid from iminodiacetonitrile by immobilized recombinant Escherichia coli harboring nitrilase[J].Journal of Molecular Microbiology and Biotechnology,2012,22:35-47.
[9]MAKSIMOVA Y G,VASILYEV D M,OVECHKINA G V,et al.Transformation of 2-and 4-cyanopyridines by free and immobilized cells of nitrile-hydrolyzing bacteria[J].Applied Biochemistry and Microbiology,2013,49:347-351.
[10]KABAIVANOVA L V,CHERNEV G E,SALVADO I M M,et al.Silica-carrageenan hybrids used for cell immobilization realizing hightemperature degradation of nitrile substrates[J].Central European Journal of Chemistry,2011,9:232-239.
[11]GONG J S,LI H,LU Z M,et al.Engineering of a fungal nitrilase for improving catalytic activity and reducing by-product formation in the absence of structural information[J].Catalysis Science&Technology,2016,6:4134-4141.
[12]KAUL P,BANERJEE A,BANERJEE U C.Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst[J].Biomacromolecules,2006,7:1536-1541.
[13]KUMAR S,MOHAN U,KAMBLE A L,et al.Cross-linked enzyme aggregates of recombinant Pseudomonas putida nitrilase for enantioselective nitrile hydrolysis[J].Bioresource Technology,2010,101:6856-6858.
[14]LIU Z Q,LU M M,ZHANG X H,,et al.Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid[J].International Journal of Biological Macromolecules,2018,116:563-571.
[15]ZHANG X H,LIU Z Q,XUE Y P,et al.Nitrilase-catalyzed conversion of(R,S)-mandelonitrile by immobilized recombinant Escherichia coli cells harboring nitrilase[J].Biotechnology and Applied Biochemistry,2016,63:479-489.
[16]ZHANG Z J,PAN J,LI C X,et al.Efficient production of(R)-(-)-mandelic acid using glutaraldehyde cross-linked Escherichia coli cells expressing Alcaligenes sp.nitrilase[J].Bioprocess and Biosystems Engineering,2014,37:1241-1248.
[17]ZHANG J F,LIU Z Q,ZHANG X H,et al.Biotransformation of iminodiacetonitrile to iminodiacetic acid by Alcaligenes faecalis cells immobilized in ACA-membrane liquid-core capsules[J].Chemical Papers,2014,68:53-64.