Diacetylchitobiose deacetylase (Dac) hydrolyzes N-acetylglucosamine (GlcNAc) into the biologically active substance glucosamine (GlcN), and efficient production of Dac is the key to realizing this catalytic process.Using a Dac-expressing Bacillus subtilis BS-P43-NMK-T0 as a starting strain, 12 signal peptides were first screened, among which, the signal peptide NprB had the strongest ability to direct Dac secretion, and after optimizing its functional region, the recombinant strain BS-P43-NMK-T4-N1 had an extracellular enzyme activity reached 69.83 U/mL.Subsequently, three inducible promoters were screened, among which the promoter Pgrac100 induced the best expression effect, and the extracellular enzyme activity of the recombinant strain BS-NMK-T4-N1-Pgrac was increased to 88.92 U/mL after optimizing the induction conditions.When the fermentation conditions were 4% inoculum, 20 g/L glucose (carbon source) and 10 g/L nutrient broth (nitrogen source), the extracellular Dac enzyme activity reached 127.59 U/mL, and the addition of glucose in the shake flasks could further increase the Dac activity to 144.25 U/mL.Finally, when the amplified culture was carried out in the 3-L fermenter, the extracellular Dac activity was 319.63 U/mL, which was 7.09 times higher than the extracellular Dac activity of the starting strain.This study provides basic research for the industrial production of GlcN by enzymatic conversion method.
[1] LIU L, LIU Y F, SHIN H D, et al.Developing Bacillus spp.as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology[J].Applied Microbiology and Biotechnology, 2013, 97(14):6113-6127.
[2] PANGESTUTI R, KIM S K.Biological activities and health benefit effects of natural pigments derived from marine algae[J].Journal of Functional Foods, 2011, 3(4):255-266.
[3] GRZANNA M W, AU R Y, AU A Y, et al.Avocado/soybean unsaponifiables, glucosamine and chondroitin sulfate combination inhibits proinflammatory COX-2 expression and prostaglandin E2 production in tendon-derived cells[J].Journal of Medicinal Food, 2020,23(2):139-146.
[4] LIU X B, MA P S.Recent development of glucosamine-6-phosphate derivatives as potential antibacterial agents[J].Chemistry Select, 2020,5(5):1718-1727.
[5] SHEN K T, CHEN M H, CHAN H Y, et al.Inhibitory effects of chitooligosaccharides on tumor growth and metastasis[J].Food and Chemical Toxicology, 2009, 47(8):1864-1871.
[6] ZHANG H K, LU Y T, WANG Y H, et al.D-Glucosamine production from chitosan hydrolyzation over a glucose-derived solid acid catalyst[J].RSC Advances, 2018,8(10):5608-5613.
[7] LIU H Y, LI N, LIU X B, et al.Poly(N-acryloyl-glucosamine-co-methylenebisacrylamide)-based hydrophilic magnetic nanoparticles for the extraction of aminoglycosides in meat samples[J].Journal of Chromatography A, 2020,1609:460517.
[8] DHUNGEL P, BHATTACHERJEE A, HRYNETS Y, et al.The effect of amino acids on non-enzymatic browning of glucosamine:Generation of butterscotch aromatic and bioactive health compounds without detectable levels of neo-formed alkylimidazoles[J].Food Chemistry, 2020,308:125612.
[9] 陈志蓉, 常思思, 董银卯, 等.氨基葡萄糖改性阿魏酸的溶解性和美白功效研究[J].中国药师, 2010, 13(12):1715-1718.
CHEN Z R, CHANG S S, DONG Y M, et al.Research of modified solubility and whitening efficacy of ferulic acid by glucosamine[J].China Pharmacist, 2010, 13(12):1715-1718.
[10] CHEN X, LIU L, LI J H, et al.Improved glucosamine and N-acetylglucosamine production by an engineered Escherichia coli via step-wise regulation of dissolved oxygen level[J].Bioresource Technology, 2012, 110:534-538.
[11] LIU L, LIU Y F, SHIN H D, et al.Microbial production of glucosamine and N-acetylglucosamine:Advances and perspectives[J].Applied Microbiology and Biotechnology, 2013, 97(14):6149-6158.
[12] JIANG Z, LV X Q, LIU Y F, et al.Biocatalytic production of glucosamine from N-acetylglucosamine by diacetylchitobiose deacetylase[J].Journal of Microbiology and Biotechnology, 2018, 28(11):1850-1858.
[13] 乔岩, 王爱勤.N-乙酰氨基葡萄糖合成方法的改进[J].化学试剂, 2002, 24(3):162-190.
QIAO Y, WANG A Q.A convenient method for the synthesis of N-acetyl-D-glucosamine[J].Chemical Reagents, 2002, 24(3):162-190.
[14] 姜竹. 几丁二糖脱乙酰酶的克隆表达与生物转化合成氨基葡萄糖的研究[D].无锡:江南大学, 2019.
JIANG Z.Cloning and expression of chitosan deacetylase and its biotransformation to glucosamine[D].Wuxi:Jiangnan University, 2019.
[15] TANAKA T, FUKUI T, ATOMI H, et al.Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1[J].Journal of Bacteriology, 2003, 185(17):5175-5181.
[16] NAKAMURA T, YONEZAWA Y, TSUCHIYA Y, et al.Substrate recognition of N,N′-diacetylchitobiose deacetylase from Pyrococcus horikoshii[J].Journal of Structural Biology, 2016, 195(3):286-293.
[17] 刘波, 倪金凤, 申玉龙.极端嗜热古菌 Pyrococcus horikoshii 几丁二糖脱乙酰酶的克隆、表达及性质研究[J].微生物学报, 2006, 46(2):255-258.
LIU B, NI J F, SHEN Y L.Cloning, expression and biochemical characterization of a novel diacetylchitobiose deacetylase from the hyperthermophilic archaeon Pyrococcus horikoshii[J].Acta Microbiologica Sinice, 2006, 46(2):255-258.
[18] SONI T, ZHUANG M C, KUMAR M, et al..Multifaceted production strategies and applications of glucosamine:A comprehensive review[J].Critical Reviews in Biotechnology,2023,43(1):100-120.
[19] HUANG Z Y, MAO X Z, LV X Q, et al.Engineering diacetylchitobiose deacetylase from Pyrococcus horikoshii towards an efficient glucosamine production[J].Bioresource Technology, 2021,334:125241.
[20] MAO X Z, HUANG Z Y, SUN G Y, et al.High level production of diacetylchitobiose deacetylase by refactoring genetic elements and cellular metabolism[J].Bioresource Technology,2021,341:125836.
[21] YANG S, KANG Z, CAO W L, et al.Construction of a novel, stable, food-grade expression system by engineering the endogenous toxin-antitoxin system in Bacillus subtilis[J].Journal of Biotechnology, 2016, 219:40-47.
[22] SUN G Y, WU Y K, HUANG Z Y, et al.Directed evolution of diacetylchitobiose deacetylase via high-throughput droplet sorting with a novel, bacteria-based biosensor[J].Biosensors and Bioelectronics, 2023, 219:114818.
[23] LIU Y H, SHI C S, LI D K, et al.Engineering a highly efficient expression system to produce BcaPRO protease in Bacillus subtilis by an optimized promoter and signal peptide[J].International Journal of Biological Macromolecules, 2019,138:903-911.
[24] 田荣臻, 刘延峰, 李江华, 等.典型模式微生物基因表达精细调控工具的研究进展[J].合成生物学, 2020, 1(4):454-469.
TIAN R Z, LIU Y F, LI J H, et al.Progress in the regulatory tools of gene expression for model microorganisms[J].Synthetic Biology, 2020, 1(4):454-469.
[25] 周勇. 嗜麦芽糖寡养单胞菌脂肪酶 Lips 在枯草芽孢杆菌中的高效分泌表达[D] 杭州:浙江大学, 2015.
ZHOU Y.High level secretion expression of a lipase from stenotrophomonas maltophilia in Bacillus subtilis[D] Hangzhou:Zhejiang University, 2015.
