富含角蛋白的羽毛废弃物是一种潜在的优质蛋白资源,但缺乏有效的回收方式。角蛋白酶可催化降解羽毛废弃物产生氨基酸和多肽等高价值水解产物,被认为是一种极具潜力的废弃角蛋白回收方式。然而,角蛋白酶较低的二硫键还原活性却成为限制羽毛水解效率的瓶颈问题。该研究通过二硫键还原酶破坏羽毛角蛋白的二硫键,与角蛋白酶协同作用加速羽毛废弃物的降解。首先,在Bacillus subtilis WB600中异源表达来源于Bacillus subtilis 168的4种二硫键还原酶,并对其酶学性质进行表征;进而通过优化二硫键还原酶MsrA的信号肽,提高了其胞外表达量;最后,探究了二硫键还原酶与角蛋白酶KerZ1协同降解羽毛的效果,分析了水解液中氨基酸的种类和含量。二硫键还原酶AhpC、DLD、MsrA和TrxR成功实现了胞外表达,比酶活力分别为0.53、1.69、16.7、2.06 U/mg;MsrA携带信号肽YxkH时胞外二硫键还原酶活性提高了4.2倍;MsrA+KerZ1降解羽毛的水解液中可溶性蛋白含量是KerZ1单独降解时的3倍;优化后的MsrA+KerZ1降解100 g/L的鸭毛和鸡毛,水解液中氨基酸含量分别为7 472.33、5 589.67 mg/L;另外,使用羽毛水解液浇灌的玉米植株拥有更粗壮的茎和更发达的根系,表明水解液中的营养物质可显著促进植物生长。二硫键还原酶显著促进了角蛋白酶降解羽毛的效率,其与角蛋白酶协同作用的降解体系为羽毛废弃物的高效回收奠定了工作基础。
Feather waste rich in keratin is a potential high-quality protein resource, but effective recycling methods are lacking. Keratinase is considered to be a promising method for recycling waste keratin because of its ability to hydrolyze feather waste to produce high-value hydrolysates such as amino acids and peptides. However, the low disulfide reduction activity restricted the hydrolysis efficiency of keratinase on feathers. In this study, disulfide reductase was introduced to reduce the disulfide bond of feather keratin, and the degradation process of feather waste was accelerated by the synergistic action of highly active disulfide reductase and keratinase. First, four disulfide reductases derived from Bacillus subtilis 168 were heterologously expressed in Bacillus subtilis WB600, and their enzymatic properties were characterized. Furthermore, the extracellular expression of the disulfide reductase MsrA was increased by optimizing the signal peptide. Finally, the system for the synergistic degradation of feathers by disulfide reductase and keratinase KerZ1 was optimized, and the soluble proteins and amino acids in the hydrolysate were analyzed. The disulfide reductases AhpC, DLD, MsrA and TrxR were successfully extracellularly expressed with specific activities of 0.53, 1.69, 16.7, and 2.06 U/mg, respectively. When MsrA carried the signal peptide YxkH, the activity of extracellular disulfide reductase increased by 4.2 times. The soluble protein content in the hydrolysate obtained from the degradation of feathers by MsrA+KerZ1 was 3 times than that of KerZ1 alone. The optimized MsrA+KerZ1 degraded 100 g/L duck feather and chicken feather to produce 7 472.33 and 5 589.67 mg/L amino acids in the hydrolysate. In addition, corn plants watered with feather hydrolysate had thicker stems and more developed root systems, indicating that the nutrients in the hydrolysate could significantly promote plant growth. In conclusion, disulfide reductase can accelerate the efficiency of keratinase to degrade feathers, and the synergistic degradation system with keratinase lays a foundation for the efficient recovery of feather waste.
[1] LI Q X.Progress in microbial degradation of feather waste[J].Frontiers in Microbiology, 2019, 10:2717.
[2] WANG J, HAO S L, LUO T T, et al.Development of feather keratin nanoparticles and investigation of their hemostatic efficacy[J].Materials Science and Engineering:C, 2016, 68:768-773.
[3] WANG B, YANG W, MCKITTRICK J, et al.Keratin:Structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration[J].Progress in Materials Science, 2016, 76:229-318.
[4] PENG Z, MAO X Z, ZHANG J, et al.Effective biodegradation of chicken feather waste by co-cultivation of keratinase producing strains[J].Microbial Cell Factories, 2019, 18(1):84.
[5] CALVARESI M, ECKHART L, ALIBARDI L.The molecular organization of the beta-sheet region in corneous beta-proteins (beta-keratins) of sauropsids explains its stability and polymerization into filaments[J].Journal of Structural Biology, 2016, 194(3):282-291.
[6] PENG Z, ZHANG J, DU G C, et al.Keratin waste recycling based on microbial degradation:Mechanisms and prospects[J].ACS Sustainable Chemistry & Engineering, 2019, 7(11):9 727-9 736.
[7] CALLEGARO K, BRANDELLI A, DAROIT D J.Beyond plucking:Feathers bioprocessing into valuable protein hydrolysates[J].Waste Management, 2019, 95:399-415.
[8] QIU J W, WILKENS C, BARRETT K, et al.Microbial enzymes catalyzing keratin degradation:Classification, structure, function[J].Biotechnology Advances, 2020, 44:107607.
[9] PENG Z, XU P, SONG Y, et al.Cysteine-mediated cyclic metabolism drives the microbial degradation of keratin[J].ACS Sustainable Chemistry & Engineering, 2021, 9(29):9 861-9 870.
[10] LIN H H, YIN L J, JIANG S T.Cloning, expression, and purification of Pseudomonas aeruginosa keratinase in Escherichia coli AD494(DE3) pLysS expression system[J].Journal of Agricultural and Food Chemistry, 2009, 57(9):3 506-3 511.
[11] JAOUADI N Z, REKIK H, BADIS A, et al.Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities[J].PLoS One, 2013, 8(10):e76722.
[12] 齐志国, 张铁鹰, 董杰丽, 等.角蛋白降解菌分离、鉴定及其降解机制研究[J].饲料工业, 2012, 33(17):19-24.
QI Z G, ZHANG T Y, DONG J L, et al.Research on isolation and identification of keratin-degrading bacterias and its degradation mechanism[J].Feed Industry, 2012, 33(17):19-24.
[13] 王德山. 地衣芽孢杆菌CP-16降解羽毛角蛋白相关蛋白酶的分离、表达和相互关系[D].北京:中国农业科学院, 2014.
WANG D S.Purification, expression and relationship of some proteases related to feather keratin degradation from B.licheniformis CP-16[D].Beijing:Chinese Academy of Agricultural Sciences, 2014.
[14] 邹林源, 张铁鹰, 齐志国, 等.嗜麦芽窄食单胞菌胞内具二硫键还原活性酶蛋白的研究[J].饲料工业, 2015, 36(13):30-34.
ZOU L Y, ZHANG T Y, QI Z G, et al.Research on enzyme with disulfide reductase in Stenotrophomonas maltophilia[J].Feed Industry, 2015, 36(13):30-34.
[15] 廖朝勇. 地衣芽孢杆菌CP-16降解羽毛关键酶高效表达研究[D].北京:中国农业科学院, 2019.
LIAO C Y.High-efficiency expression of key hydrolysis enzymes of feathers from Bacillus licheniformis CP-16[D].Beijing:Chinese Academy of Agricultural Sciences, 2019.
[16] PRAKASH D, WALTERS K A, MARTINIE R J, et al.Toward a mechanistic and physiological understanding of a ferredoxin:Disulfide reductase from the domains archaea and bacteria[J].The Journal of Biological Chemistry, 2018, 293(24):9 198-9 209.
[17] PENG Z, MAO X Z, ZHANG J, et al.Biotransformation of keratin waste to amino acids and active peptides based on cell-free catalysis[J].Biotechnology for Biofuels, 2020, 13:61.
[18] 冒鑫哲. Bacillus licheniformis BBE11-1角蛋白酶底物特异性改造及发酵优化[D].无锡:江南大学, 2020.
MAO X Z.Substrate specificity and fermentation optimization of Bacillus licheniformis BBE11-1 keratinase[D].Wuxi:Jiangnan University, 2020.
[19] 何周凤. 枯草芽孢杆菌BS8对羽毛角蛋白的降解机制初探[D].雅安:四川农业大学, 2017.
HE Z F.Exploration of the degradation mechanism of feather keratin by Bacillus subtilis 8[D].Ya'an:Sichuan Agricultural University, 2017.
[20] ARMENTEROS A J J, TSIRIGOS K D, SØNDERBY C K, et al.SignalP 5.0 improves signal peptide predictions using deep neural networks[J].Nature Biotechnology, 2019, 37(4):420-423.
[21] LAU W, HOAD G R, JIN V, et al.PSORTdb 4.0:Expanded and redesigned bacterial and archaeal protein subcellular localization database incorporating new secondary localizations[J].Nucleic Acids Research, 2021, 49(D1):D803-D808.
[22] TSIRIGOTAKI A, GEYTER J D, SˇOSˇTARIC N, et al.Protein export through the bacterial Sec pathway[J].Nature Reviews Microbiology, 2017, 15(1):21-36.
[23] MÜLLER M, KLÖSGEN R B.The Tat pathway in bacteria and chloroplasts[J].Molecular Membrane Biology, 2005, 22(1-2):113-121.
[24] 张健, 李燕婷, 袁亮, 等.氨基酸发酵尾液可促进樱桃番茄对水溶肥料氮素的吸收利用[J].植物营养与肥料学报, 2018, 24(1):114-121.
ZHANG J, LI Y T, YUAN L, et al.Tail liquid from amino acid fermentation could improve the uptake and utilization of water soluble fertilizer nitrogen by cherry tomato[J].Journal of Plant Nutrition and Fertilizers, 2018, 24(1):114-121.
