|
|
|
| Cloning and Heterologous Expression of the Keratinase from Thermophilic Bacillus licheniformis Y6 |
|
|
|
|
Abstract Bacillus licheniformis could secrete a kind of keratinase which can hydrolyze keratin such as feather, hair, nail and so on. In previous work, the results indicated that a thermophilic Bacillus licheniformis Y6 was able to live at elevated temperature, it also could degrade feather rapidly, and lived on feather as the only source of carbon and nitrogen. Based on these properties, this paper finally extracted a kind of thermostable keratinase with excellent characteristics was extracted. Hence, it possess the potential to manage poultry waste and can be exploited in some specific industries. aiming at cloning a keratinase gene from Bacillus licheniformis Y6, this paper designed a pair of primers, and finally obtained the gene (GenBank No. MF327392) which included a complete Open Reading Frame with the size of 1 140 bp. Aligned in NCBI, this keratianse remained to be conserved during evolution, and shared the same catalytic triad Ser-His-Asp with other serine proteases. Analyzed by some online software, this entire gene encoded 379 amino acids with pre-, pro- and mature protein regions, and its mature protein, with the molecular weight 27.27 kD and the theory isoelectric point 6.57, was classified as stable protein. After cloning the gene into vector pET-32a, The recombinant vector pET-32a kerY6 was transformed into Escherichia coli BL21(DE3) induced with 0.5 mmol/L isopropyl β-D-thiogalactoside(IPTG), then the precipitation and culture supernatant were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis which indicated that the recombinant keratinase was successfully induced with a band at 58 kD corresponding to expectation, but could not be detected in the broth. In order to detect whether it developed into inclusion body, precipitation and supernatant of bacteria lysate were subjected to SDS-PAGE, results showed no band in the supernatant lane. Subsequently, the recombinant keratinase was purified by Ni-chelating affinity chromatography. Followed by characterization, the recombinant keratinase displayed a wide range of reaction temperature 60~80 ℃, and retained 85% maximum activity for a long time which indicated an excellent thermost ability. As the time accumulation, deactivation of enzyme was observed by incubating the recombinant keratinase over 80 ℃. Incubated with a variety of pH values, the maximum activity was obtained at pH value 8.5. The results of pH values assay demonstrated that the recombinant keratinase was a typical alkaline protease. Ethylene diaminete traacetic acid (EDTA), sodium dodecyl sulfate (SDS), phenylmethanesulfonyl fluoride (PMSF), Mn2+ and Fe3+ could remarkly inhibited its activity by 76%, 49%, 59%, 34%, 75%, respectively; Ca2+ and Mg2+ prompted its activity to 118%, 125%, respectively. Organic solvent, for instance, isopropylalcohol, glycerin and dimethyl sulfoxide (DMSO) almost had no influence on its activity. In conclusion, the keratinase from thermophilic bacillus licheniformis Y6 has great potential to apply to industry.
|
|
Received: 10 July 2017
Published: 04 February 2018
|
|
|
|
| 贺光 2002. 生物信息学在蛋白质研究中的应用. 国际遗传学杂志 [J], 25: 156-158.（He G. 2002. The application of bioinformatics in protein research. International Journal of Genetics [J], 25: 156-158.）
刘艳海, 姚大伟, 刘建新,等. 2017. 1株嗜热性羽毛降解菌的分离鉴定及其酶学性质. 江苏农业科学 [J], 45: 232-237.（Liu Y H, Yao D W, Liu J X. 2017. Isolation and Characterization of a Thermotolerant Feather-degrading Bacterium and Its Enzymatic Properties. JIANGSU AGRICULTRAL SCIENCES [J], 45: 232-237.）
董旭, 辛毅 2007. 重组蛋白在大肠杆菌中可溶性表达的策略. 大连医科大学学报 [J], 29: 393-395.（Dong X, Xin Y. 2007 Strategies for soluble expression of recombinant protein in Escherichia coli J Journal of Dalian Medical University [J], 29: 393-395.）
汪世华, 王文勇, 黄益洲 2007. 丝氨酸蛋白酶研究进展. 福建农业学报 [J], 22: 453-456.（Wang S H, Wang W Y, Huang Y Z 2007 Research Progress of Serine Protease J Fujian Journal of Agricultural Sciences [J], 22: 453-456.）
BRANDELLI A, DAROIT D J, RIFFEL A 2010. Biochemical features of microbial keratinases and their production and applications. Applied Microbiology and Biotechnology [J], 85: 1735-1750.
GOUSTEROVA A, BRAIKOVA D, GOSHEV I, et al. 2005. Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis. Letters in Applied Microbiology [J], 40: 335–340.
GUPTA R, RAMNANI P 2006. Microbial keratinases and their prospective applications: an overview. Applied Microbiology and Biotechnology [J], 70: 21.
GUPTA R, SHARMA R, BEG Q K 2013. Revisiting microbial keratinases: next generation proteases for sustainable biotechnology. Critical Reviews in Biotechnology [J], 33: 216.
HU H, HE J, YU B, et al. 2013. Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes. Biotechnology Letters [J], 35: 239.
KIM J M, LIM W J, SUH H J 2001. Feather-degrading Bacillus species from poultry waste. Process Biochemistry [J], 37: 287-291.
KORNI??OWICZ-KOWALSKA T, BOHACZ J 2011. Biodegradation of keratin waste: Theory and practical aspects. Waste Management [J], 31: 1689-1701.
LIN X, KELEMEN D W, MILLER E S, et al. 1995. Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1. Applied & Environmental Microbiology [J], 61: 1469-1474.
LIN X, LEE C G, CASALE E S, et al. 1992. Purification and Characterization of a Keratinase from a Feather-Degrading Bacillus licheniformis Strain. Applied & Environmental Microbiology [J], 58: 3271-3275.
LIN X, WONG S L, MILLER E S, et al. 1997. Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis. Journal of Industrial Microbiology & Biotechnology [J], 19: 134-138.
LIU B, ZHANG J, FANG Z, et al. 2013a. Expression, Characterization and Fermentation Optimization of Keratinase from Bacillus licheniformis in Escherichia coli. Chinese Journal of Applied & Environmental Biology [J], 19: 997.
LIU B, ZHANG J, LI B, et al. 2013b. Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing. World Journal of Microbiology and Biotechnology [J], 29: 825-832.
MALVIYA H K, RAJAK R C, HASIJA S K 1992. Synthesis and regulation of extracellular keratinase in three fungi isolated from the grounds of a gelatin factory, Jabalpur, India. Mycopathologia [J], 120: 1-4.
MOLYNEUX G 1959. The Digestion of Wool by a Keratinolytic Bacillus. Australian Journal of Biological Sciences [J], 12: 274-281.
NICKERSON W J, NOVAL J J, ROBISON R S 1963. KERATINASE. I. PROPERTIES OF THE ENZYME CONJUGATED ELABORATED BY STREPTOMYCES FRADIAE. Biochimica Et Biophysica Acta [J], 77: 73-86.
NOVAL J J, NICKERSON W J 1959. Decomposition of native keratin by Streptomyces fradiae. Journal of Bacteriology [J], 77: 251-263.
PAPADOPOULOS M C 1986. The effect of enzymatic treatment on amino acid content and nitrogen characteristics of feather meal. Animal Feed Science and Technology [J], 16: 151-156.
PORRES J M, BENITO M J, LEI X G 2002. Functional expression of keratinase (kerA) gene from Bacillus licheniformis in Pichia pastoris. Biotechnology Letters [J], 24: 631-636.
SAEKI K, OZAKI K, KOBAYASHI T, et al. 2007. Detergent alkaline proteases: enzymatic properties, genes, and crystal structures. Journal of Bioscience & Bioengineering [J], 103: 501-508.
SARKER P K, TALUKDAR S A, DEB P, et al. 2013. Optimization and partial characterization of culture conditions for the production of alkaline protease from Bacillus licheniformis P003. SpringerPlus [J], 2: 506.
SUNTORNSUK W, TONGJUN J, ONNIM P, et al. 2005. Purification and Characterisation of Keratinase from a Thermotolerant Feather-degrading Bacterium. World Journal of Microbiology and Biotechnology [J], 21: 1111-1117.
TIWARY E, GUPTA R 2010. Extracellular expression of keratinase from Bacillus licheniformis ER-15 in Escherichia coli. Journal of Agricultural & Food Chemistry [J], 58: 8380-8385.
|
|
|
|
