Expression of Cellobiohydrolase Gene (cbh) from Cattle (Bos taurus domestica) Rumen Microbes in Lactic Acid Bacteria (Lactococcus lactis)
SUN Kang-Yong-Jie1, YANG Yu-Ze2, WEI Ya-Qin3, WAN Xue-Rui1, ZHANG Zhao1, MA Chun-Juan1, CAO Lei1, ZOU Ai-Ai1, WANG Chuan1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Beijing Animal Husbandry Station, Beijing 100107, China; 3 Key Laboratory of Microbial Resources Exploitation and Application of Gansu Province/Center of Anaerobic Microbes, Institute of Biology, Gansu Academy of Sciences, Lanzhou 730000, China
Abstract：Corn (Zea mays) stalk is the main source of feed for cattle, sheep and other livestock, and contains a large amount of cellulose. Exogenous cellobiohydrolase (CBH) can promote the degradation of cellulose during silage. As probiotics, lactic acid bacteria (Lactococcus lactis) can improve the quality of silage. In present study, mixed DNA of the cattle (Bos taurus domestica) rumen microbes was obtained by crude extraction, and the cbh gene was amplified and cloned into food-grade vector pMG36e, which was used to construct a secreted expression vector pMG36e::CBH. The expression vector was transformed into L. lactis NZ9000, and the corresponding enzyme activity was determined by 3,5-dinitrosalicylic acid (DNS) method, and the enzymatic properties of the recombinant enzyme were analyzed. The results showed that a sequence with 1 600 bp was cloned from cattle rumen, the molecular weight of the recombinant enzyme was about 56 kD, the filter paper enzyme activity was (2.218 1±0.834 3) U/mL, and the exoglucanase activity was (10.499 2±1.113 5) U/mL. The recombinant enzyme had the highest specificity for regenerated amorphous cellulose (RAC), followed by microcrystalline cellulose, lower specificity for filter paper and absorbent cotton, but almost no activity for sodium carboxymethyl cellulose (CMC-Na); the optimal pH and temperature were pH 6 and 60 ℃; 1 mmol/L Mn2+, Zn2+, Fe2+, Cu2+, Co2+, and 1% Tween-20 promoted the enzyme activity of recombinant CBH, while 1 mmol/L Ba2+ and Hg2+ had almost no effect on the enzyme activity, but 1 mmol/L K+, Mg2+, and EDTA inhibited the enzyme activity, 5 mmol/L Mn2+, Fe2+, Ca2+, Cu2+, and Co2+ promoted the enzyme activity, 10% Tween-20 and 5 mmol/L Zn2+, Ba2+, Hg2+, K+, Mg2+ and EDTA inhibited the enzyme activity. The recombinant lactobacillus with cbh gene from cattle rumen microbes constructed in this study is conducive to promoting the enzymatic hydrolysis of straw during silage and improving the nutritional value of silage.
[1] 程抒劼, 郑兰娟, 林俊芳, 等. 2009. 纤维素酶活力测定研究进展[J]. 食品工业科技, 30(07): 334-336, 342. (Cheng S J, Zheng L J, Lin J F, et al.2009. Research progress of determination of cellulase activity[J]. Science and Technology of Food Industry, 30(07): 334-336, 342.) [2] 刘原子, 王艳, 万学瑞, 等. 2017. 多粘类芽孢杆菌纤维素酶基因bglA, bglB和EG在乳酸菌中的分泌表达[J]. 草原与草坪, 37(01): 8-13. (LIU Y Z, Wang Y, Wan X R, et al.2017. Secretory expression of Bacillus polymyxa cellulase gene bglA, bglB and EG in lactic acid bacteria[J]. Grassland and Turf, 37(01): 8-13.) [3] 王艳, 马亚茹, 万学瑞, 等. 2017. 多粘类芽孢杆菌β-葡萄糖苷酶bglA、bglB和bgl基因在大肠杆菌中的表达[J]. 草业学报, 26(05): 189-196. (Wang Y, Ma Y R, Wan X R, et al.2017. Expression of β-glucosidase genes bglA, bglB, and bgl from Bacillus polymyxa in Escherichia coli[J]. Acta Prataculturae Sinica, 26(05): 189-196.) [4] 杨海峰, 魏亚琴, 孙康永杰, 等. 2020. 烟曲霉纤维二糖水解酶cbh基因在大肠杆菌中的表达[J] .草业科学, 37(12): 2457-2462. (Yang H F, Wei Y Q, Sun K Y J, et al.2020. Expression of the cellobiohydrolase gene of Aspergillus fumigatus in E. coli[J]. Pratacultural Science, 37(12): 2457-2462.) [5] 杨瑞红, 王加启, 罗淑萍, 等. 2005. 奶牛瘤胃胃液微生物总DNA的提取和纯化[J]. 新疆农业大学学报, (02): 39-42. (Yang R H ,Wang J Q, Luo S P, et al. 2005. Extration and purification of DNA from evironmental rumen samples[J]. Journal of Xinjiang Agricultural University, (02): 39-42.) [6] 袁茂翼, 叶发银, 雷琳, 等. 2017. 纤维二糖水解酶的研究进展[J] .食品与发酵工业, 43(10): 248-255. (Yuan M Y, Ye F Y, Lei L, et al.2017. Research progress of cellobiohydrolases[J]. Food and Fermentation Industries, 43(10): 248-255.) [7] Abrão F O, Duarte ER, Pessoa MS, et al.2017. Notable fibrolytic enzyme production by Aspergillus spp. isolates from the gastrointestinal tract of beef cattle fed in lignified pastures[J]. PLOS ONE, 12(8): e0183628. [8] Davison S A, Keller N T, van Zyl W H, et al.2019. Improved cellulase expression in diploid yeast strains enhanced consolidated bioprocessing of pretreated corn residues[J]. Enzyme and Microbial Technology, 131: 109382. [9] Enari T M, Niku-Paavola M L.1987. Enzymatic hydrolysis of cellulose: Is the current theory of the mechanisms of hydrolysis valid[J]. Critical Reviews in Biotechnology, 5(1): 67-87. [10] Garvey M, Klose H, Fischer R,et al.2013. Cellulases for biomass degradation: Comparing recombinant cellulase expression platforms[J]. Trends in Biotechnology, 31(10): 581-593. [11] Grimson M J, Haigler C H, Blanton R L.1996. Cellulose microfibrils, cell motility, and plasma membrane protein organization change in parallel during culmination in Dictyostelium discoideum[J]. Journal of Cell Science, 109(Pt 13): 3079-3087. [12] Haq I U, Tahir S F, Aftab M N, et al.2018. Purification and characterization of a thermostable cellobiohydrolase from Thermotoga petrophila[J]. Protein and Peptide Letters, 25(11): 1003-1014. [13] Hassan M, Ding W, Umar M, et al.2017. Methane enhancement and asynchronism minimization through co-digestion of goose manure and NaOH solubilized corn stover with waste activated sludge[J]. Energy, 118: 1256-1263. [14] Hess M, Sczyrba A, Egan R, et al.2011. Metagenomic discovery of biomass-degrading genes and genomes from cow rumen[J]. Science, 331(6016): 463-467. [15] Himmel M E, Xu Q, Luo Y, et al.2010. Microbial enzyme systems for biomass conversion: Emerging paradigms[J]. Biofuels, 1(2): 323-341. [16] Hu Y, Pang Y, Yuan H, et al.2015. Promoting anaerobic biogasification of corn stover through biological pretreatment by liquid fraction of digestate (LFD)[J]. Bioresource Technology, 175: 167-173. [17] Huws S A, Creevey C J, Oyama L B. Mizrahi I, et al.2018. Addressing global ruminant agricultural challenges through understanding the rumen microbiome: Past, present, and future[J]. Frontiers in Microbiology, 9: 2161. [18] Jeoh T, Cardona M J, Karuna N, et al.2017. Mechanistic kinetic models of enzymatic cellulose hydrolysis-A review[J]. Biotechnology and Bioengineering, 114(7): 1369-1385. [19] Kimura S, Ohshima C, Hirose E, et al.2001. Cellulose in the house of the appendicularian Oikopleura rufescens[J]. Protoplasma, 216(1-2): 71-74. [20] Klemm D, Heublein B, Fink H P, et al.2005. Cellulose: Fascinating biopolymer and sustainable raw material[J]. Angewandte Chemie, 44(22): 3358-3393. [21] Kumar R, Hu F, Hubbell C A, et al.2013. Comparison of laboratory delignification methods, their selectivity, and impacts on physiochemical characteristics of cellulosic biomass[J]. Bioresource Technology, 130(13): 372-381. [22] Latgé J P.1999. Aspergillus fumigatus and aspergillosis[J]. Clinical Microbiology Reviews, 12(2): 310-350. [23] Li H, Dai M, Dai S, Dong X, et al.2018. Current status and environment impact of direct straw return in China's cropland - A review[J]. Ecotoxicology and Environmental Safety, 159: 293-300. [24] Li M, Zhou H, Zi X, et al.2017. Silage fermentation and ruminal degradation of stylo prepared with lactic acid bacteria and cellulase[J]. Animal Science Journal, 88(10): 1531-1537. [25] Liu Q, Li J, Zhao J,et al.2019. Enhancement of lignocellulosic degradation in high-moisture alfalfa via anaerobic bioprocess of engineered Lactococcus lactis with the function of secreting cellulase[J]. Biotechnology and Bioengineering, 12(1): 88. [26] Mahmood R T, Asad M J, Mehboob N, et al.2013. Production, purification, and characterization of exoglucanase by Aspergillus fumigatus[J]. Applied Biochemistry and Biotechnology, 170(4): 895-908. [27] McFarlane H E, Döring A, Persson S.2014. The cell biology of cellulose synthesis[J]. Annual Review of Plant Biology, 65(1): 69-94. [28] Moroz O V, Maranta M, Shaghasi T, et al.2015. The three-dimensional structure of the cellobiohydrolase Cel7A from Aspergillus fumigatus at 1.5 Å resolution[J]. Acta Crystallographica Section F-Structural Biology Communications, 71(Pt 1): 114-120. [29] Niu L, Zhang H, Wu Z, et al.2018. Modified TCA/acetone precipitation of plant proteins for proteomic analysis[J]. PLOS ONE, 13(12): e0202238. [30] Ozkose E, Akyol I, Kar B, et al.2009. Expression of fungal cellulase gene in Lactococcus lactis to construct novel recombinant silage inoculants[J]. Folia Microbiologica, 54(4): 335-342. [31] Pauly M, Keegstra K.2008. Cell-wall carbohydrates and their modification as a resource for biofuels[J]. Plant Journal, 54(4): 559-568. [32] Payen A.1838. Mémoire sur la composition du tissu propre des plantes et du ligneux[J]. Comptes Rendus Hebdomadaires des Séances de l Académie des Sciences, 7(1): 1052. [33] Pear J R, Kawagoe Y, Schreckengost W E, et al.1996. Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase[J]. Proceedings of the National Academy of Science of the USA, 93(22): 12637-12642. [34] Peterbauer C, Maischberger T, Haltrich D.2011. Food-grade gene expression in lactic acid bacteria[J]. Biotechnology Journal, 6(9): 1147-1161. [35] Popova M, Martin C, Morgavi D P.2010 .Improved protocol for high-quality co-extraction of DNA and RNA from rumen digesta[J]. Folia Microbiologica, 55(4): 368-372. [36] Römling U.2002. Molecular biology of cellulose production in bacteria[J]. Research in Microbiology, 153(4): 205-212. [37] Segato F, Damásio A R, de Lucas R C, et al.2014. Genomics review of holocellulose deconstruction by aspergilli[J]. Microbiology and Molecular Biology Reviews, 78(4): 588-613. [38] Taylor N G.2008. Cellulose biosynthesis and deposition in higher plants[J]. New Phytologist, 178(2): 239-252. [39] van Asseldonk M, Rutten G, Oteman M, et al.1990. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363[J]. Gene, 95(1): 155-160. [40] Wood T M.1971. The cellulase of Fusarium solani. Purification and specificity of the β-(1-4)-glucanase and the β-D-glucosidase components[J]. Biochemical Journal, 121(3): 353-362. [41] Zhang F, Wang X, Lu W, et al.2019. Improved quality of corn silage when combining cellulose-decomposing bacteria and Lactobacillus buchneri during silage fermentation[J]. Biomed Research International, 2019: 4361358. [42] Zhang Y H, Hong J, Ye X.2009. Cellulase assays[J]. Methods in Molecular Biology, 581(14): 213-231. [43] Zhao C, Wang L, Ma G, et al.2021. Cellulase interacts with lactic acid bacteria to affect fermentation quality, microbial community, and ruminal degradability in mixed silage of soybean residue and corn stover[J]. Animals (Basel), 11(2): 334. [44] Zhong W, Zhang Z, Luo Y, et al.2011. Effect of biological pretreatments in enhancing corn straw biogas production[J]. Bioresource Technology, 102(24): 11177-11182. [45] Zhou H, Gao Y, Gao G, et al.2015. Oral administration of recombinant Lactococcus lactis expressing the cellulase gene increases digestibility of fiber in geese[J]. Current Microbiology, 71(6): 693-698.
