牛瘤胃纤维素酶<i>eg</i>基因在乳酸菌中的克隆表达及酶学性质分析

邹爱爱; 魏亚琴; 杨宇泽; 曹磊; 孙康永杰; 万学瑞; 王川

doi:10.11829/j.issn.1001-0629.2021-0001

牛瘤胃纤维素酶eg基因在乳酸菌中的克隆表达及酶学性质分析

1.
甘肃农业大学动物医学院, 甘肃兰州 730070
2.
甘肃省科学院生物研究所/甘肃省微生物资源开发利用重点实验室 / 厌氧微生物中心, 甘肃兰州 730000
3.
北京市畜牧总站，北京 100107

基金项目: 甘肃省科技计划项目重点研发计划(18YF1NA077)；甘肃农业大学动物医学院学科建设基金(GSAU-XKJS-2018-074)；甘肃省科学院科技产业化项目(牦牛瘤胃厌氧真菌基因重组工程菌发酵生产木质纤维素降解酶的研究)；甘肃农业大学2020年学生科研训练计划(SRTP-202003018)

摘要: 为构建高效表达的内切葡聚糖酶基因工程菌，本研究以牛瘤胃液中微生物全基因组为模板，通过PCR扩增方法得到eg片段，将其克隆至表达载体pMG36e中获得分泌型表达载体pMG36e::eg；将测序正确后的重组质粒电转导到乳酸菌(Lactococcus lactis NZ9000)中，得到L. lactis NZ9000/pMG36e::eg重组菌株，将发酵上清液通过10% 三氯乙酸(Trichloroacetic acid, TCA)/丙酮沉淀法浓缩蛋白后，用刚果红染色法和3, 5-二硝基水杨酸(3, 5-Dinitrosalicylic acid, DNS)法检测该蛋白酶活性，使用滤纸酶活力(filter paper enzyme activity, FPA)法检测该蛋白酶的总酶活，并对重组内切葡聚糖酶进行酶学性质研究。结果表明：从牛瘤胃微生物中克隆得到一个基因大小为1 500 bp左右的条带，该酶分子量为50 kDa左右，经刚果红染色有明显水解圈，水解圈直径为2.32 cm；采用DNS法测定该重组蛋白的酶活为12.401 9 U·mL⁻¹，用FPA法测定总酶活为12.246 9 U·mL⁻¹；重组蛋白酶对羧甲基纤维素钠、滤纸、微晶纤维素、脱脂棉均有酶活性；最适反应温度为90 ℃；最适反应pH为6；其中Cu^{2 +}、Mn^{2 +}、Ba^{2 +}、Zn^{2 +}、Co^{2 +}等均可以提高重组酶的酶活力，而Fe^{2 +}可抑制重组内切葡聚糖酶活力。本试验纤维素酶eg基因在L. lactis NZ9000中的稳定高效表达为提高青贮饲料的营养价值和消化率提供了技术支持。

关键词:

English

Expression of the eg gene of cellulase from cattle rumen in Lactobacillus and analysis of enzymatic properties

1.
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, Gansu, China
2.
Key Laboratory of Microbial Resources Exploitation and Application of Gansu Province, Institute of Biology, Gansu Academy of Sciences / Center of Anaerobic Microbes, Lanzhou 730000, Gansu, China
3.
Beijing Animal Husbandry Station, Beijing 100107, China

Received Date: 2021-01-04
Accepted Date: 2021-04-06
Available Online: 2021-12-09
Publish Date: 2021-12-14

Abstract: In order to construct highly expressing endoglucanase genetically engineered bacteria, the whole genome of the microorganism in bovine rumen juice was used as a template in this study, and the eg fragment was obtained by PCR amplification In order to construct genetically engineered bacteria with high endoglucanase expression, we used the whole genomes of microorganisms in bovine rumen juice as a template. We obtained a fragment containing the eg gene of cellulase by PCR amplification, which was cloned into the expression vector pMG36e to yield the expression vector pMG36e::eg. Recombinant plasmids containing the pMG36e::eg construct were electrotransduced into lactic acid bacteria (Lactococcus lactis NZ9000) to obtain an L. lactis NZ9000/pMG36e::eg recombinant strain, and the fermentation supernatant of the recombinant strain was concentrated using the 10% trichloroacetic acid/acetone precipitation method. Enzyme activity of the recombinant endoglucanase was determined using the 3, 5-dinitrosalicylic acid (DNS) and Congo red staining methods, total enzyme activity was determined using the filter paper enzyme activity (FPA) method, and enzymatic properties were examined. A gene of approximately 1 500 bp was cloned from a bovine rumen microorganism and the molecular weight of the encoded enzyme was approximately 50 kDa. Congo red staining analysis revealed that the recombinant enzyme caused a clear 2.32 cm zone of hydrolysis. The enzyme activity of the recombinant protein was 12.401 9 U·mL⁻¹ based on the DNS method and 12.246 9 U·mL⁻¹ using the FPA method. Furthermore, the recombinant protease has enzymatic activity on CMC-Na, filter paper, microcrystalline cellulose, and absorbent cotton. Enzyme activity was found to be optimal at a temperature of 90 °C and pH 6. Metal ions, including Cu^{2 +}, Mn^{2 +}, Ba^{2 +}, Zn^{2 +}, and Co^{2 +}, were found to promote activity of the recombinant enzyme, whereas Fe^{2 +} inhibited recombinant endoglucanase activity. In this study, we thus demonstrated the stable high-efficiency expression of the cellulase eg gene in L. lactis NZ9000, the practical utilization of which will contribute to improving the nutritional value of silage and digestibility of cattle feed.

Keywords:

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参考文献

[1]	SINGHANIA R R, PATEL A K, SUKUMARAN R K, LARROCHE C, PANDEY A. Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technology, 2013, 127: 500-507. doi: 10.1016/j.biortech.2012.09.012
[2]	HAO F, LI M X. Cellulase production by recombinant Trichoderma reesei and its application in enzymatic hydrolysis of agricultural residues. Fuel Guildford, 2015, 143: 211-216. doi: 10.1016/j.fuel.2014.11.056
[3]	杨天龙, 王淑玲, 顾招兵, 朱仁俊, 刘旭川, 张春勇, 杨舒黎, 毛华明, 冷静. 独龙牛瘤胃细菌纤维素酶基因克隆. 南方农业学报, 2017, 48(5): 901-906. doi: 10.3969/j.issn.2095-1191.2017.05.024 YANG T L, WANG S L, GU Z B, ZHU R J, LIU X C, ZHANG C Y, YANG S L, MAO H M, LENG J. Cloning of cellulase gene from rumen bacteria of Dulong cattle. Journal of Southern Agriculture, 2017, 48(5): 901-906. doi: 10.3969/j.issn.2095-1191.2017.05.024
[4]	WANG H, SQUINA F, SEGATO F, MORT A, LEE D, PAPPAN K, PRADE R. High-temperature enzymatic breakdown of cellulose. Applied and Environmental Microbiology, 2011, 77(10): 5199-5206.
[5]	赵士萍, 周敏, 蒋林树. 青贮饲料添加剂的研究进展. 中国农学通报, 2016, 32(20): 6-10. doi: 10.11924/j.issn.1000-6850.casb15120078 ZHAO S P, ZHOU M, JIANG L S. Research progress of silage additives. Chinese Agricultural Science Bulletin, 2016, 32(20): 6-10. doi: 10.11924/j.issn.1000-6850.casb15120078
[6]	YU Y, YUAN J, WANG Q, FAN X, WANG P, CUI L. Noncovalent immobilization of cellulases using the reversibly soluble polymers for biopolishing of cotton fabric. Biotechnology and Applied Biochemistry, 2015, 62(4): 494-501. doi: 10.1002/bab.1289
[7]	GOMES D, DOMINGUES L, GAMA M. Valorizing recycled paper sludge by a bioethanol production process with cellulase recycling. Bioresource Technology, 2016, 216: 637-644. doi: 10.1016/j.biortech.2016.06.004
[8]	黄国昌, 熊大维, 顾斌涛. 内切葡聚糖酶基因克隆和表达研究进展. 江西科学, 2016, 34(1): 24-28, 51. HUANG G C, XIONG D W, GU B T. Progress in cloning and expression of endoglucanase gene. Jiangxi Science, 2016, 34(1): 24-28, 51.
[9]	LIU Z L, LI H N, SONG H T, XIAO W J, XIA W C, LIU X P, JIANG Z B. Construction of a trifunctional cellulose and expression in Saccharomyces cerevisiae using a fusion protein. BMC Biotechnology, 2018, 18(1): 43. doi: 10.1186/s12896-018-0454-x
[10]	郝凤奇, 杨桂连, 叶丽萍, 王春凤. “食品级”乳酸菌表达载体系统的研究进展. 中国预防兽医学报, 2008, 30(10): 824-830. HAO F Q, YANG G L, YE L P, WANG C F. Research progress of “food grade” Lactobacillus expression vector system. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(10): 824-830.
[11]	黄佳明, 姜宁, 张爱忠. 乳酸菌作为基因工程菌的研究进展. 中国饲料, 2019(13): 16-20. HUANG J M, JIANG N, ZHANG A Z. Research progress of lactic acid bacteria as genetic engineering bacteria. China Feed, 2019(13): 16-20.
[12]	李争明, 张娟, 邓中洋, 卢凡, 秦文胜. 纤维素酶产生菌的筛选、鉴定及发酵产酶条件优化. 生物技术通报, 2015, 31(5): 146-152. LI Z M, ZHANG J, DENG Z Y, LU F, QIN W S. Screening and identification of cellulase producing strains and optimization of fermentation conditions. Biotechnology Bulletin, 2015, 31(5): 146-152.
[13]	WANG B, XIA L. High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei. Bioresource Technology, 2011, 102(6): 4568-4572. doi: 10.1016/j.biortech.2010.12.099
[14]	HOLO H, NES I F. High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Applied and Environmental Microbiology, 1989, 55(12): 3119-3123. doi: 10.1128/aem.55.12.3119-3123.1989
[15]	NUC P, NUC K. Recombinant protein production in Escherichia coli. Postepy Biochemii, 2006, 52(4): 448-456.
[16]	丁轲, 罗伟光, 丁盼盼, 李旺, 李元晓, 余祖华, 恒子钤, 贾艳艳, 程相朝. 双纤维素酶基因在乳酸杆菌中的融合分泌表达. 食品科学, 2014, 35(15): 127-131. doi: 10.7506/spkx1002-6630-201415026 DING K, LUO W G, DING P P, LI W, LI Y X, YU Z H, HENG Z Q, JIA Y Y, CHENG X C. Expression of the fusion and secretion of the double cellulase gene in Lactobacillus. Food Science, 2014, 35(15): 127-131. doi: 10.7506/spkx1002-6630-201415026
[17]	王婷婷. 饲用纤维素酶活力的测定方法. 养殖技术顾问, 2014(10): 233-234. WANG T T. Determination method of feed cellulase activity. Technical Advisor for Animal Husbandry, 2014(10): 233-234.
[18]	BEDFORD M R, SCHULZE H. Exogenous enzymes for pigs and poultry. Nutrition Research Reviews, 1998, 11(1): 91-114. doi: 10.1079/NRR19980007
[19]	SELINGER L B, FORSBERG C W, CHENG K J. The rumen: A unique source of enzymes for enhancing livestock production. Anaerobe, 1996, 2(5): 263-284. doi: 10.1006/anae.1996.0036
[20]	汤斌, 张莹莹, 杨亚平. 匍枝根霉TP-02内切葡聚糖酶基因eg2的克隆表达及功能分析. 食品与发酵工业, 2013, 39(7): 13-17. TANG B, ZHANG Y Y, YANG Y P. Cloning, expression and functional analysis of endoglucanase gene eg2 from Rhizopus stolonifera TP-02. Food and Fermentation Industries, 2013, 39(7): 13-17.
[21]	宝力德, 秦伟, 杨嘉萌, 邵影. Bacillus. sp. bs-1的内切葡聚糖酶在大肠杆菌中表达条件的优化及其活性分析. 内蒙古农业大学学报(自然科学版), 2013, 34(5): 77-82. BAO L D, QIN W, YANG J M, SHAO Y. Bacillus. sp. bs-1 optimization of expression conditions and activity analysis of endoglucanase. Journal of Inner Mongolia Agricultural University (Natural Science Edition), 2013, 34(5): 77-82.
[22]	刘原子, 王艳, 万学瑞, 王川, 吴润, 刘岗, 吴自祥. 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. 草原与草坪, 2017, 37(1): 8-13. doi: 10.3969/j.issn.1009-5500.2017.01.003 LIU Y Z, WANG Y, WAN X R, WANG C, WU R, LIU G, WU Z X. Secretory expression of cellulase genes bglA, bglB and eg of Bacillus polymyxa in lactic acid bacteria. Grassland and Turf, 2017, 37(1): 8-13. doi: 10.3969/j.issn.1009-5500.2017.01.003
[23]	ZUTZ A, NIELSEN L H, PEDERSEN L E, KASSEM M M, NIELSEN A T. A dual-reporter system for investigating and optimizing protein translation and folding in E. coli. Nature Communications, 2020, 12: 6093.
[24]	OZKOSE E, AKYOL I, KAR B, COMLEKCIOGLU U, EKINCI M S. Expression of fungal cellulase gene in Lactococcus lactis to construct novel recombinant silage inoculants. Folia Microbiologica, 2009, 54(4): 335-342. doi: 10.1007/s12223-009-0043-4
[25]	丁寅寅, 马会勤, 左芳雷, 郝彦玲, 陈尚武. 乳酸菌载体pMG36e的应用现状. 中国生物工程杂志, 2009, 29(11): 106-111. DING Y Y, MA H Q, ZUO F L, HAO Y L, CHEN S W. Application status of pMG36e carrier for lactic acid bacteria. Chinese Biotechnology, 2009, 29(11): 106-111.
[26]	向华, 刘敬忠. 欧芹苯丙氨酸脱氨酶cDNA在乳酸乳球菌中的表达研究. 微生物学报, 1999, 39(3): 196-204. XIANG H, LIU J Z. Expression of phenylalanine deaminase cDNA from parsley in Lactococcus lactis. Acta Microbiologica Sinica, 1999, 39(3): 196-204.
[27]	VAN DE GUCHTE M, KODDE J, VAN DER VOSSEN J M, KOK J, VENEMA G. Heterologous gene expression in Lactococcus lactis subsp. lactis: Synthesis, secretion, and processing of the Bacillus subtilis neutral protease. Applied and Environmental Microbiology, 1990, 56(9): 2606-2611. doi: 10.1128/aem.56.9.2606-2611.1990
[28]	向华, 郭顺星. 人铜锌超氧化物歧化酶基因的克隆和在乳酸乳球菌中的表达. 生物工程学报, 2000, 16(1): 6-9. doi: 10.3321/j.issn:1000-3061.2000.01.003 XIANG H, GUO S X. Cloning and expression of human copper zinc superoxide dismutase gene in Lactococcus lactis. Chinese Journal of Biotechnology, 2000, 16(1): 6-9. doi: 10.3321/j.issn:1000-3061.2000.01.003
[29]	王翠艳, 王玉华, 朴春红, 刘俊梅, 胡耀辉, 任大勇, 于寒松. 绿色木霉纤维二糖水解酶基因的克隆及其在乳酸菌中的表达与鉴定. 食品科学, 2016, 37(19): 141-146. doi: 10.7506/spkx1002-6630-201619024 WANG C Y, WANG Y H, PIAO C H, LIU J M, HU Y H, REN D Y, YU H S. Cloning of Trichoderma viride cellobiohydrolase gene and its expression and identification in lactic acid bacteria. Food Science, 2016, 37(19): 141-146. doi: 10.7506/spkx1002-6630-201619024
[30]	王瑾, 邬敏辰, 周晨妍. 内切葡聚糖酶基因在大肠杆菌与毕赤酵母中的表达. 生物技术通报, 2008(3): 110-114. WANG J, WU M C, ZHOU C Y. Expression of endoglucanase gene in Escherichia coli and Pichia pastoris. Biotechnology Bulletin, 2008(3): 110-114.
[31]	NAKAZAWA H, OKADA K, KOBAYASHI R, KUBOTA T, ONODERA T, OCHIAI N, OMATA N, OGASAWARA W, OKADA H, MORIKAWA Y. Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli. Applied Microbiology and Biotechnology, 2008, 81(4): 681-689. doi: 10.1007/s00253-008-1667-z
[32]	岳林芳, 成立新, 李蕴华, 凤英, 高爱武, 乔健敏, 王志铭, 于朝晖, 宝华. 益生菌在反刍动物生产中应用的研究进展. 畜牧与饲料科学, 2019, 40(9): 54-62. doi: 10.12160/j.issn.1672-5190.2019.09.011 YUE L F, CHENG L X, LI Y H, FENG Y, GAO A W, QIAO J M, WANG Z M, YU C H, BAO H. Research progress of probiotics in ruminant production. Animal Husbandry and Feed Science, 2019, 40(9): 54-62. doi: 10.12160/j.issn.1672-5190.2019.09.011
[33]	谢骜李畅, 杨雨鑫, 王小龙, 陈玉林. 桑天牛肠道微生物内切葡聚糖酶基因的克隆及在乳酸杆菌中的表达. 动物营养学报, 2020, 32(3): 1344-1352. XIE A L C, YANG Y X, WANG X L, CHEN Y L. Cloning and expression of endoglucanase gene from intestinal microorganism of Apriona germari in Lactobacillus. Chinese Journal of Animal Nutrition, 2020, 32(3): 1344-1352.
[34]	PANDEY S, KUSHWAH J, TIWARI R, KUMAR R, SOMVANSHI V S, NAINA L, SAXENA K A. Cloning and expression of β-1, 4-endoglucanase gene from Bacillus subtilis isolated from soil long term irrigated with effluents of paper and pulp mill. Microbiological Research, 2014, 169(9/10): 693-698.
[35]	ANNAMALAI N, RAJESWARI M V, ELAYARAJA S, BALASUBRAMANIAN T. Thermostable, haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes. Carbohydrate Polymers, 2013, 94(1): 409-415. doi: 10.1016/j.carbpol.2013.01.066
[36]	LEE Y J, KIM B K, LEE B H, JO K I, LEE N K, CHUNG C H, LEE Y C, LEE J W. Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresource Technology, 2008, 99(2): 378-386. doi: 10.1016/j.biortech.2006.12.013
[37]	朱泾, 赵述淼, 彭楠, 梁运祥. 冰岛硫化叶菌β-1, 4-内切葡聚糖酶的同源表达、纯化与性质. 华中农业大学学报, 2011, 30(6): 674-679. ZHU J, ZHAO S M, PENG N, LIANG Y X. Homologous expression, purification and characterization of β-1, 4-endoglucanase from Sulfurophyllum icelandicum. Journal of Huazhong Agricultural University, 2011, 30(6): 674-679.
[38]	HAKAMADA Y, KOIKE K, YOSHIMATSU T, MORI H, KOBAYASHI H, ITO S. Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237. Extremophiles, 1997, 1(3): 151-156. doi: 10.1007/s007920050028

图 1 目的基因的PCR扩增产物及重组质粒双酶切鉴定

M：DNA分子质量标准；1：eg基因PCR产物；2：pMG36e::eg重组质粒双酶切；3：pMG36e空质粒双酶切。

Figure 1. PCR amplification products of the target gene and identification of the recombinant plasmid by double enzyme digestion

M: DL5 000 DNA marker; 1: PCR products of the eg gene; 2: Double digestion of pMG36e::eg recombinant plasmid; 3: Double digestion of pMG36e empty plasmid.

下载: 全尺寸图片幻灯片

图 2 eg序列系统发育树

Figure 2. A phylogenetic tree of the eg gene in different microbial species

下载: 全尺寸图片幻灯片

图 3 SDS-PAGE电泳

M：蛋白质分子质量标准；1、2、3分别为NZ9000/pMG36e::eg重组菌的菌液、培养上清液的浓缩蛋白、菌体超声破碎后的沉淀；4、5、6分别为NZ9000/pMG36e空质粒重组菌的菌液、培养液的上清液、菌体超声破碎后的沉淀。

Figure 3. SDS-PAGE electrophoresis

M: Protein markers; 1, 2, and 3 are NZ9000/pMG36e::eg recombinant bacterial suspension, protein concentrate in culture supernatant, and bacterial suspension after ultrasonic disruption, respectively; 4, 5, and 6 are NZ9000/pMG36e empty plasmid recombinant bacterial suspension, supernatant of the culture medium, and sediment after ultrasonic disruption, respectively.

下载: 全尺寸图片幻灯片

图 4 刚果红染色水解圈

1、2、3分别为NZ9000/pMG36e空质粒重组菌的菌液、培养液的上清液、菌体超声破碎后的沉淀；4：ddH₂O；5、6、7分别为NZ9000/pMG36e::eg重组菌超声破碎后的沉淀、培养液的上清液、重组蛋白。

Figure 4. Congo red stain zones of hydrolysis

1, 2, and 3 are NZ9000/pMG36e empty plasmid recombinant bacteria suspension, culture medium supernatant, and cell sediment after ultrasonic disruption, respectively; 4: ddH₂O; 5, 6, and 7 are NZ000/pMG36e::eg recombinant bacteria sediment after ultrasonic disruption, culture medium supernatant, and recombinant protein, respectively.

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图 5 重组内切葡聚糖酶酶学性质分析

A：重组内切葡聚糖酶在不同pH下的相对酶活力；B：重组内切葡聚糖酶在不同温度下的相对酶活力；C：重组内切葡聚糖酶对不同底物的水解能力。不同小写字母表示不同处理间差异显著(P < 0.05)。

Figure 5. Analysis of the enzymatic properties of the recombinant endoglucanase

A: Relative enzyme activity of recombinant endoglucanase at different pH values; B: Relative enzyme activity of the recombinant endoglucanase at different reaction temperatures; C: Hydrolysis capacity of the recombinant endoglucanase on different substrates. Different lowercase letters indicate significant difference between the different treatments at the 0.05 level.

下载: 全尺寸图片幻灯片

表 1 二硝基水杨酸法和滤纸法测定酶活力值

Table 1 Determination of enzyme activity using the 3,5-dinitrosalicylic acid (DNS) and filter paper methods

方法 Method	酶活力 Enzyme activity/(U·mL⁻¹)
方法 Method	试验组酶活值 Enzyme activities in the test group	对照组酶活值 Enzyme activity in the control group
DNS法 DNS method	12.401 9 ± 0.044 5a	0.573 4 ± 0.140 2b
滤纸酶活力法 Enzyme activity of filter paper	12.246 9 ± 0.447 5a	0.579 0 ± 0.118 0b
同行不同小写字母表示试验组与对照组间差异显著(P < 0.05)。　Different lowercase letters within the same row indicate significant difference between the test and control groups at the 0.05 level.

下载: 导出CSV

表 2 各种金属离子对重组内切葡聚糖酶相对酶活力的影响

Table 2 Effects of different metal ions on the relative enzyme activity of the recombinant endoglucanase

金属离子 Metal ions	相对酶活力 Relative enzyme activity/%
Cu^{2 +}	151.54 ± 0.82
Mn^{2 +}	138.06 ± 0.47
Ba^{2 +}	127.86 ± 0.43
Mg^{2 +}	117.30 ± 0.98
Fe^{2 +}	90.35 ± 0.50
Hg^{2 +}	118.76 ± 0.13
K⁺	109.83 ± 0.20
Zn^{2 +}	129.32 ± 0.08
Co^{2 +}	135.15 ± 0.26
对照 Control	100.00 ± 0.03

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参考文献(38)

[1]	SINGHANIA R R, PATEL A K, SUKUMARAN R K, LARROCHE C, PANDEY A. Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technology, 2013, 127: 500-507. doi: 10.1016/j.biortech.2012.09.012
[2]	HAO F, LI M X. Cellulase production by recombinant Trichoderma reesei and its application in enzymatic hydrolysis of agricultural residues. Fuel Guildford, 2015, 143: 211-216. doi: 10.1016/j.fuel.2014.11.056
[3]	杨天龙, 王淑玲, 顾招兵, 朱仁俊, 刘旭川, 张春勇, 杨舒黎, 毛华明, 冷静. 独龙牛瘤胃细菌纤维素酶基因克隆. 南方农业学报, 2017, 48(5): 901-906. doi: 10.3969/j.issn.2095-1191.2017.05.024 YANG T L, WANG S L, GU Z B, ZHU R J, LIU X C, ZHANG C Y, YANG S L, MAO H M, LENG J. Cloning of cellulase gene from rumen bacteria of Dulong cattle. Journal of Southern Agriculture, 2017, 48(5): 901-906. doi: 10.3969/j.issn.2095-1191.2017.05.024
[4]	WANG H, SQUINA F, SEGATO F, MORT A, LEE D, PAPPAN K, PRADE R. High-temperature enzymatic breakdown of cellulose. Applied and Environmental Microbiology, 2011, 77(10): 5199-5206.
[5]	赵士萍, 周敏, 蒋林树. 青贮饲料添加剂的研究进展. 中国农学通报, 2016, 32(20): 6-10. doi: 10.11924/j.issn.1000-6850.casb15120078 ZHAO S P, ZHOU M, JIANG L S. Research progress of silage additives. Chinese Agricultural Science Bulletin, 2016, 32(20): 6-10. doi: 10.11924/j.issn.1000-6850.casb15120078
[6]	YU Y, YUAN J, WANG Q, FAN X, WANG P, CUI L. Noncovalent immobilization of cellulases using the reversibly soluble polymers for biopolishing of cotton fabric. Biotechnology and Applied Biochemistry, 2015, 62(4): 494-501. doi: 10.1002/bab.1289
[7]	GOMES D, DOMINGUES L, GAMA M. Valorizing recycled paper sludge by a bioethanol production process with cellulase recycling. Bioresource Technology, 2016, 216: 637-644. doi: 10.1016/j.biortech.2016.06.004
[8]	黄国昌, 熊大维, 顾斌涛. 内切葡聚糖酶基因克隆和表达研究进展. 江西科学, 2016, 34(1): 24-28, 51. HUANG G C, XIONG D W, GU B T. Progress in cloning and expression of endoglucanase gene. Jiangxi Science, 2016, 34(1): 24-28, 51.
[9]	LIU Z L, LI H N, SONG H T, XIAO W J, XIA W C, LIU X P, JIANG Z B. Construction of a trifunctional cellulose and expression in Saccharomyces cerevisiae using a fusion protein. BMC Biotechnology, 2018, 18(1): 43. doi: 10.1186/s12896-018-0454-x
[10]	郝凤奇, 杨桂连, 叶丽萍, 王春凤. “食品级”乳酸菌表达载体系统的研究进展. 中国预防兽医学报, 2008, 30(10): 824-830. HAO F Q, YANG G L, YE L P, WANG C F. Research progress of “food grade” Lactobacillus expression vector system. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(10): 824-830.
[11]	黄佳明, 姜宁, 张爱忠. 乳酸菌作为基因工程菌的研究进展. 中国饲料, 2019(13): 16-20. HUANG J M, JIANG N, ZHANG A Z. Research progress of lactic acid bacteria as genetic engineering bacteria. China Feed, 2019(13): 16-20.
[12]	李争明, 张娟, 邓中洋, 卢凡, 秦文胜. 纤维素酶产生菌的筛选、鉴定及发酵产酶条件优化. 生物技术通报, 2015, 31(5): 146-152. LI Z M, ZHANG J, DENG Z Y, LU F, QIN W S. Screening and identification of cellulase producing strains and optimization of fermentation conditions. Biotechnology Bulletin, 2015, 31(5): 146-152.
[13]	WANG B, XIA L. High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei. Bioresource Technology, 2011, 102(6): 4568-4572. doi: 10.1016/j.biortech.2010.12.099
[14]	HOLO H, NES I F. High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Applied and Environmental Microbiology, 1989, 55(12): 3119-3123. doi: 10.1128/aem.55.12.3119-3123.1989
[15]	NUC P, NUC K. Recombinant protein production in Escherichia coli. Postepy Biochemii, 2006, 52(4): 448-456.
[16]	丁轲, 罗伟光, 丁盼盼, 李旺, 李元晓, 余祖华, 恒子钤, 贾艳艳, 程相朝. 双纤维素酶基因在乳酸杆菌中的融合分泌表达. 食品科学, 2014, 35(15): 127-131. doi: 10.7506/spkx1002-6630-201415026 DING K, LUO W G, DING P P, LI W, LI Y X, YU Z H, HENG Z Q, JIA Y Y, CHENG X C. Expression of the fusion and secretion of the double cellulase gene in Lactobacillus. Food Science, 2014, 35(15): 127-131. doi: 10.7506/spkx1002-6630-201415026
[17]	王婷婷. 饲用纤维素酶活力的测定方法. 养殖技术顾问, 2014(10): 233-234. WANG T T. Determination method of feed cellulase activity. Technical Advisor for Animal Husbandry, 2014(10): 233-234.
[18]	BEDFORD M R, SCHULZE H. Exogenous enzymes for pigs and poultry. Nutrition Research Reviews, 1998, 11(1): 91-114. doi: 10.1079/NRR19980007
[19]	SELINGER L B, FORSBERG C W, CHENG K J. The rumen: A unique source of enzymes for enhancing livestock production. Anaerobe, 1996, 2(5): 263-284. doi: 10.1006/anae.1996.0036
[20]	汤斌, 张莹莹, 杨亚平. 匍枝根霉TP-02内切葡聚糖酶基因eg2的克隆表达及功能分析. 食品与发酵工业, 2013, 39(7): 13-17. TANG B, ZHANG Y Y, YANG Y P. Cloning, expression and functional analysis of endoglucanase gene eg2 from Rhizopus stolonifera TP-02. Food and Fermentation Industries, 2013, 39(7): 13-17.
[21]	宝力德, 秦伟, 杨嘉萌, 邵影. Bacillus. sp. bs-1的内切葡聚糖酶在大肠杆菌中表达条件的优化及其活性分析. 内蒙古农业大学学报(自然科学版), 2013, 34(5): 77-82. BAO L D, QIN W, YANG J M, SHAO Y. Bacillus. sp. bs-1 optimization of expression conditions and activity analysis of endoglucanase. Journal of Inner Mongolia Agricultural University (Natural Science Edition), 2013, 34(5): 77-82.
[22]	刘原子, 王艳, 万学瑞, 王川, 吴润, 刘岗, 吴自祥. 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. 草原与草坪, 2017, 37(1): 8-13. doi: 10.3969/j.issn.1009-5500.2017.01.003 LIU Y Z, WANG Y, WAN X R, WANG C, WU R, LIU G, WU Z X. Secretory expression of cellulase genes bglA, bglB and eg of Bacillus polymyxa in lactic acid bacteria. Grassland and Turf, 2017, 37(1): 8-13. doi: 10.3969/j.issn.1009-5500.2017.01.003
[23]	ZUTZ A, NIELSEN L H, PEDERSEN L E, KASSEM M M, NIELSEN A T. A dual-reporter system for investigating and optimizing protein translation and folding in E. coli. Nature Communications, 2020, 12: 6093.
[24]	OZKOSE E, AKYOL I, KAR B, COMLEKCIOGLU U, EKINCI M S. Expression of fungal cellulase gene in Lactococcus lactis to construct novel recombinant silage inoculants. Folia Microbiologica, 2009, 54(4): 335-342. doi: 10.1007/s12223-009-0043-4
[25]	丁寅寅, 马会勤, 左芳雷, 郝彦玲, 陈尚武. 乳酸菌载体pMG36e的应用现状. 中国生物工程杂志, 2009, 29(11): 106-111. DING Y Y, MA H Q, ZUO F L, HAO Y L, CHEN S W. Application status of pMG36e carrier for lactic acid bacteria. Chinese Biotechnology, 2009, 29(11): 106-111.
[26]	向华, 刘敬忠. 欧芹苯丙氨酸脱氨酶cDNA在乳酸乳球菌中的表达研究. 微生物学报, 1999, 39(3): 196-204. XIANG H, LIU J Z. Expression of phenylalanine deaminase cDNA from parsley in Lactococcus lactis. Acta Microbiologica Sinica, 1999, 39(3): 196-204.
[27]	VAN DE GUCHTE M, KODDE J, VAN DER VOSSEN J M, KOK J, VENEMA G. Heterologous gene expression in Lactococcus lactis subsp. lactis: Synthesis, secretion, and processing of the Bacillus subtilis neutral protease. Applied and Environmental Microbiology, 1990, 56(9): 2606-2611. doi: 10.1128/aem.56.9.2606-2611.1990
[28]	向华, 郭顺星. 人铜锌超氧化物歧化酶基因的克隆和在乳酸乳球菌中的表达. 生物工程学报, 2000, 16(1): 6-9. doi: 10.3321/j.issn:1000-3061.2000.01.003 XIANG H, GUO S X. Cloning and expression of human copper zinc superoxide dismutase gene in Lactococcus lactis. Chinese Journal of Biotechnology, 2000, 16(1): 6-9. doi: 10.3321/j.issn:1000-3061.2000.01.003
[29]	王翠艳, 王玉华, 朴春红, 刘俊梅, 胡耀辉, 任大勇, 于寒松. 绿色木霉纤维二糖水解酶基因的克隆及其在乳酸菌中的表达与鉴定. 食品科学, 2016, 37(19): 141-146. doi: 10.7506/spkx1002-6630-201619024 WANG C Y, WANG Y H, PIAO C H, LIU J M, HU Y H, REN D Y, YU H S. Cloning of Trichoderma viride cellobiohydrolase gene and its expression and identification in lactic acid bacteria. Food Science, 2016, 37(19): 141-146. doi: 10.7506/spkx1002-6630-201619024
[30]	王瑾, 邬敏辰, 周晨妍. 内切葡聚糖酶基因在大肠杆菌与毕赤酵母中的表达. 生物技术通报, 2008(3): 110-114. WANG J, WU M C, ZHOU C Y. Expression of endoglucanase gene in Escherichia coli and Pichia pastoris. Biotechnology Bulletin, 2008(3): 110-114.
[31]	NAKAZAWA H, OKADA K, KOBAYASHI R, KUBOTA T, ONODERA T, OCHIAI N, OMATA N, OGASAWARA W, OKADA H, MORIKAWA Y. Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli. Applied Microbiology and Biotechnology, 2008, 81(4): 681-689. doi: 10.1007/s00253-008-1667-z
[32]	岳林芳, 成立新, 李蕴华, 凤英, 高爱武, 乔健敏, 王志铭, 于朝晖, 宝华. 益生菌在反刍动物生产中应用的研究进展. 畜牧与饲料科学, 2019, 40(9): 54-62. doi: 10.12160/j.issn.1672-5190.2019.09.011 YUE L F, CHENG L X, LI Y H, FENG Y, GAO A W, QIAO J M, WANG Z M, YU C H, BAO H. Research progress of probiotics in ruminant production. Animal Husbandry and Feed Science, 2019, 40(9): 54-62. doi: 10.12160/j.issn.1672-5190.2019.09.011
[33]	谢骜李畅, 杨雨鑫, 王小龙, 陈玉林. 桑天牛肠道微生物内切葡聚糖酶基因的克隆及在乳酸杆菌中的表达. 动物营养学报, 2020, 32(3): 1344-1352. XIE A L C, YANG Y X, WANG X L, CHEN Y L. Cloning and expression of endoglucanase gene from intestinal microorganism of Apriona germari in Lactobacillus. Chinese Journal of Animal Nutrition, 2020, 32(3): 1344-1352.
[34]	PANDEY S, KUSHWAH J, TIWARI R, KUMAR R, SOMVANSHI V S, NAINA L, SAXENA K A. Cloning and expression of β-1, 4-endoglucanase gene from Bacillus subtilis isolated from soil long term irrigated with effluents of paper and pulp mill. Microbiological Research, 2014, 169(9/10): 693-698.
[35]	ANNAMALAI N, RAJESWARI M V, ELAYARAJA S, BALASUBRAMANIAN T. Thermostable, haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes. Carbohydrate Polymers, 2013, 94(1): 409-415. doi: 10.1016/j.carbpol.2013.01.066
[36]	LEE Y J, KIM B K, LEE B H, JO K I, LEE N K, CHUNG C H, LEE Y C, LEE J W. Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresource Technology, 2008, 99(2): 378-386. doi: 10.1016/j.biortech.2006.12.013
[37]	朱泾, 赵述淼, 彭楠, 梁运祥. 冰岛硫化叶菌β-1, 4-内切葡聚糖酶的同源表达、纯化与性质. 华中农业大学学报, 2011, 30(6): 674-679. ZHU J, ZHAO S M, PENG N, LIANG Y X. Homologous expression, purification and characterization of β-1, 4-endoglucanase from Sulfurophyllum icelandicum. Journal of Huazhong Agricultural University, 2011, 30(6): 674-679.
[38]	HAKAMADA Y, KOIKE K, YOSHIMATSU T, MORI H, KOBAYASHI H, ITO S. Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237. Extremophiles, 1997, 1(3): 151-156. doi: 10.1007/s007920050028

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牛瘤胃纤维素酶eg基因在乳酸菌中的克隆表达及酶学性质分析

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Expression of the eg gene of cellulase from cattle rumen in Lactobacillus and analysis of enzymatic properties

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