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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (9): 27-38    DOI: 10.13523/j.cb.2205014
研究报告     
温泉宏基因组来源的D-来苏糖异构酶性质研究*
苏雅丽1,陈凤贞1,石贤爱1,2,王国增1,2,**()
1.福州大学生物科学与工程学院 福州 350108
2.福州大学福建省医疗器械和医药技术重点实验室 福州 350108
Molecular Characterization of Novel D-lyxose Isomerases from a Hot Spring Metagenome
SU Ya-li1,CHEN Feng-zhen1,SHI Xian-ai1,2,WANG Guo-zeng1,2,**()
1. College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China
2. Fujian Key Laboratory of Medical Instrument and Pharmaceutical Technology, Fuzhou University, Fuzhou 350108, China
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摘要：

目的: 从高温温泉宏基因组中挖掘能够高效催化合成稀有糖的新耐高温D-来苏糖异构酶。方法: 从云南昌宁鸡飞温泉底泥中提取宏基因组DNA并进行高通量测序,经基因注释及序列比对鉴定D-来苏糖异构酶基因,构建大肠杆菌异源表达载体并诱导表达,通过亲和层析纯化重组蛋白并对其性质研究。结果: 从温泉底泥宏基因组测序结果中鉴定得到8个D-来苏糖异构酶基因,选择4个基因进行异源表达,其中JF-LI1和JF-LI4在大肠杆菌中成功表达并检测到酶活性。研究表明,JF-LI1和JF-LI4的最适温度分别为70℃和75℃。JF-LI4具有较宽的作用温度和良好的热稳定性,在30~100℃的温度范围内剩余40%以上的酶活力。JF-LI1和JF-LI4的最适pH分别为7.0和7.5,在中性偏酸性条件下具有较高的活力和较强的稳定性。重组JF-LI1和JF-LI4具有较宽的底物谱,除了对D-来苏糖活性最高外,对L-核糖、L-核酮糖、D-果糖和D-甘露糖均具有活性。重组JF-LI1和JF-LI4对L-核糖的催化效率分别为0.56 L/(mmol·s-1)和0.61 L/(mmol·s-1),是目前已知的D-来苏糖异构酶中最高的。结论: 从高温温泉宏基因组中获得8个新的D-来苏糖异构酶基因,对JF-LI1和JF-LI4进行异源表达和性质研究,具有pH稳定性好、热稳定性强以及底物特异性宽泛的特点,在制药、食品、化妆品等工业领域有重要的应用潜力。
关键词： D-来苏糖异构酶;  高温温泉;  宏基因组;  稀有糖;  功能糖    
Abstract:

Objective: To explore new thermostable D-lyxose isomerases that can efficiently catalyze the synthesis of rare sugars from the metagenomes of high temperature hot springs. Methods: Metagenomic DNA was extracted from the sediment of Jifei Hot Spring in Changning,Yunnan and subjected to high-throughput sequencing. The D-lyxose isomerase genes were identified by gene annotation and sequence alignment,and heterologous expression vectors were constructed and induced in E. coli. Two recombinant D-lyxose isomerases were purified by affinity chromatography and were characterized. Results: Eight D-lyxose isomerase genes were identified from the metagenomic sequencing results of the Jifei Hot Spring sediment. Four genes were selected for heterologous expression,among which JF-LI1 and JF-LI4 were successfully expressed in E. coli and their enzymatic activities were detected. The optimum temperatures of recombinant JF-LI1 and JF-LI4 were 70℃ and 75℃,respectively. JF-LI4 has a wide action temperature and high thermal stability,which retained more than 40% of its enzymatic activity in the temperature range of 30℃ to 100℃. The optimum pH of recombinant JF-LI1 and JF-LI4 were 7.0 and 7.5,respectively,and they have high activity and stability under neutral and slightly acidic conditions. Recombinant JF-LI1 and JF-LI4 have a broad substrate spectrum and are active on L-ribose,L-ribulose,D-fructose and D-mannose in addition to their most active effect on D-lyxose. The catalytic efficiencies of recombinant JF-LI1 and JF-LI4 for L-ribose were 0.56 and 0.61 L/(mmol·s-1),respectively,which were the highest among the known D-lyxose isomerases. Conclusion: Eight new D-lyxose isomerase genes were obtained from the high temperature hot spring metagenome,and two of them were heterologously expressed and characterized. They have high pH stability,strong thermal stability and wide substrate specificity,which enable them to have important application potential in pharmaceutical industry, food, cosmetics and other industrial fields.
Key words: D-lyxose isomerase    High temperature hot springs    Metagenome    Rare sugars    Functional sugars
收稿日期: 2022-05-06 出版日期: 2022-10-10
ZTFLH:  Q78  
基金资助: * 福建省自然科学基金(2019J01237)
通讯作者: 王国增     E-mail: wanggz@fzu.edu.cn
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苏雅丽
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引用本文:

苏雅丽,陈凤贞,石贤爱,王国增. 温泉宏基因组来源的D-来苏糖异构酶性质研究*[J]. 中国生物工程杂志, 2022, 42(9): 27-38.

SU Ya-li,CHEN Feng-zhen,SHI Xian-ai,WANG Guo-zeng. Molecular Characterization of Novel D-lyxose Isomerases from a Hot Spring Metagenome. China Biotechnology, 2022, 42(9): 27-38.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2205014    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I9/27

引物名称 序列(5'-3') 长度/碱基
JF-LI1-F CTTTAAGAAGGAGATATACCATGATTAAAAGAAGTAAGGCAGAGG 49
JF-LI1-R CAGTGGTGGTGGTGGTGGTGATCTTCTACTATTTCAGTTATTCTTTTTATATC 53
JF-LI4-F CTTTAAGAAGGAGATATACCATGAAGAGAAGAGAATATGAAATGG 45
JF-LI4-R CAGTGGTGGTGGTGGTGGTGATCTTCTATTATTGGAATCCTTTTTATC 48
JF-LI6-F CTTTAAGAAGGAGATATACCATGATTACCGAAAGCCAGTTGCGTCAG 47
JF-LI6-R CAGTGGTGGTGGTGGTGGTGCCCTGGCACCACCTTGGTGATG 42
JF-LI7-F CTTTAAGAAGGAGATATACCATGTTAAGTAAACAAGAAAAAAAGAAAGC 49
JF-LI7-R CAGTGGTGGTGGTGGTGGTGCTTCTCTACTCTAATGATGTTAGG 44
表1  本研究所用的引物
蛋白名称 氨基酸个数 氨基酸序列一致性最高菌 一致性/ %
JF-LI1 182 Thermoprotei archaeon(RLE72808) 72
JF-LI2 181 Dictyoglomus turgidum(HGB31030) 91
JF-LI3 177 Armatimonadetes bacterium(HGB59748) 86
JF-LI4 180 Dictyoglomus turgidum(PNV80534) 79
JF-LI5 181 Anaerolineae bacterium(MBC7224383) 96
JF-LI6 182 Armatimonadetes bacterium(HHS07368) 99
JF-LI7 179 Thaumarchaeota archaeon(NHV06097) 68
JF-LI8 181 Dictyoglomus thermophilum(WP_149122603) 100
表2  鸡飞温泉底泥宏基因组来源的D-来苏糖异构酶序列信息
图1  鸡飞温泉来源的8个D-来苏糖异构酶与已报道的D-来苏糖异构酶的进化关系及一致性
图2  鸡飞温泉来源的8个D-来苏糖异构酶与已做过性质研究的D-来苏糖异构酶蛋白序列比对
图3  重组JF-LI1和JF-LI4蛋白SDS-PAGE分析
底物 JF-LI1/% JF-LI4/%
D-来苏糖 100 100
L-核糖 94 76
L-核酮糖 38 21
D-甘露糖 21 3.5
D-果糖 12 1.5
D-木糖 0 0
D-葡萄糖 0 0
D-半乳糖 0 0
表3  重组JF-LI1和JF-LI4对不同种类糖的相对活性
图4  pH对重组JF-LI1和JF-LI4活性和稳定性的影响
图5  温度对重组JF-LI1和JF-LI4活性和稳定性的影响
微生物来源 最适pH 最适温度/ ℃ 最适金属
离子		 Km(D-来
苏糖/L-核糖,
mmol·L-1)		 kcat(D-来
苏糖/L-核糖,
1·s-1)		 kcat/Km[D-
来苏糖/L-核糖,
L/(mmol·s-1)]
C. laevoribosii[13] 6.5 70 Mn2+ 22.4/121.7 1 902.0/26.4 84.91/0.22
P. stuartii[14] 7.5 45 Mn2+ 47.0/- 31 340.0/- 666.81/-
S. proteamaculans[15] 7.5 40 Mn2+ 13.3/- 30 810.0/- 2 316.54/-
D. turgidum[19] 7.5 75 Co2+ 39.0/- 59.5/- 1.53/-
T. oceani[21] 6.5 65 Mn2+ 15.5/- 51.8/- 3.34/-
T. dichotomicum[23] 7.5 60 Mn2+ 43.5/98.5 42.7/5.4 0.98/0.05
B. velezensis[20] 6.5 55 Co2+ 33.1/151 22.3/16.3 0.67/0.11
B. licheniformis[18] 7.5~8.0 40~45 Mn2+ 30.4/- 98.0/- 3.22/-
P. bacterium[22] 7.0 70 Co2+ 51.0/1 130.1 36.1/68.3 0.71/0.06
T. archaeon[24] 6.5 85 Ni2+ 76.1/1 427.9 57.6/466.9 0.76/0.33
C. polysaccharolyticus[25] 6.5 65 Mn2+ 87.1/178 14.4/3.1 0.17/0.02
Thermofilum sp.[26] 7.0 >95 Mn2+ 74/- 129.6/- 1.75/-
E. coli O157:H7[16] 7.5 50 Mn2+ 16.1/- 13.7/- 0.85/-
温泉宏基因组(JF-LI1) 7.0 70 Co2+ 15.5/19.6 63.0/11.0 4.06/0.56
温泉宏基因组(JF-LI4) 7.5 75 Co2+ 15.8/36.7 88.6/22.6 5.61/0.62
表4  不同微生物来源的D-来苏糖异构酶及其性质
图6  金属离子对重组JF-LI1和JF-LI4活性的影响
图7  重组JF-LI1和JF-LI4对D-来苏糖和L-核糖的动力学常数
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