生物技术通报 ›› 2022, Vol. 38 ›› Issue (1): 157-167.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0129
岑潇龙1(), 雷曦1, 马诗云1, 陈倩茹1, 黄遵锡1,2,3,4, 周峻沛1,2,3,4, 张蕊1,2,3,4()
收稿日期:
2021-02-02
出版日期:
2022-01-26
发布日期:
2022-02-22
作者简介:
岑潇龙,男,硕士研究生,研究方向:微生物学;E-mail: 基金资助:
CEN Xiao-long1(), LEI Xi1, MA Shi-yun1, CHEN Qian-ru1, HUANG Zun-xi1,2,3,4, ZHOU Jun-pei1,2,3,4, ZHANG Rui1,2,3,4()
Received:
2021-02-02
Published:
2022-01-26
Online:
2022-02-22
摘要:
透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖。微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点。通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化性分析。纯化后的重组酶rHylS的最适pH和温度分别为5.0和45℃;专一性降解透明质酸,比活是(1.6×105±5.4)U/mg;降解透明质酸产生低分子量的不饱和寡糖,属于内切透明质酸裂解酶;酶解产物对ABTS、DPPH、超氧阴离子和羟自由基清除能力显著高于未酶解的高分子量透明质酸,且与浓度呈正相关。金黄色葡萄球菌来源的透明质酸裂解酶HylS酶学性质优良,可用于生产具有抗氧化性低分子量的不饱和透明质酸寡糖。
岑潇龙, 雷曦, 马诗云, 陈倩茹, 黄遵锡, 周峻沛, 张蕊. 金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究[J]. 生物技术通报, 2022, 38(1): 157-167.
CEN Xiao-long, LEI Xi, MA Shi-yun, CHEN Qian-ru, HUANG Zun-xi, ZHOU Jun-pei, ZHANG Rui. Heterologous Expression and Characterization of the Hyaluronic Acid Lyase HylS from Staphylococcus aureus[J]. Biotechnology Bulletin, 2022, 38(1): 157-167.
透明质酸裂解酶 Hyaluronic acid lyase | HylS | HAase | - | HAase-B | HCLaseM | HCLase |
---|---|---|---|---|---|---|
序列一致性a Sequence identity a | 100 | 27.4 | 24.1 | 22.4 | 20.7 | 19.6 |
带电荷氨基酸残基 Charged amino acids(RKHDE) | 31.0 | 26.0 | 25.5 | 24.6 | 21.5 | 21.6 |
酸性氨基酸残基 Acidic amino acids(DE) | 14.7 | 12.4 | 11.6 | 13.2 | 10.5 | 9.5 |
碱性氨基酸残基 Basic amino acids(KR) | 14.4 | 11.3 | 12.0 | 10.1 | 8.7 | 9.4 |
极性氨基酸残基 Polar amino acids(NCQSTY) | 32.5 | 28.7 | 36.0 | 29.8 | 24.0 | 28.6 |
疏水氨基酸残基 Hydrophobic amino acids(AILFWV) | 26.4 | 35.4 | 28.9 | 32.4 | 39.2 | 35.8 |
D Asp | 8.8 | 6.2 | 6.4 | 6.3 | 5.9 | 5.8 |
E Glu | 5.9 | 6.3 | 5.1 | 6.9 | 4.6 | 3.7 |
C Cys | 0.2 | 0.5 | 0 | 0.1 | 0.6 | 0.3 |
Y Tyr | 4.6 | 4.3 | 4.0 | 3.9 | 1.9 | 4.0 |
H His | 1.9 | 2.3 | 1.9 | 1.3 | 2.4 | 2.7 |
K Lys | 12.4 | 7.8 | 9.2 | 6.2 | 1.0 | 4.4 |
R Arg | 2.0 | 3.5 | 2.8 | 3.9 | 7.7 | 4.9 |
蛋白序列登陆号Protein accession numbers in GenBank database | AYU99970 | AKM20831 | CAD46929 | AHB61202 | QGL52623 | AIL54323 |
来源Source | Staphylococcus aureus STA | Streptococcus zooe-pidemicus MF002 | Streptococcus aga- lactiae NEM316 | Bacillus sp. A50 | Microbacterium sp. H14 | Vibrio sp. FC509 |
表1 PL8家族透明质酸裂解酶的氨基酸残基组成比率
Table 1 Amino acid residues frequencies of hyaluronic acid(HA)lyases in PL8 family
透明质酸裂解酶 Hyaluronic acid lyase | HylS | HAase | - | HAase-B | HCLaseM | HCLase |
---|---|---|---|---|---|---|
序列一致性a Sequence identity a | 100 | 27.4 | 24.1 | 22.4 | 20.7 | 19.6 |
带电荷氨基酸残基 Charged amino acids(RKHDE) | 31.0 | 26.0 | 25.5 | 24.6 | 21.5 | 21.6 |
酸性氨基酸残基 Acidic amino acids(DE) | 14.7 | 12.4 | 11.6 | 13.2 | 10.5 | 9.5 |
碱性氨基酸残基 Basic amino acids(KR) | 14.4 | 11.3 | 12.0 | 10.1 | 8.7 | 9.4 |
极性氨基酸残基 Polar amino acids(NCQSTY) | 32.5 | 28.7 | 36.0 | 29.8 | 24.0 | 28.6 |
疏水氨基酸残基 Hydrophobic amino acids(AILFWV) | 26.4 | 35.4 | 28.9 | 32.4 | 39.2 | 35.8 |
D Asp | 8.8 | 6.2 | 6.4 | 6.3 | 5.9 | 5.8 |
E Glu | 5.9 | 6.3 | 5.1 | 6.9 | 4.6 | 3.7 |
C Cys | 0.2 | 0.5 | 0 | 0.1 | 0.6 | 0.3 |
Y Tyr | 4.6 | 4.3 | 4.0 | 3.9 | 1.9 | 4.0 |
H His | 1.9 | 2.3 | 1.9 | 1.3 | 2.4 | 2.7 |
K Lys | 12.4 | 7.8 | 9.2 | 6.2 | 1.0 | 4.4 |
R Arg | 2.0 | 3.5 | 2.8 | 3.9 | 7.7 | 4.9 |
蛋白序列登陆号Protein accession numbers in GenBank database | AYU99970 | AKM20831 | CAD46929 | AHB61202 | QGL52623 | AIL54323 |
来源Source | Staphylococcus aureus STA | Streptococcus zooe-pidemicus MF002 | Streptococcus aga- lactiae NEM316 | Bacillus sp. A50 | Microbacterium sp. H14 | Vibrio sp. FC509 |
图1 HylS与PL8家族透明质酸裂解酶的部分氨基酸序列比对 序列的名称为GenBank数据库蛋白序列登陆号:AAK99090来自天蓝色链霉菌A3(2),PDB登陆号为2WCO、2WDA和2X03;AHB61202来自芽孢杆菌属A50;AAK74491来自肺炎链球菌TIGR4;CAD46929来自无乳链球菌NEM316,PDB登陆号为1F1S、1I8Q和1LXM;QGL52623来自细小杆菌属H14;相似氨基酸用方框标注;一致氨基酸用黑影标注;催化氨基酸残基用“*”标记;结合底物透明质酸的芳香族氨基酸残基用“#”标记
Fig. 1 Partial amino acid sequences alignment of HylS and PL8 HA lyases Sequences are named as the protein accession numbers in GenBank database. AAK99090 from Streptomyces coelicolor A3(2)(PDB ID:2WCO,2WDA and 2X03). AHB61202 from Bacillus sp. A50. AAK74491 from Streptococcus pneumoniae TIGR4. CAD46929 from S. agalactiae NEM316(PDB ID:1F1S,1I8Q and 1LXM). QGL52623 from Microbacterium sp. H14. Identical and similar amino acids are shaded in black and framed,respectively. The catalytic group is marked using “*”. The hydrophobic patch in hyaluronic acid of bound substrate is marked using “#”
图2 纯化的rHylS的SDS-PAGE分析 M:蛋白标准品;1:未诱导的重组菌破碎液上清;2:诱导后的重组菌破碎液上清;3:纯化后的重组酶HylS
Fig. 2 SDS-PAGE analysis of the purified rHylS M:Protein markers. 1:Cell lysate of uninduced transformant. 2:Cell lysate of induced transformant. 3:Purified recombinant HylS
试剂 Reagent | 相对酶活力 Relative activity /% | 试剂 Reagent | 相对酶活力 Relative activity/% | |
---|---|---|---|---|
None | 100.0±1.4 | CaCl2 | 60.7±2.0 | |
MnSO4 | 178.3±1.6 | NiSO4 | 40.7±0.9 | |
KCl | 123.1±2.8 | PbAc | 39.8±0.9 | |
FeSO4 | 103.8±2.3 | ZnSO4 | 29.8±0.5 | |
NaCl | 99.9±0.8 | AlCl3 | 0 | |
MgSO4 | 97.9±1.1 | β-Mercaptoethanol | 162.7±1.8 | |
LiCl | 93.8±2.4 | SDS | 91.7±2.1 | |
CuSO4 | 86.5±1.5 | CTAB | 65.2±1.0 | |
CoCl2 | 77.4±1.0 | EDTA | 42.8±0.7 |
表2 金属离子和化学试剂对纯化rHylS酶活力影响
Table 2 Effects of metal ions and chemical reage-nts on the purified rHylS
试剂 Reagent | 相对酶活力 Relative activity /% | 试剂 Reagent | 相对酶活力 Relative activity/% | |
---|---|---|---|---|
None | 100.0±1.4 | CaCl2 | 60.7±2.0 | |
MnSO4 | 178.3±1.6 | NiSO4 | 40.7±0.9 | |
KCl | 123.1±2.8 | PbAc | 39.8±0.9 | |
FeSO4 | 103.8±2.3 | ZnSO4 | 29.8±0.5 | |
NaCl | 99.9±0.8 | AlCl3 | 0 | |
MgSO4 | 97.9±1.1 | β-Mercaptoethanol | 162.7±1.8 | |
LiCl | 93.8±2.4 | SDS | 91.7±2.1 | |
CuSO4 | 86.5±1.5 | CTAB | 65.2±1.0 | |
CoCl2 | 77.4±1.0 | EDTA | 42.8±0.7 |
图5 rHylS酶解透明质酸所得产物的TLC和ESI-MS分析 TLC:M1和M2分别为壳二糖和壳四糖;1-7:分别为rHylS在0.5、1、2、4、6、8和10 h下降解2%HA的酶解产物;8-13:分别为rHylS在0.5、2、4、6、8和10 h下降解5% HA的酶解产物;ESI-MS:rHylS在2 h下降解2%HA的酶解产物
Fig. 5 TLC and ESI-MS analysis of the degraded products of HA by rHylS TLC:M1 and M2 are chitobiose and chitotetraose,respectively. 1-7:rHylS degraded 2% HA for 0.5,1,2,4,6,8 and 10 h,respectively. 8-13:rHylS degraded 5% HA for 0.5,2,4,6,8 and 10 h,respectively. ESI-MS:rHylS degraded 2% HA for 2 h
图6 rHylS酶解透明质酸所得产物的抗氧化活性 rHylS酶解产物对各自由基的清除能力采用最小差异检验(P<0.01)进行标记,各自由基实验组分别标记,不同字母代表差异显著
Fig. 6 Antioxidant activity of the degraded products of HA by rHylS The scavenging ability of the degraded products by rHylS to each free radical is labeled individually according to the least significant difference test among values(P<0.01),and different letters refer to significant differences
透明质酸裂解酶 HA lyase | HylS | HAase | - | HAase-B | HCLaseM | HCLase |
---|---|---|---|---|---|---|
最适pH Optimal pH | 5.0 | 6.0 | 6.3 | 6.5 | 7.0 | 8.0 |
pH稳定性a pH stabilitya | 100%/ pH 5.0/60 min | 95% pH 5.0/60 min | - | 75% pH 5.0/60 min | 70% pH 5.0/12 h | - |
最适温度 Optimal temperature/℃ | 45 | 37 | 40 | 44 | 35 | 30 |
热稳定性b Thermostabilityb | 60%/50℃/60 min | 70%/50℃/60 min | - | 50%/50℃/10 min | 50%/40℃/60 min | 50%/40℃/60 min |
激活剂 Activators | Mn2+/K+ β-Mercaptoethanol | Ca2+/Mg2+/Co2+ | Mg2+ | Ca2+/Mg2+/Ni2+ | - | Li+/Na+/K+ |
抑制剂 Inhibitors | Ni2+/Zn2+/Al3+/Pb2+ EDTA | Zn2+/Cu2+ | Zn2+/Al3+/Cu2+ /Fe2+/Mn2+ | Zn2+/ Cu2+/ SDS/EDTA | Hg2+/SDS | Ag+/Co2+/Hg2+/ Ni2+/Cu2+/Zn2+/Fe3+/Cr3+ |
底物特异性c Substrate specificityc | HA | - | HA/CS/DS | HA/CS | HA/CS/DS | HA/CS |
比活d Specific activity / (U·mg-1)d | 1.6×105 | 10.5 | 8.1×104 | 1.0×106 | 278.3 | 4.5×105 |
检测方法e Measurement methode | 紫外法 UV spectrophotometry | 二硝基水杨酸比色法 DNS colorimetry | CATB浊度法 CATB turbidimetry | BSA浊度法 BSA turbidimetry | 紫外法 UV spectroph-otometry | 紫外法 UV spectrophotometry |
降解模式 Degrading pattern | 内切 Endo | - | 外切 Exo | 内切 Endo | 内切 Endo | 内切 Endo |
来源 Source | S. aureus STA | S. zooepidemicus MF002 | S. agalactiae NEM316 | Bacillus sp. A50 | Microbacterium sp.H14 | Vibrio sp. FC509 |
参考文献References | This study | [17] | [18-19] | [20] | [21] | [22] |
表3 PL8家族透明质酸裂解酶的酶学特性
Table 3 Characteristics of HA lyases in PL8 family
透明质酸裂解酶 HA lyase | HylS | HAase | - | HAase-B | HCLaseM | HCLase |
---|---|---|---|---|---|---|
最适pH Optimal pH | 5.0 | 6.0 | 6.3 | 6.5 | 7.0 | 8.0 |
pH稳定性a pH stabilitya | 100%/ pH 5.0/60 min | 95% pH 5.0/60 min | - | 75% pH 5.0/60 min | 70% pH 5.0/12 h | - |
最适温度 Optimal temperature/℃ | 45 | 37 | 40 | 44 | 35 | 30 |
热稳定性b Thermostabilityb | 60%/50℃/60 min | 70%/50℃/60 min | - | 50%/50℃/10 min | 50%/40℃/60 min | 50%/40℃/60 min |
激活剂 Activators | Mn2+/K+ β-Mercaptoethanol | Ca2+/Mg2+/Co2+ | Mg2+ | Ca2+/Mg2+/Ni2+ | - | Li+/Na+/K+ |
抑制剂 Inhibitors | Ni2+/Zn2+/Al3+/Pb2+ EDTA | Zn2+/Cu2+ | Zn2+/Al3+/Cu2+ /Fe2+/Mn2+ | Zn2+/ Cu2+/ SDS/EDTA | Hg2+/SDS | Ag+/Co2+/Hg2+/ Ni2+/Cu2+/Zn2+/Fe3+/Cr3+ |
底物特异性c Substrate specificityc | HA | - | HA/CS/DS | HA/CS | HA/CS/DS | HA/CS |
比活d Specific activity / (U·mg-1)d | 1.6×105 | 10.5 | 8.1×104 | 1.0×106 | 278.3 | 4.5×105 |
检测方法e Measurement methode | 紫外法 UV spectrophotometry | 二硝基水杨酸比色法 DNS colorimetry | CATB浊度法 CATB turbidimetry | BSA浊度法 BSA turbidimetry | 紫外法 UV spectroph-otometry | 紫外法 UV spectrophotometry |
降解模式 Degrading pattern | 内切 Endo | - | 外切 Exo | 内切 Endo | 内切 Endo | 内切 Endo |
来源 Source | S. aureus STA | S. zooepidemicus MF002 | S. agalactiae NEM316 | Bacillus sp. A50 | Microbacterium sp.H14 | Vibrio sp. FC509 |
参考文献References | This study | [17] | [18-19] | [20] | [21] | [22] |
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