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

doi:10.13560/j.cnki.biotech.bull.1985.2021-0129

摘要/Abstract

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。

关键词: 金黄色葡萄球菌, 透明质酸, 裂解酶, 酶学性质, 不饱和寡糖, 抗氧化性

Abstract:

Hyaluronidases degrade hyaluronic acid into low molecular weight oligosaccharides with biological activities such as antioxidant. Hyaluronidases from microorganisms have been a hot area of research in the development of hyaluronidase,because of their diverse enzymatic properties and easily recombinant expression. A potential hyaluronic acid（HA）lyase（hylS）gene from Staphylococcus aureus was identified by genome sequencing. It was successfully expressed by Escherichia coli BL21（DE3）. The characteristics of the recombinant enzyme were analyzed,and the antioxidation of its enzymolysis products was determined. The optimal pH and temperature of the purified recombinant enzyme rHylS was 5.0 and 45℃,respectively. It showed specific degradation of HA,and specific activity towards the substrate was（1.6×10⁵±5.4）U/mg. It was an endo-hyaluronate lyase that degraded HA to produce low molecular weight unsaturated oligosaccharides（HAP）. The scavenging abilities of HAP on ABTS,DPPH,superoxide anions and hydroxyl radicals were significantly higher than that of high molecular weight HA without degradation,and there was a positive correlation with the concentration. All these excellent characters indicate that rHylS from S. aureus is of enzymatic property,and it may be used to produce unsaturated hyaluronic acid oligosaccharides of low molecular weight and antioxidant properties.

Key words: Staphylococcus aureus, hyaluronic acid, lyase, enzymatic characterization, unsaturated oligosaccharide, antioxidant

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.

图/表 9

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

图3 纯化的rHylS的最适反应pH（A）和pH稳定性（B）

Fig. 3 Optimal pH（A）and pH stability（B）of the purified rHylS

图4 纯化的rHylS的最适温度（A）和热稳定性（B）

Fig. 4 Optimal temperature（A）and thermostability（B）of the purified rHylS

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

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 stability^a	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 Thermostability^b	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 specificity^c	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 method^e	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
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