Heterologous Expression and Characterization of the Hyaluronic Acid Lyase HylS from Staphylococcus aureus

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

Abstract

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

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.

Figures/Tables 9

References 31

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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

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

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

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

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
参考文献References	This study	[17]	[18-19]	[20]	[21]	[22]