壳寡糖化学修饰对云芝漆酶催化活性和酶学性质的影响

doi:10.7503/cjcu20140771

摘要/Abstract

摘要：

利用壳寡糖(COS)对纯化后的云芝漆酶(EC 1.10.3.2)进行化学修饰以改善其活性和稳定性. 最优化学修饰条件: 温度5 ℃, pH=4.0, 100 mg NaIO₄, 20 mg COS. 在此条件下得到的修饰酶(COS-TvL)的活性(456.00 U/mg)为天然酶(TvL)活性(314.39 U/mg)的145.04%. 十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)结果表明, TvL的3条肽链相对分子质量为58100, 45200和21600, 而 COS-TvL的3条肽链相对分子质量为62900, 53300和39700, 说明酶分子修饰成功, 氨基修饰率为30.7%. TvL的最适pH值在4.0附近, 而COS-TvL的最适pH值在3.5~4.0之间. TvL 的最适温度为40 ℃, 而COS-TvL的最适温度为45 ℃. 动力学研究结果显示, TvL的K_m=9.929 mmol/L, v_max=714.29 mmol/(mg·min); 而COS-TvL的K_m=8.989 mmol/L, v_max= 1250 mmol/(mg·min). 通过红外光谱、紫外吸收光谱、紫外差示光谱和荧光发射光谱对TvL和COS-TvL进行了结构表征.

关键词: 漆酶, 化学修饰, 酶活性, 壳寡糖

Abstract:

Chemical modification is an important pathway to transform the catalytic activity and enzymological properties of enzymes. In order to improve the catalytic activity and enzymological stability of laccase from Trametes versicolor(EC 1.10.3.2, TvL), the enzyme was purified by DEAE 52 weak anion exchange resin chromatography. The pure enzyme was activated by NaIO₄. The activated enzyme was chemically modified with chitooligosaccharides(COS, average relative molecular weight 5000). The optimal modification conditions by single factor and orthogonal experiments were: temperature 5 ℃, pH=4.0, 100 mg NaIO₄, 20 mg COS. Under these conditions the activity of modified enzyme(COS-TvL, 456.00 U/mg) was 145.04% of that of unmodified enzyme(TvL, 314.39 U/mg). The results of SDS-PAGE experiments indicated that the relative molecular weights of 3 polypeptides of TvL were 58100, 45200 and 21600, respectively. But the relative molecular weights of 3 polypeptides of COS-TvL were 62900, 53300 and 39700, respectively. This meant that the modification experiments were successful. The amino modification rates changed from 0 of TvL to 30.7% of COS-TvL. The optimal pH values of TvL and COS-TvL were 4.0 and 3.5—4.0, respectively. The optimal temperatures of TvL and COS-TvL were 40 and 45 ℃, respectively. The results of kinetic study indicated that K_m and v_max of TvL were 9.929 mmol/L and 714.29 mmol/(mg·min). But the K_m and v_max of COS-TvL were 8.989 mmol/L and 1250 mmol/(mg·min), respectively. The infrared spectra, the ultraviolet absorption spectra, the ultraviolet differential spectra, and fluorescence emission spectra of TvL and COS-TvL were also studied and analyzed.

Key words: Laccase, Chemical modification, Enzyme activity, Chitooligosaccharide

中图分类号:

O629.8
Q554

TrendMD:

黎春怡, 黄卓烈, 王春晓. 壳寡糖化学修饰对云芝漆酶催化活性和酶学性质的影响. 高等学校化学学报, 2015, 36(4): 704.

LI Chunyi, HUANG Zhuolie, WANG Chunxiao. Effects of Chemical Modification with Chitooligosaccharides on Catalytic Activity and Enzymological Properties of Laccase from Trametes versicolor^†. Chem. J. Chinese Universities, 2015, 36(4): 704.

图/表 15

Fig.1 Chromatographic profile of TvL on DEAE 52 colum(ϕ 2.5 cm×40 cm)

Table 1 Data of TvL purification

Purification step	Total volume/mL	Total protein/mg	Total activity/U	Specific activity/ (U·mg^-1)	Activity recovery(%)	Purification multiple
Coarse TvL	6	26.3	3111.55	118.31	100	1
Purified TvL	4	2.31	726.24	314.39	23.3	2.66

Fig.2 PAGE diagram of TvL Lane 1: Coarse TvL; lane 2: Purified TvL of tube 24;lane 3: Purified TvL of tube 25.

Fig.3 Results of single factor experiments The letters in Fig.3 were the results of Duncan’s new multiple range test(P<0.05). The same letters in the figure mean not significant difference. The different letters in the figure meant significant difference.

Fig.4 Chromatographic profile of COS-TvL on DEAE 52 colume(ϕ 2.5 cm×40 cm)

Fig.5 PAGE diagram of COS-TvL Lane 1: Unpurified COS-TvL; lane 2: Purified COS-TvL of tube 20; lane 3: Purified TvL.

Table 2 Data of COS-TvL purification

Purification step	Total volume/mL	Total protein/mg	Total activity/U	Specific activity/ (U·mg^-1)	Activity recovery(%)	Purification multiple
Before purification	4.2	9.66	4404.96	456.00	100	1
After purification	4	1.39	699.39	503.16	15.9	1.10

Fig.6 SDS-PAGE diagram of TvL and COS-TvL Lane 1: Protein markers; lane 2: Natural TvL; lane 3: TvL modified by COS(COS-TvL).

Fig.7 Optimal pH of TvL(a) and COS-TvL(b)

Fig.8 Effect of incubated time at different pH on specific activity of TvL(a, b, c) and COS-TvL(d, e, f) a, d. pH=3.0; b, e. pH=4.0; c, f. pH=5.0.

Fig.9 Optimal temperature of TvL(a) and COS-TvL(b)

Fig.10 Incubated time-specific activity curves of TvL(a, b) and COS-TvL(c, d) at 50 ℃(b, d) and 60 ℃(a, c)

Fig.11 Storage stability of TvL(a, b) and COS-TvL(c, d) a, d. Cold storage; b, c. room storage.

Fig.12 Lineweaver-Burk curves of TvL(a) and COS-TvL(b) in 0.05 mol/L citrate buffer sollution(pH=4.0 )

Fig.13 Fluorescent three-dimensional spectra of TvL(A) and COS-TvL(B) in the buffer

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