Effects of Chemical Modification with Chitooligosaccharides on Catalytic Activity and Enzymological Properties of Laccase from Trametes versicolor†

doi:10.7503/cjcu20140771

Abstract

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

CLC Number:

O629.8
Q554

TrendMD:

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

Figures/Tables 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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