Effects of Carbohydrate-binding Modules on the Enzymatic Properties of Xylanase

doi:10.13560/j.cnki.biotech.bull.1985.2023-1032

Abstract

Abstract:

【Objective】This study is aimed to explore the binding ability of different sources of CBM to beech-xylanan, and to fuse exogenous CBM with high binding ability to the C-terminal and N-terminal of Streptomyces L10904 xylanase（XYN）, to explore the effects of exogenous CBM on the enzymatic properties of xylanase. 【Method】First, through the substrate adsorption method, the concentration of CBM in the solution before and after adsorption was detected by Coomassie Brilliant blue G250 method, and the substrate binding rate of CBM was calculated. CBM1 and CBM4 with better xylan binding ability were screened. In order to explore the effect of the fusion location of CBM with high substrate binding ability on the enzymatic properties of xylanase, CBM1 and CBM4 were fused with the C-terminal and N-terminal of XYN by flexible binding peptide, and four recombinant enzymes were obtained by expression in Escherichia coli BL21（DE3）. They were named CBM1-XYN, XYN-CBM1, CBM4-XYN, and XYN-CBM4.【Result】The binding rates of CBM1 and CBM4 to xylan were 89% and 95%, respectively. The specific activities of XYN, CBM1-XYN, XYN-CBM1, CBM4-XYN and XYN-CBM4 were 32 274.81, 49 342.21, 602.48, 230.42 and 2 362.24 U/mg, respectively, measured at 60℃ and pH 7.0. The specific activities of CBM1-XYN were 1.5 times higher than the specific activities of XYN. The analysis of enzymatic properties showed that CBM1 improved the temperature stability and pH stability of XYN, XYN and CBM1-XYN were incubated at 60℃ for 1 h, and the residual enzyme activity of CBM1-XYN and XYN was 81% and 28%, respectively. In the pH range of 3-11, CBM1-XYN maintained more than 90% enzyme activity after incubation at 4℃ for 12 h. 【Conclusion】Streptomyces derived xylanase was heterogeneically expressed in E. coli BL21（DE3）, and CBM1 and CBM4 with high substrate binding rates were screened. CBM was successfully fused to XYN by protein fusion technology, and CBM1-XYN with improved enzymatic properties was obtained, which improved the temperature stability, pH tolerance and specific enzyme activity of xylanase.

Key words: carbohydrate-binding modules, xylanase, fusion protein, soluble expression, enzyme activity, temperature stability, pH stability

JIANG Wen-ping, RAN Qiu-ping, LIU Jia-shu, ZHANG Hui-min, ZHANG Di, JIANG Zheng-bing, LI Hua-nan. Effects of Carbohydrate-binding Modules on the Enzymatic Properties of Xylanase[J]. Biotechnology Bulletin, 2024, 40(5): 269-279.

Figures/Tables 10

References 41

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代号Code name	名称 Name	生物信息数据库GenBank	来源 Source
代号Code name	名称 Name	生物信息数据库GenBank	菌属 Genus	酶 Enzyme
1	CBM1 1号	BAD01163.1	Trametes hirsuta	Endoglucanase
2	CBM1 2号	AAF35251.1	T. versicolor	Cellobiohydrolase
3	CBM1 3号	CAM98445.1	Acremonium thermophilum	Cellulose 1,4-beta-cellobiosidase
4	CBM1 4号	CAA83846.1	Trichoderma reesei	Endo-1,4-beta-glucanase V
5	CBM1 5号	CAA37878.1	T. viride	Cellobiohydrolase
6	CBM1 6号	AAQ21383.1	T. viride	Endoglucanase III
7	CBM1 7号	AAQ76092.1	T. viride	Eellobiohydrolase I
8	CBM1 8号	AAQ76094.1	T. viride	Cellobiohydrolase II
1#	CBM4	AF039030.1	Hungateiclostridium thermocellum JW20	Cellulose 1,4-beta-cellobiosidase
2#	CBM3 1号	ABN54273.1	H. thermocellum ATCC 27405	Cellulosome anchoring protein cohesin region
3#	CBM3 2号	ABN51281.1	H. thermocellum ATCC 27405	Glycoside hydrolase family 9
4#	CBM2	AAB42115.1	Thermobifida fusca YX	Beta-1,4-endoglucanase precursor

代号Code name	名称 Name	生物信息数据库GenBank	来源 Source
代号Code name	名称 Name	生物信息数据库GenBank	菌属 Genus	酶 Enzyme
1	CBM1 1号	BAD01163.1	Trametes hirsuta	Endoglucanase
2	CBM1 2号	AAF35251.1	T. versicolor	Cellobiohydrolase
3	CBM1 3号	CAM98445.1	Acremonium thermophilum	Cellulose 1,4-beta-cellobiosidase
4	CBM1 4号	CAA83846.1	Trichoderma reesei	Endo-1,4-beta-glucanase V
5	CBM1 5号	CAA37878.1	T. viride	Cellobiohydrolase
6	CBM1 6号	AAQ21383.1	T. viride	Endoglucanase III
7	CBM1 7号	AAQ76092.1	T. viride	Eellobiohydrolase I
8	CBM1 8号	AAQ76094.1	T. viride	Cellobiohydrolase II
1#	CBM4	AF039030.1	Hungateiclostridium thermocellum JW20	Cellulose 1,4-beta-cellobiosidase
2#	CBM3 1号	ABN54273.1	H. thermocellum ATCC 27405	Cellulosome anchoring protein cohesin region
3#	CBM3 2号	ABN51281.1	H. thermocellum ATCC 27405	Glycoside hydrolase family 9
4#	CBM2	AAB42115.1	Thermobifida fusca YX	Beta-1,4-endoglucanase precursor

引物 Primers	序列 Sequence（5'-3'）
pET23a-F	gctagcatgactggtggacagcaaatgggt
pET23a-R	gtatatctccttcttaaagttaaacaaaattatttctagagggaaaccgtt
sfgfp-sumo-F	aactttaagaaggagatatacatgcatcatcaccatcaccatatggtgagcaagggc
sfgfp-sumo-R1	ctgccgcttaccgtcctgctgtccaccaatctgttctctgtgagc
sfgfp-sumo-R2	accgcactggccccagacggctccaccaatctgttctctgtg
sfgfp-sumo-R3	tctttcatacaaaaggtcgtTtccaccaatctgttctctgtgagcctc
xyn-F	gctcacagagaacagattggtggacagcaggacggtaagcggcag
xyn-R	tttgctgtccaccagtcatgctagccatcagccgctgaccgtgatgttcga
cbm1-xyn-F	cacagagaacagattggtggagccgtctggggccagtgcggt
cbm1-xyn-R	gctgtccaccagtcatgctagctcagccgctgaccgtgat
cbm4-xyn-F	gaggctcacagagaacagattggtggaAacgaccttttgtatgaaaga
cbm4-xyn-R	tccaccagtcatgctagctcagccgctgaccgtgatgttcga
xyn-cbm1-R	tttgctgtccaccagtcatgctagcctggcactgcgagtagt
xyn-cbm4-R	tttgctgtccaccagtcatgctagcaggatcgtagagagatacatcatcaagg
cbm1-F	ggcggtggtgggtcgggtggcggtggctcggccgtctggggccag
cbm1-R	accgcactggccccagacggctccaccaatctgttctctgtg
cbm4-F	ggcggtggtgggtcgggtggcggtggctcgAacgaccttttgtat
cbm4-R	cgacccaccaccgcccgagccaccgccaccaggatcgtagagagatac

引物 Primers	序列 Sequence（5'-3'）
pET23a-F	gctagcatgactggtggacagcaaatgggt
pET23a-R	gtatatctccttcttaaagttaaacaaaattatttctagagggaaaccgtt
sfgfp-sumo-F	aactttaagaaggagatatacatgcatcatcaccatcaccatatggtgagcaagggc
sfgfp-sumo-R1	ctgccgcttaccgtcctgctgtccaccaatctgttctctgtgagc
sfgfp-sumo-R2	accgcactggccccagacggctccaccaatctgttctctgtg
sfgfp-sumo-R3	tctttcatacaaaaggtcgtTtccaccaatctgttctctgtgagcctc
xyn-F	gctcacagagaacagattggtggacagcaggacggtaagcggcag
xyn-R	tttgctgtccaccagtcatgctagccatcagccgctgaccgtgatgttcga
cbm1-xyn-F	cacagagaacagattggtggagccgtctggggccagtgcggt
cbm1-xyn-R	gctgtccaccagtcatgctagctcagccgctgaccgtgat
cbm4-xyn-F	gaggctcacagagaacagattggtggaAacgaccttttgtatgaaaga
cbm4-xyn-R	tccaccagtcatgctagctcagccgctgaccgtgatgttcga
xyn-cbm1-R	tttgctgtccaccagtcatgctagcctggcactgcgagtagt
xyn-cbm4-R	tttgctgtccaccagtcatgctagcaggatcgtagagagatacatcatcaagg
cbm1-F	ggcggtggtgggtcgggtggcggtggctcggccgtctggggccag
cbm1-R	accgcactggccccagacggctccaccaatctgttctctgtg
cbm4-F	ggcggtggtgggtcgggtggcggtggctcgAacgaccttttgtat
cbm4-R	cgacccaccaccgcccgagccaccgccaccaggatcgtagagagatac

金属离子和化学试剂 Metal ion and chemical reagent	相对酶活Relative enzyme activity/%
金属离子和化学试剂 Metal ion and chemical reagent	XYN	CBM1-XYN
FeCl₃	92.6±1.67	101.9±5.07
NaCl	95.4±4.14	109.6±3.29
CaCl₂	93.4±1.84	106.8±7.74
MnSO₄	73.1±1.89	92.6±1.22
KCl	101.7±4.14	104.9±2.85
FeSO₄	75.3±0.66	81±2.11
CoCl₂	98.8±1.71	101.4±1.13
MgCl₂	94.8±2.20	108.7±1.66
（NH₄）₂SO₄	98.4±1.45	101±1.02