碳水化合物结合域对木聚糖酶酶学性质的影响

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

摘要/Abstract

摘要：

【目的】旨在探究不同来源碳水化合物结合域（CBM）对山毛榉木聚糖的结合能力，并将具有较高结合能力的外源CBM融合到链霉菌L10904木聚糖酶（XYN）的C端和N端，以探究外源CBM对木聚糖酶酶学性质的影响。【方法】通过底物吸附方法，利用考马斯亮蓝G250法检测溶液中CBM在吸附前后的浓度，计算CBM的底物结合率，筛选到了结合木聚糖能力较好的CBM1和CBM4。为了探究对底物结合能力高的CBM融合位置对木聚糖酶酶学性质的影响，将CBM1和CBM4通过柔性连接肽分别与XYN的C端和N端融合，并在大肠杆菌中表达获得4种重组酶，分别命名为CBM1-XYN、XYN-CBM1、CBM4-XYN和XYN-CBM4。【结果】CBM1和CBM4与木聚糖结合率分别为89%和95%。在60℃, pH 7.0反应条件下，XYN、CBM1-XYN、XYN-CBM1、CBM4-XYN和XYN-CBM4的比活力分别是32 274.81、49 342.21、602.48、230.42和2 362.24 U/mg，CBM1-XYN比活力较XYN比活力提高了1.5倍。酶学性质分析表明，CBM1使XYN温度稳定性和pH稳定性得到了提高，将XYN和CBM1-XYN分别在60℃孵育1 h，CBM1-XYN残余酶活力和XYN残余酶活力分别为81%和28%；在pH 3-11范围内，CBM1-XYN在4℃孵育12 h后能够保持90%以上的酶活力。【结论】在大肠杆菌中成功异源表达了链霉菌来源的木聚糖酶，筛选到了对底物结合率高的两种CBM1和CBM4，并通过蛋白质融合技术成功将CBM融合到XYN上，获得酶学性质得到改良的CBM1-XYN，能够提高木聚糖酶的温度稳定性、pH耐受性及比酶活。

关键词: 碳水化合物结合域, 木聚糖酶, 融合蛋白, 可溶性表达, 比活力, 温度稳定性, pH稳定性

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

蒋文萍, 冉秋萍, 刘家书, 张慧敏, 张迪, 江正兵, 李华南. 碳水化合物结合域对木聚糖酶酶学性质的影响[J]. 生物技术通报, 2024, 40(5): 269-279.

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.

图/表 10

参考文献 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