具有辅助降解纤维素功能的大斑刚毛座腔菌糖苷水解酶GH61的鉴定、异源表达及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0996

摘要/Abstract

摘要：

糖苷水解酶61家族（GH61）属于一类同时具有氧化作用和水解作用的纤维素降解酶类，既具有微弱的纤维素内切酶活性，也可通过氧化作用破坏纤维素晶体结构促进纤维素酶对木质纤维素的降解，在生物质资源的利用方面具有潜在的应用价值。对大斑刚毛座腔菌Setosphaeria turcica的GH61家族基因进行鉴定及生物信息学分析，通过转录组数据分析及荧光定量PCR验证，筛选出受玉米秸秆木质纤维素底物诱导表达的GH61家族基因StGH61-11。将StGH61-11进行重组表达，使用2,6-二甲氧基苯酚氧化反应测定其酶活力并优化诱导条件，表征其酶学性质并检测其对纤维素酶水解木质纤维素的促进作用。结果表明，在S. turcica基因组中存在21个GH61家族基因，且S. turcica在玉米秸秆的诱导下滤纸酶活明显增加，对其进行转录组分析发现，在以玉米秸秆为碳源时GH61家族基因中有11个基因的表达量增加。将其中StGH61-11基因在大肠杆菌中诱导表达，最佳诱导条件为25℃、1 mmol/L IPTG诱导9 h，此时获得的蛋白比活力可达到（54.08±1.67）U/g。重组蛋白StGH61-11的最佳催化条件为温度50℃、pH 5，在最佳催化条件下可以显著提高纤维素酶水解玉米秸秆的活性，协同度最高可达2.5，糖化率最高可达46.5%。

关键词: GH61糖苷水解酶家族, 多糖单加氧酶, 纤维素酶, AA9, 大斑刚毛座腔菌, 协同降解, 异源表达

Abstract:

Glycoside hydrolase 61 family（GH61）belongs to the category of both oxidation and hydrolysis of cellulose degradation enzymes, it has a weak cellulose endonuclease, also can promote cellulase by oxidative damage to cellulose crystal structure on the degradation of lignocellulose, in the use of biomass resources it has potential application value. The GH61 family gene of Setosphaeria turcica was identified and bioinformatics to it was analyzed. Through transcriptome data analysis and fluorescence quantitative PCR verification, the GH61 family gene StGH61-11, induced by the carbon source corn stalk, was screened out. StGH61-11 was heterologous expressed, and its enzyme activity was determined by oxidation reaction with 2,6-dimethoxy-phenol, and the induction conditions were optimized. Its enzymatic properties were explored and the effect of StGH61-11 on promoting cellulase hydrolysis of cellulose was studied. The results showed that there were 21 GH61 family genes in S. turcica genome, and the filter paper enzyme activity of S. turcica significantly increased under the induction of corn stover. Transcriptome analysis of S. turcica showed that the expression levels of 11 genes in GH61 family increased when corn stover was used as carbon source. StGH61-11 was heterologous expressed in Escherichia coli. The optimal induction condition was 1 mmol/L IPTG at 25℃ for 9 h, and the specific activity reached（54.08±1.67）U/g. The optimal catalytic conditions were 50℃ and pH 5 for the recombinant protein StGH61-11 and the recombinant protein significantly improved the activity of cellulase hydrolysis of corn stover, with the highest degree of synergy up to 2.5 and the highest glycosylation rate up to 46.5%.

Key words: GH61 glycoside hydrolase family, lytic polysaccharide monooxygenases, cellulase, AA9, Setosphaeria turcica, synergistic degradation, heterologous expression

马玉倩, 孙东辉, 岳浩峰, 辛佳瑜, 刘宁, 曹志艳. 具有辅助降解纤维素功能的大斑刚毛座腔菌糖苷水解酶GH61的鉴定、异源表达及功能分析[J]. 生物技术通报, 2023, 39(4): 124-135.

MA Yu-qian, SUN Dong-hui, YUE Hao-feng, XIN Jia-yu, LIU Ning, CAO Zhi-yan. Identification, Heterologous Expression and Functional Analysis of a GH61 Family Glycoside Hydrolase from Setosphaeria turcica with the Assisting Function in Degrading Cellulose[J]. Biotechnology Bulletin, 2023, 39(4): 124-135.

图/表 7

表1 实时荧光定量PCR引物序列

Table 1 Primer sequences used in RT-qPCR

引物名称Primer name	序列Sequence（5'-3'）	引物名称Primer name	序列Sequence（5'-3'）
StGH61_1-F	GGCTCTGGAACTGGCAAGAT	StGH61_12-F	CTCCTGGTCCGTCACTGTTC
StGH61_1-R	TAGTACTCGAGGTCCCTGGC	StGH61_12-R	GGCTGGAGAGATCGGTCATG
StGH61_2-F	GTCAGTTTCCCGGGTGCTTA	StGH61_13-F	GGCAGAAGGACCAGATCGAG
StGH61_2-R	GACCACGCTGTTAGTCCCAA	StGH61_13-R	CGCTGTAGAGACCGGGAATC
StGH61_3-F	ACATCAACACGTGGGATCCC	StGH61_14-F	CGACGCCATCCTAGACACTC
StGH61_3-R	TGCGCACGACAGGTAGAATT	StGH61_14-R	TTGAAGTCGATGCCTGGGTC
StGH61_4-F	CGACTTCCGTTGCAACAAGG	StGH61_15-F	CACCAAAGTCGAGCCCTTCT
StGH61_4-R	AACATCACCAGGAGCCTTGG	StGH61_15-R	TTGAGGGAGAGGGAGACGAG
StGH61_5-F	TTGCGCAAATCACAACCTCG	StGH61_16-F	GTGCCAAGGGAGGTCTCTTC
StGH61_5-R	TCGTGGCATGTCATGTTGGA	StGH61_16-R	GGTCGGCAGTGAAAGAGACA
StGH61_6-F	CACCCAGACTGTCACCATCC	StGH61_17-F	GGTTCAAGGTGCAGGAGGAA
StGH61_6-R	CACCCAGACTGTCACCATCC	StGH61_17-R	CTACCCGTCACCTTGAGCTG
StGH61_7-F	GGTCCAAGTTCTCGCAGGAA	StGH61_18-F	AGCTCGACTGCCATGATCTG
StGH61_7-R	CATTCACATTCGAGCCGCTG	StGH61_18-R	GGGGAGCTTGACGTTGATGA
StGH61_8-F	CCACAAGAATGCTAGCCCCA	StGH61_19-F	CAAGGTCTCCAACGCAGCTA
StGH61_8-R	GCACCCTCAATCTTGGTCCA	StGH61_19-R	AAAATTGAGCACCGCCAACC
StGH61_9-F	TGGTTCAAGGTTTCCAGCGA	StGH61_20-F	CATCCCTGCTTGTATTGCGC
StGH61_9-R	AAATGTAGAATTGCGCGCCG	StGH61_20-R	GGAACGAGACGGTTGATGGT
StGH61_10-F	GGGCAGTGATGTGAAGAGCT	StGH61_21-F	AAGATCGACGAGCAAGGCAT
StGH61_10-R	GGAGCACGCGAGGTAGAATT	StGH61_21-R	GACGCATACCCAGGAATCGT
StGH61_11-F	AGCCTCTCTTCCTCGGACAT	β-tubulin-F	GTGCGCAAGGAGGCTGAGGG
StGH61_11-R	TTTTGGCGTCGCTGACTTTG	β-tubulin-R	CATGAAGAAATGGAGACGGGGGAA

表2 大斑刚毛座腔菌糖苷水解酶GH61家族蛋白性质预测

Table 2 Prediction of the protein properties of the glycoside hydrolase GH61 family from S. turcica

基因 Gene	检索号 Accession number	氨基酸数目 Number of amino acids	分子量 Molecular weight/kD	等电点 pI	信号肽长度 Signal peptide length/aa	蛋白定位 Protein location
StGH61-1	XP_008027730	250	26.69	8.26	16	胞外Extracellular
StGH61-2	XP_008031219	334	36.13	5.77	18	胞外Extracellular
StGH61-3	XP_008024527	221	23.46	8.33	17	胞外Extracellular
StGH61-4	XP_008025833	418	43.38	7.63	17	胞外Extracellular
StGH61-5	XP_008021808	266	28.81	8.24	21	胞外Extracellular
StGH61-6	XP_008023212	291	32.06	6.16	22	膜结合溶酶体Lysosomes
StGH61-7	XP_008024957	248	26.32	7.69	20	胞外Extracellular
StGH61-8	XP_008025253	329	34.56	5.98	22	分泌到胞外 Extracellular（Secreted）
StGH61-9	XP_008030985	149	15.76	7.83	—	分泌到胞外 Extracellular（Secreted）
StGH61-10	XP_008022828	236	25.55	9.00	20	膜结合线粒体Mitochondria
StGH61-11	XP_008023697	229	23.52	8.49	16	胞外Extracellular
StGH61-12	XP_008023696	258	27.49	5.47	22	胞外Extracellular
StGH61-13	XP_008028494	325	34.02	6.31	18	胞外Extracellular
StGH61-14	XP_008028319	292	30.73	6.28	21	胞外Extracellular
StGH61-15	XP_008025489	472	49.64	7.44	19	胞外Extracellular
StGH61-16	XP_008027303	222	23.26	7.69	20	胞外Extracellular
StGH61-17	XP_008027295	246	25.92	8.44	18	胞外Extracellular
StGH61-18	XP_008024355	344	34.75	6.49	17	胞外Extracellular
StGH61-19	XP_008022590	243	25.06	8.90	17	胞外Extracellular
StGH61-20	XP_008026441	233	24.21	7.67	18	胞外Extracellular

图1 大斑刚毛座腔菌糖苷水解酶GH61家族蛋白结构域及进化分析 A：大斑刚毛座腔菌糖苷水解酶GH61家族蛋白结构域；B：大斑刚毛座腔菌糖苷水解酶GH61家族蛋白进化关系

Fig. 1 Protein domains and evolutionary analysis of the GH61 family of glycoside hydrolases from S. turcica A: Domain of GH61 family of glycoside hydrolases from S. turcica. B: Evolutionary relationship of glycoside hydrolases GH61 family proteins from S. turcica

图2 大斑刚毛座腔菌纤维素酶的滤纸酶活及GH61家族基因表达分析 A：大斑刚毛座腔菌纤维素酶的滤纸酶活；B：大斑刚毛座腔菌中GH61家族糖苷水解酶基因的表达图谱；C：RT-qPCR验证大斑刚毛座腔菌中GH61家族糖苷水解酶基因相对表达量。不同小写字母表示同一基因在不同处理下存在显著差异（P<0.05）

Fig. 2 Filter paper enzymatic activity and GH61 family gene expression analysis of cellulase from S. turcica A: Filter paper enzymatic activity of cellulase from S. turcica. B: Expression map of GH61 family glycoside hydrolase genes in S. turcica. C: RT-qPCR verifies the relative expression of GH61 family glycoside hydrolase genes in S. turcica. Different normal letters indicate significant differences among different treatments at 0.05 level

图3 StGH61-11重组蛋白异源表达及优化诱导条件 A：目的片段StGH61-11的PCR扩增产物琼脂糖凝胶电泳结果, 1-3：StGH61-11的cDNA片段, M：DNA marker；B：PCR验证重组转化子的琼脂糖凝胶电泳结果, 1-3：Pet32a-StGH61-11 重组质粒, M：DNA marker；C：SDS-PAGE 检测pET32a-StGH61-11（BL21）重组菌表达蛋白, 0：Pet32a-StGH61-11-BL21（DE3）重组菌未诱导对照, 1 ：Pet32a-StGH61-11-BL21（DE3）重组菌经IPTG诱导后, M ：预染蛋白分子量标准；D：诱导时间对重组蛋白StGH61-11酶活的影响；E：IPTG浓度对重组蛋白StGH61-11酶活的影响

Fig. 3 Heterologous expression of StGH61-11 recombinant protein and optimized induction conditions A: Agarose gel electrophoresis results of the target gene StGH61-11 PCR products, 1-3: GH61-11 cDNA fragment. M：DNA marker. B: Agarose gel electrophoresis results of PCR for recombinant transformants, 1-3: Pet32a-StGH61-11 recombinant plasmid, M：DNA marker. C: SDS-PAGE detection of whole bacterial lysate expressed by recombinant pET32a-StGH61-11（BL21）, 0 : Pet32a-StGH61-11-BL21（DE3）not induce control, 1 : Pet32a-StGH61-11-BL21（DE3）the recombinant bacteria were induced by IPTG, M : Standard for molecular weight of pre-dyed protein. D: Effect of induction time on the enzymatic activity of recombinant protein StGH61-11. E: Effect of IPTG concentration on the enzymatic activity of recombinant protein StGH61-11

图4 StGH61-11重组蛋白的最适反应温度和稳定性及pH和稳定性 A：温度对StGH61-11纤维素酶活力的影响；B：温度对StGH61-11纤维素酶活力的稳定性影响；C：pH对StGH61-11纤维素酶活力的影响；D：pH对StGH61-11纤维素酶活力稳定性的影响

Fig. 4 Optimal reaction temperature and stability and pH and stability of StGH61-11 recombinant protein A: Effects of temperature on the cellulase activity of StGH61-11. B: Effect of temperature on the stability of StGH61-11 cellulase activity. C: Effects of pH on StGH61-11 cellulase activity. D: Effect of pH on the viability stability of StGH61-11 cellulase

图5 StGH61-11 与纤维素酶水解玉米秸秆的协同反应 A：StGH61-11对纤维素酶活性的促进作用；B：StGH61-11与纤维素酶共同作用的协同度

Fig. 5 Synergistic reaction of StGH61-11 with cellulase hydrolysis of corn stover A: Promoting effect of StGH61-11 on cellulase activity. B: Synergism degree of StGH61-11 and cellulase

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