基于计算设计的GH11家族木聚糖酶CDBFV的热稳定性改造及潜在机制研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0384

生物技术通报 ›› 2024, Vol. 40 ›› Issue (10): 305-314.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0384

基于计算设计的GH11家族木聚糖酶CDBFV的热稳定性改造及潜在机制研究

韩雪¹^,²(), 张阿娜³, 王海燕⁴, 辛凤姣¹^,², 谷天一¹^,²(), 王钰璐¹^,²()

1.中国农业科学院农产品加工研究所，北京 100193
2.中国农业科学院农产品加工与营养健康研究院（沧州），沧州 061001
3.山西农业大学食品学院，晋中 030600
4.北京挑战生物技术有限公司，北京 100081

收稿日期:2024-04-15 出版日期:2024-10-26 发布日期:2024-11-20
通讯作者: 王钰璐，女，博士，助理研究员，研究方向：生物大分子结构与功能；E-mail: wnewyx@163.com；
谷天一，男，助理研究员，研究方向：食品酶学与合成生物学；E-mail: 18501151081@163.com
作者简介:韩雪，女，博士研究生，研究方向：食品酶学；E-mail: njhanxue1995@163.com
第一联系人：
张阿娜为本文共同第一作者
基金资助:
企业合作项目（饲用酶产品开发与升级），中央级公益性科研院所基本科研业务费专项(S2022JBKY-13);中国农业科学院农产品加工研究所创新工程院所重点任务(CAAS-ASTIP-G2022-IFST-07)

Computer-aided Thermostability Engineering and Underlying Mechanism Investigation of the GH11 Family Xylanase CDBFV

HAN Xue¹^,²(), ZHANG A-na³, WANG Hai-yan⁴, XIN Feng-jiao¹^,², GU Tian-yi¹^,²(), WANG Yu-lu¹^,²()

1. Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193
2. Institute of Food Science Technology Nutrition and Health（Cangzhou）, Chinese Academy of Agricultural Sciences, Cangzhou 061001
3. College of Food Science, Shanxi Agricultural University, Jinzhong 030600
4. Beijing Challenge Biotechnology Co., Ltd., Beijing 100081

Received:2024-04-15 Published:2024-10-26 Online:2024-11-20

摘要/Abstract

摘要：

【目的】瘤胃真菌Neocallimastix patriciarum GH11家族木聚糖酶CDBFV在饲料、食品等领域具有良好应用前景，提高其热稳定性对其生产应用十分重要。【方法】使用分子动力学模拟、机器学习等策略设计潜在CDBFV热稳定性突变体，在大肠杆菌和毕赤酵母中进行异源表达纯化，测定最适反应条件、比酶活和85℃孵育3 min后相对剩余活力，通过结构分析明确热稳定性提高机制。【结果】位于CDBFV N端基序³⁶GNNS³⁹具有较高柔性，对其改造设计的单突变体N37P和N38V在85℃孵育3 min后，相对活力分别降低至70.3%和55.1%，较野生型（48.7%）提升了21.6%和6.5%；进而，在相对活力提升显著的N37P基础上叠加已报道优势突变体N88G，构建双突变体N37P/N88G，其相对活力达到了73.4%，较野生型提高了24.7%；此外，将N37P/N88G在毕赤酵母中进行了分泌表达，在85℃处理3 min后，相对活力达到了88.8%；结构分析表明，N37P突变的引入使得CDBFV形成了新的氢键相互作用，降低活性位点附近柔性，并干预了糖基化形成，进而提高了热稳定性。【结论】成功得到了高耐热双突变体N37P/N88G，为提高GH11家族木聚糖酶的热稳定性改造提供了新的思路和方法，有望推动CDBFV在饲料工业等高温环境下的广泛应用。

关键词: 木聚糖酶, 柔性区域, 热稳定性, 毕赤酵母, 理性设计

Abstract:

【Objective】 The rumen fungus Neocallimastix patriciarum GH11 family xylanase CDBFV has good application prospects in feed, food and related industries. Improving its thermal stability is crucial for optimizing its production and utilization. 【Method】 Potential thermostable mutants of CDBFV were designed using strategies such as molecular dynamics simulation and machine learning. And then heterologous expression and purification were carried out in Escherichia coli and Pichia pastoris. The optimal reaction conditions, specific enzyme activity and the relative residual activity after incubation at 85℃ for 3 min were determined, and the mechanisms for improving thermal stability were clarified through structural analysis. 【Result】 The ³⁶GNNS³⁹ motif at the N-terminus of CDBFV was highly flexible. Single mutant N37P and N38V created by modifying this motif showed the relative activites of 70.3% and 55.1% after incubation at 85℃ for 3 min, representing increases of 21.6% and 6.5% respectively compared to the wild-type（48.7%）. Based on the significant increase in relative activity observed in N37P, combined with the previously reported beneficial mutant N88G, the double mutant N37P/N88G was constructed. This double mutant presented a relative activity of 73.4%, and 24.7% improvement over the wild-type. In addition, when N37P/N88G was expressed in Pichia pastoris, it had a relative activity of 88.8% after treatment at 85℃ for 3 min. Structural analysis indicated that the N37P mutation introduced new hydrogen bonds in CDBFV, decreased the flexibility of the active site and disrupted glycosylation, thereby improving its thermal stability. 【Conclusion】 This study successfully generated the high-temperature-resistance double mutant N37P/N88G, offering new insights and approaches for improving the thermal stability of GH11 family xylanases. This advancement is anticipated to facilitate the extensive utilization of CDBFV in high-temperature settings like the feed industry.

Key words: xylanase, flexible region, thermostability, Pichia pastoris, rational design

韩雪, 张阿娜, 王海燕, 辛凤姣, 谷天一, 王钰璐. 基于计算设计的GH11家族木聚糖酶CDBFV的热稳定性改造及潜在机制研究[J]. 生物技术通报, 2024, 40(10): 305-314.

HAN Xue, ZHANG A-na, WANG Hai-yan, XIN Feng-jiao, GU Tian-yi, WANG Yu-lu. Computer-aided Thermostability Engineering and Underlying Mechanism Investigation of the GH11 Family Xylanase CDBFV[J]. Biotechnology Bulletin, 2024, 40(10): 305-314.

图/表 9

表1 定点突变所用引物

Table 1 Primers used by site-directed mutagenesis

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
N37P-F	CGGATAGCGGCCCGAATAGCGCGACCTTTTATA-G
N37P-R	CGCGCTATTCGGGCCGCTATCCGCCCACAGTTCATAG
N38V-F	CGGATAGCGGCAATGTTAGCGCGACCTTTTATAGCGATGGC
N38V-R	GGTCGCGCTGTTATTGCCGCTATCCGCCCACAGTTCATAG
N88G-F	CTGGTGAAACAGGGTAGCAGCAATGTGGGCTATAGCTATG
N88G-R	GCCCACATTGCTGCTACCCTGTTTCACCAGTTTAAAATCCGC

图1 序列分析及突变位点的筛选 A：野生型CDBFV在298 K和360 K下不同残基的RMSF值分析；B：野生型CDBFV与同源木聚糖酶的N端序列比对，XynSW1、Xyn11B、Xyn11NX和PVX分别来自链霉菌、黑曲霉、新疆涅斯捷连科氏菌和拟青霉属，绿色框为基序36GNNS39；C：柔性区域在结构中的位置，黄色表示RMSF值较高的柔性区域151SIDGD155和36GNNS39

Fig. 1 Sequence analysis and screening of mutation sites A: Analysis of RMSF values of wild-type CDBFV at 298 K and 360 K. B: N-terminal sequence alignment of wild-type CDBFV with homologous xylanases, XynSW1, Xyn11B, Xyn11NX and PVX from Streptomyces sp., Aspergillus niger, Nesterenkonia xinjiangensis and Paecilonyces variotii, respectively. Green box refers to motif 36GNNS39. C: The flexible region 151SIDGD155和36GNNS39 with high RMSF value is highlighted in yellow in the structure

图2 机器学习模型预测能量排名机器学习模型预测后高温条件下最保守氨基酸分值减去野生型氨基酸分值的大小排序

Fig. 2 Machine learning-based energy ranking prediction The figure illustrates the ranking of the most conserved amino acid scores at high temperature minus the wild-type amino acid scores predicted by a machine learning model

图3 野生型及单突变体SDS-PAGE及酶学性质 A：野生型CDBFV及单突变体的SDS-PAGE分析，M：标准分子量蛋白Marker；B：野生型及单突变体的最适pH；C：野生型及单突变体的最适温度；D：野生型及单突变体在85℃处理3 min较处理前相对酶活力

Fig. 3 SDS-PAGE analysis and enzymatic properties of wild-type and single mutants A: SDS-PAGE analysis of wild type CDBFV and single mutant; M: standard molecular weight protein marker. B: The optimal pH of wild type and single mutants. C: The optimal temperature of wild type and single mutants. D: The relative enzyme activity of wild type and single mutants after treatment at 85℃ for 3 min compared to their pre-treatment states

图4 野生型及双突变体SDS-PAGE及酶学性质 A：野生型CDBFV及双突变体N37P/N88G的SDS-PAGE分析，M：标准分子量蛋白Marker；B：野生型及双突变体的最适pH；C：野生型及双突变体的最适温度；D：野生型及双突变体在85℃处理3 min较处理前相对酶活力

Fig. 4 SDS-PAGE analysis and enzymatic properties of wild-type and the double mutant A: SDS-PAGE analysis of wild type CDBFV and N37P/N88G double mutant; M: standard molecular weight protein marker. B: The optimal pH of wild type and the double mutant. C: The optimal temperature of wild type and the double mutant. D: The relative enzyme activity of wild type and the double mutant after treatment at 85℃ for 3 min compared to their pre-treatment states

表2 CDBFV野生型和突变体的最适条件及酶活测定

Table 2 Optimal condition and enzymatic activity determination of CDBFV wild-type and mutants

酶Enzyme	最适pH Optimal pH	最适温度Optimal temperature/℃	绝对酶活Specific activity/（U·mg^-1）	相对酶活^a Relative activity/%
WT（E. coli）	6	60	5 216.2	48.7
N37P（E. coli）	6	60	1 108	70.3
N38V（E. coli）	6	60	2 130.45	55.1
N37P/N88G（E. coli）	6	60	807.2	73.4
WT（P. pastoris）	5	70	1 050	76.6
N37P/N88G（P. pastoris）	5	70	900	88.8

图5 毕赤酵母表达的野生型及双突变体SDS-PAGE及酶学性质 A：野生型及双突变体的SDS-PAGE分析，M：标准分子量蛋白Marker；B：野生型及双突变体的最适pH；C：野生型及双突变体的最适温度；D：野生型及双突变体在85℃处理3 min较处理前相对酶活力；E：野生型及双突变体在70℃处理0-60 min的温度稳定性；F：野生型及双突变体在80℃处理0-60 min的温度稳定性

Fig. 5 SDS-PAGE analysis and enzymatic properties of wild-type and the double mutant expressed in Pichia pastoris A: SDS-PAGE analysis of wild type and double mutant; M: standard molecular weight protein marker. B: Optimal pH of wild type and the double mutant. C: Optimal temperature of wild type and the double mutant. D: The relative enzyme activity of wild type and the double mutant after treatment at 85℃ for 3 min compared to their pre-treatment states. E: Temperature stability of wild type and the double mutant after treatment at 70℃ for 0-60 min. F: Temperature stability of wild type and the double mutant after treatment at 80℃ for 0-60 min

图6 野生型和双突变体N88G/N37P的结构分析 A：野生型和突变体N88G的表面电荷分析；蓝色为正电荷，红色为负电荷，白色不带电荷；B：野生型和突变体N37P的形成的相互作用分析；蓝色线表示氨基酸之间的氢键相互作用

Fig. 6 Structural analysis of the wild-type and double mutant N88G/N37P A: Analysis of the surface potential of the wild-type and mutant N88G mutant; blue, red and white colors correspond to positive, negative and uncharged regions, respectively. B: Amino acid interaction analysis of the wild-type and mutant N37P; the blue line indicates the hydrogen bond interactions

图7 野生型及双突变体360 K的分子动力学模拟 A：野生型及双突变体在360 K下的模拟50 ns RMSD值分析；B：野生型及双突变体在360 K下不同残基的RMSF值分析

Fig. 7 Molecular dynamic simulation of the wild-type and double mutant at 360 K A: Analysis of the RMSD values for the wild-type and double mutant during a 50 ns simulation at 360 K. B: Analysis of the RMSF values for different residues in the wild-type and double mutant at 360 K

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基于计算设计的GH11家族木聚糖酶CDBFV的热稳定性改造及潜在机制研究

Computer-aided Thermostability Engineering and Underlying Mechanism Investigation of the GH11 Family Xylanase CDBFV

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