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

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

Abstract

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

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.

Figures/Tables 9

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

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

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

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

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

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

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

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

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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