| 4,6-α-Glucanotransferase（4,6-α-Gtf,EC 2.4.1.-）is a novel transglycosidase that converts starch intoα-1,6-rich bonds low-calorie dextrin,potentially replacing traditional maltodextrin in food processing.Our previous studies identified two 4,6-α-Gtfs:Bs Gtf from Bacillus sporothermodurans and Lf Gtf from Limosilactobacillus fermentum NCC 3057.Among them,Bs Gtf produced low-calorie dextrin with the highest ratio ofα-1,6 bonds,providing enhanced resistance.On the other hand,Lf Gtf generated low molecular weight dextrins with excellent solubility.However,both Bs Gtf and Lf Gtf face challenges in their application for low-calorie dextrin production,including low optimum temperature,poor thermal stability,and limited soluble expression,which hinder their industrial use.Therefore,this study aimed to improve the thermal stability of Bs Gtf and Lf Gtf through rational design and directed evolution and explore their recombinant expression in Bacillus subtilis.Subsequently,the dominant mutant strains of Bs Gtf and Lf Gtf were evaluated in shake flasks and 3-L tank fermentation studies by comparing different protease knockout hosts of B.subtilis and optimizing fermentation conditions.The main findings are outlined below:（1）Thermal stability modification of Bs Gtf.Firstly,thermal stability of Bs Gtf was improved by multiple sequence alignment,surface charge optimization,and directed evolution strategies,and a total of 8 mutants with improved thermal stability were obtained.Secondly,the combination mutation was carried out through iterative combination,and mutant combinations with synergistic effects was obtained（E110P/S209＿K210ins P/D30K/E31I/K327E/K328E/G331S/L333K/S405N/N406D/E407L/E408N/K455P,CMVI）.Compared to the wild type,CMVI exhibited a 10℃increase in optimum temperature（55℃）,a 12.01℃increase in Tm（64.5℃）,and a 16.4-fold increase in half-life(t1/2)at 50°C（144.4 min）.Molecular dynamics simulation（MD）analysis of CMVI revealed lower RMSD and SASA values compared to the wild type,indicating a more compact and rigid structure.Surface charge optimization was identified as the primary factor contributing to the improved thermal stability of CMVI.Finally,the dominant mutant was employed for low-calorie dextrin production at 50°C,resulting in a 1.46-fold increase in the content of resistant components compared to the wild type,demonstrating the superior application potential of the dominant mutant.（2）Thermal stability enhancement of Lf Gtf:Initially,11 mutants with improved thermostability were obtained by modifying the Loop region of Lf Gtf using computer-aided design,homologous sequence alignment,and directed evolution strategies.Subsequently,an iterative combination method was employed to generate nine mutant combinations（Y134V/Y241P/N247P/D420P/A473P/T517M/M765L/L777P/S813P）with synergistic effects,named CM9.Compared to the wild type,CM9 exhibited a 5°C increase in optimum temperature（50°C）,a 5.8°C increase in Tm（59.5°C）,and a 12.0-fold increase in t1/2at 50°C（330.1 min）.MD simulation analysis of CM9 revealed significantly lower RMSD and SASA values compared to the wild type,indicating a more compact and rigid structure,which contributed to the improved thermal stability.Finally,the dominant mutant was employed for low-calorie dextrin production at 50°C,resulting in a 1.43-fold increase in the content of the resistant component compared to the wild type,highlighting the improved application potential of the dominant mutant at high temperatures.（3）Recombinant expression of Bs Gtf in B.subtilis:Firstly,the most stable structure of the mutant BsΔGtf1-705（abbreviated as BsΔGtf）was obtained by truncating the C-terminus of Bs Gtf by 158 amino acids.Expression in B.subtilis yielded activity that was 3.4 times that of the control group.Secondly,among 11 endogenous promoters,the optimal single promoter Pnprewas selected,and the double promoter Phag-npreshowed the highest suitability for recombinant expression of BsΔGtf,resulting in an activity that was 4.83 times that of the control group.Additionally,the signal peptide SPywe Aexhibited the most significant effect,with an activity 1.41 times that of the control group.This strategy was also applicable to the recombinant expression of the mutant（BsΔm Gtf）with improved thermostability,reaching an activity of 1035.9 U·m L-1,which was 24.84 times that before optimization.Furthermore,the recombinant expression of BsΔm Gtf was validated in 3-L tank fermentation,confirming its suitability for large-scale production.Host strain WS9,which knocked out six protease genes（Δnpr B,Δmpr,Δbpr,Δepr,Δapr E,andΔnpr E）,demonstrated the highest suitability for recombinant expression of BsΔm Gtf,with an activity of 1204.1 U·m L-1in shake flask.After optimization of components and parameters,the extracellular activity of BsΔm Gtf from the recombinant strain WS9BM reached 3556.5 U·m L-1,which was 2.9 times that before optimization.The optimum conditions for the expression of BsΔm Gtf in the recombinant strain WS9BM were determined through 3-L tank fermentation optimization,resulting in an extracellular activity of 36232.8 U·m L-1,which was 3.0 times that before optimization.（4）Recombinant expression of Lf Gtf in B.subtilis.Firstly,according to the principle that the greater the free energy of the hairpin structure of the m RNA 5’-end sequence,the more favorable the translation,the N-terminal 68 amino acids of Lf Gtf were truncated to obtain the truncated mutant LfΔN69-949Gtf（abbreviated as LfΔGtf）,its activity of LfΔGtf in B.subtilis was 3.1 times that of the control group.Secondly,the optimal single promoter Pahp Fwas screened out from 11 endogenous promoters and the double promoter Pamy Q-ahp Fwas most conducive to the recombinant expression of LfΔGtf by combining with other promoters in tandem,and the enzyme activity was 13.7 times that in control group.The above strategy was also applicable to the recombinant expression of the mutant with optimal thermostability（LfΔm Gtf）,and its enzyme activity reached 1194.1 U·m L-1,which was 54.28 times that before optimization.Then,the recombinant expression of LfΔm Gtf was further enhanced by using the chemical chaperone trehalose.MD results show that trehalose molecules were bound to a specific region of 4,6-α-Gtf,thereby stabilizing the local conformation,and improving the stability of the enzyme,which may be the reason for increasing its soluble expression.Finally,the fermentation system of LfΔm Gtf was scaled up.After multi-strategy optimization,the enzyme activity of LfΔm Gtf synthesized by the recombinant strain WS9LM in the 3-L tank fermentation was only 30.3%of that in the shake flask,which needs further study. |
