Secretion And Expression Of The 1,4-α-glucan Branching Enzyme Gene From Rhodothermus Obamensis In Bacillus Subtilis

1,4-α-Glucan branching enzyme（1,4-α-glucan branching enzyme,GBE,EC 2.4.1.18）is a glycosyltransferase that hydrolyzes theα-1,4 glycosidic bonds in starch molecules,and then connects the donor chain to the acceptor chain throughα-1,6 glucosidic linkages to form a branch.After modified by GBEs,the high-branched starch product becomes more stable and more prominent in slow-digesting property.Due to these,it has shown great application potential in the fields of food and medicine industry.In recent years,all the GBEs reported so far have achieved the intracellular heterologous expression in Escherichia coli.However,the difficulty of separation and purification and the safety of the expression system greatly limit the industrial application of GBEs.In this paper,the study focused on a gbe gene derived from Rhodothermus obamensis STB05（Ro-GBE）.First,its secretion pathway in Bacillus subtilis WB600 was studied,and its culture conditions in shake flasks were systematically optimized.Then the effect of N-terminal hydrophilic/hydrophobic modification on the secretion and expression of Ro-GBE was explored,and the scale-up production of the optimal mutant in the fermenter was realized.The application performance of the mutant was characterized in detail,either.Finally,the application value of this modification method for enhancing the secretion capacity of other amylases was evaluated.This study provides a new idea for the secretion and expression of amylases in B.subtilis WB600.The main research contents and results are as follows:Firstly,the gbe gene derived from R.obamensis STB05 was inserted into the expression vector p ST to construct a recombinant plasmid p ST/gbe.The plasmid was verified by sequence determination,and then was transformed into the host B.subtilis WB600 to obtain the genetically engineered bacteria B.subtilis WB600（p ST/gbe）.By replacing different types of signal peptide sequences,the effects of different secretion pathways in B.subtilis on the secretion and expression of Ro-GBE were explored.It was found that the two classical secretion pathways,Sec and Tat,were not suitable for the secretion and expression of Ro-GBE.The secretion and expression of Ro-GBE can only be achieved in the absence of signal peptides.After fermenting in TB medium at 37°C for 84 h,the extracellular activity of Ro-GBE can reach730.15 U/m L,which is much higher in the extracellular activity than other GBEs that has been expressed in B.subtilis in the literature.Secondly,the effects of fermentation conditions on the secretion and expression of Ro-GBE were investigated,and the optimal fermentation medium was determined.The results showed that the most suitable medium components for fermentation were:yeast extract powder30 g/L,soytone 12 g/L,corn starch 4 g/L,glucose 4 g/L,KH₂PO₄ 2.3136 g/L,K₂HPO₄ 16.4318g/L.The initial p H was 7.0 and the fermentation temperature was 37°C.After fermentation for84 h under the above conditions,the extracellular enzyme activity of Ro-GBE can be increased to 1196.28 U/m L,which was 1.64 times that of the control.Thirdly,to explore the mechanism of the secretion and expression without signal peptide of Ro-GBE,and to promote its extracellular secretion by utilizing this feature,the hydrophobicity analysis of the N-terminal sequences of Ro-GBE that may have signal peptide functions was carried out,and the regulatory effect of N-terminal hydrophilicity/hydrophobicity changes on Ro-GBE extracellular secretion was explored by means of regional mutation.The results showed that,within a certain range,with the increase of N-terminal hydrophilicity,the extracellular secretion of Ro-GBE gradually increased,either.After fermentation at 37°C for84 h,the extracellular enzyme activity of the mutant W3R/L4R was 75.53%higher than that of the wild-type Ro-GBE.It was speculated that the loop structure at the N-terminal of Ro-GBE has the function to identify and search for the protein transporter on the cell membrane as an"exit".The increase in the N-terminal hydrophilicity is conducive to improving the flexibility of the loop,which in turn accelerates the speed of the loop moving and searching for the"exit".At the same time,the addition of N-terminal arginine leads to an increase in the positive charge of the mutant W3R/L4R,which further promotes the combination of the N-terminal loop and the negatively charged phosphate group on the cell membrane,so that the Ro-GBE protein can aggregate massively inside the cell membrane,and then secret outside the cell more quickly.Subsequently,the extracellular secretion ability and the possibility of large-scale fermentative production of the N-terminal hydrophilic mutant W3R/L4R were verified in a 5 L fermenter.Under the condition of secondary inoculation,10%inoculation amount,and no feeding,the enzyme production level of the N-terminal hydrophilic mutant W3R/L4R in the fermenter could reach 1430.75 U/m L,which is 53.99%higher than that of the wild-type Ro-GBE,and the fermentation time in the fermenter was shortened by 57.14%compared with the shake flask fermentation.Finally,the enzymatic properties of the N-terminal hydrophilic mutant W3R/L4R of Ro-GBE were characterized and its application potential was evaluated.The results showed that the N-terminal hydrophilic mutation did not significantly change the specific enzyme activity and thermal stability of Ro-GBE.It also had no effect on the transglycosidic properties.The light transmittance of the maltodextrin solution modified by the mutant W3R/L4R was still above 90%after stored at room temperature for 180 d,which was different from the original maltodextrin solution（completely turbid after 4-5 d storage at room temperature）.Compared with this,its transparency stability was significantly improved.This modification effect was consistent with that of the wild-type Ro-GBE.Since the N-terminal hydrophilic mutation only increased the total amount of protein secreted into the fermentation supernatant,the catalytic properties of the enzyme were not significantly affected.The corresponding mutants were not only incomparable to the wild-type Ro-GBE in the modification effect of maltodextrin,but also the actual additional volume of enzyme solution was far lower than the wild-type Ro-GBE,which indicated that this molecular modification method has good application value and can provide a certain theoretical reference for the modification of the secretion capacity of other amylases.