Study On The Cold Adaptation And Product Specificity Of Maltopentaose-forming Amylase From Saccharophagus Degradans

Maltopentaose(G5)consists of five α-D-glucopyranosyl units linked by α-1,4 glycosidic bonds.It has various applications in food,medical and pharmaceutical fields due to its relatively low sweetness,and the ability to control glycemic response and improve colonic conditions.It has also served as a dietary nutrient for patients with renal failure or calorie deprivation and acted as a diagnostic reagent for the detection of α-amylase in serum and urine.Therefore,the synthesis of G5 is of high value for research and industry.G5 is commonly synthesized through an enzymatic reaction,in which maltopentaose-forming amylases(G5As,EC 3.2.1.X)hydrolyzed the specific α-1,4 glycosidic bonds of starch,producing a mixture of maltooligosaccharides with G5 as the major product.Applying G5 A to produce G5 has faced challenges of poor product specificity and stringent requirements for reaction conditions,which restrained the economic production of G5 and limited its broader applications.This drawback was mainly attributed to the lack of understanding of the catalytic mechanism of G5 As and guidance of molecular modification.To address these problems,we firstly identified a G5 A from Saccharophagus degradans(Sd G5A)and constructed an efficient,stable,and toxic-free expression system in Bacillus subtilis.We further analyzed the enzymatic properties of the purified recombinant Sd G5 A,especially its cold adaptation and product specificity.The main results are listed as follows:(1)We successfully constructed a B.subtilis expression system and optimized the medium component and culture conditions.The highest extracellular hydrolytic activity of the recombinant Sd G5 A reached up to 30.6 U/m L when it was incubated in the optimized medium containing 36 g/L yeast extract and 5 g/L maltodextrin(p H 6.0)at 25°C for 72 h.(2)In order to characterize the recombinant Sd G5 A,we purified the enzyme and measured the enzymatic properties of purified Sd G5 A.Sd G5 A showed the highest activity at 45°C but also moderate activity at 0°C and 25°C.It retained greater than 70% of its maximal activity in presence of 3 M Na Cl,indicating an outstanding cold adaptation and salt tolerance.In addition,Sd G5 A enabled the convertion of 77.7% of corn starch into maltooligosaccharides at 4°C within36 h,with 78.5% of the yield being G5,indicating a good product specificity of Sd G5 A.(3)We firstly predicted the structure of Sd G5 A using Rose TTAFold and obtained its structural information.The results showed that Sd G5 A comprised catalytic domain(Q1～A427),linker(I428～K445)and CBM(V446～F542).The catalytic residues(D163,E189 and D254)were located in the center of the catalytic domain,which was also called Domain A.CBM was located in the vicinity of Domain A,facing towards the groove oriented to the catalytic residues.The linker connecting the catalytic domain and carbohydrate-binding module(CBM)allowed the adjustment of their relative position.(4)To understand the potential mechanisms that might underlie the cold adaptation of Sd G5 A,we analyzed its structural features.Firstly,the analysis of the surface charges showed a high ratio of acidic residues on the surface of the linker and CBM regions with the density of net charges being-22.2% and-4.1%,respectively.Molecular dynamics simulations(MD)showed that those acidic residues could form H-bonds with water molecules in solvent and form a hydration layer,which was conducive to the solubility of the protein and the protection of the protein internal structure.In addition,the high density of identically charged residues promoted electrostatic repulsion between proteins,which prevented protein aggregation and inactivation.Secondly,we investigated the static and dynamic flexibility of Sd G5 A and found an extremely high flexibility in the linker-CBM region.This allowed an active molecular motion,which guaranteed to capture substrates for catalysis in the cold environment.To validate the role of the linker-CBM region on Sd G5 A adapting cold,we truncated the linker-CBM region to construct Sd G5A-CD.The optimum temperature of Sd G5A-CD was raised to 70°C and the hydrolytic activity at 0°C was only 2.6% of that of wild type,indicating the great importance of the linker-CBM region on the cold adaptation of Sd G5 A.In addition,cysteine contributed to the highest ratio(22.2%)of the total amino acid residues in the linker region of Sd G5 A.Those cysteines tended to have deprotonation reaction and were exposed to the surface of the protein in the form of thiols with strong nucleophilicity.MD showed that the deprotonated cysteines contributed to the formation of H-bonds with solvent and conferred more solubility.We further mutated the cysteines to alanines to construct mutants C432 A,C437A,C438 A and C441 A.The four mutants showed 98.4%,66.0%,50.4% and 55.4% of the activity of wild type at 0°C.All results indicated that the linker-CBM acted as a critical component allowing cold adaptation of Sd G5 A.(5)Considering Sd G5 A showed an outstanding specificity of G5 and contained a special CBM structure,we firstly investigated the effects of CBM on the product specificity of Sd G5 A,and then moved towards its effects on the endo-/exo-action pattern and processivity.We truncated linker-CBM from Sd G5 A and fused it to a maltooligosaccharides-forming amylase from B.megaterium to construct mutants Sd G5A-CD and Bs MFA.Compared to their wild type,Sd G5A-CD showed a 66.6% lower G5 production while Bs MFA showed 39.5% higher G5 production,indicating the capacity of CBM to improve the product specificity.We also compared the product specificities of Sd G5 A and Sd G5A-CD under different reaction conditions and found the modulation of the product specificity by CBM was influenced by reaction temperature and substrate structure.Sd G5 A produced 80.5%,80.8% and 55.0% of G5 by hydrolyzing amylopectin at 0°C,25°C and 45°C,respectively,which was 3.5-,4.4-and 2.1-fold higher than the G5 ratio arising from the hydrolysis by Sd G5A-CD.However,when amylose was used as substrate,Sd G5 A produced a similar amount of G5 as Sd G5A-CD,indicating the little effect of CBM on the product specificity at low temperatures or when reacting with amylose.Furthermore,CBM could contribute to the exo-action of Sd G5 A on amylopectin or short dextrin but did not affect the endo-action of Sd G5 A on the long chain of amylose.Additionally,Sd G5 A showed greater processivity than Sd G5A-CD at relatively low temperature(0°C or 25°C).Together,CBM could efficiently modulate product specificity,action pattern and processivity of Sd G5 A at low temperatures and when there were more nonreducing ends existing in substrates.(6)We further investigated the underlying mechanism of CBM affecting the product specificity of Sd G5 A by utilizing molecular docking and MD to analyze and compare the interactions within catalytic domain-substrate and CBM-substrate.We firstly docked maltooctaose(G8)to the active center(AC)and the predicted CBM pocket to construct AC-G8 and CBM-G8 complexes,respectively.The docking results showed that P194 in the CBM pocket made an H-bond with the non-reducing end of the ligand,indicating CBM promoted the recognition of the non-reducing ends of substrates and the decrease of the probability of random initial substrate-binding.In addition,the complexes revealed a head-to-tail formation with a potential preference for the substrate transport pathway from CBM to AC.We then docked G8 to the substrate-binding groove in Sd G5A-CD to construct CD-G8 complex.MD illustrated that only six residues could interact with the ligand and four of them(Y117,K166,G195 and E196)tightly bound the ligand with H-bond and hydrophobic interactions.This might interfere substrates with processive sliding to AC.In comparison,G8 interacted with 26 amino acid residues such as T457,G460 and Q513 in CBM-G8 complex.The increase in the number of binding sites weakened the interactions between the ligand and each site,which promoted substrates to processively slide towards AC.(7)To illuminate the reasons that the lower temperature was preferred for the specific production of G5 by Sd G5 A,we investigated the temperature effects on CBM binding substrates.We first developed Restricted-state(R-state)and Free-state(F-state)of substrates in MD.The Root Mean Square Fluctuation(RMSF)and conformational changes in R-state and F-state were further analyzed and compared at 0°C,25°C and 45°C using CBM-G8 complex as a model.The transition of R-state to F-state induced an average 1.9-fold increase of RMSF value in CBM at 25°C,among which the RMSF values of L508,W521,K509,R510,C507,V520,V463 increased by more than 2.8 times,indicating the substrate-binding caused the activation of CBM.The co-movement of the multiple sites in CBM might promote lowering the free energy barrier for substrate sliding from CBM to AC.A tendency of G8 ligand moving towards AC was also observed in MD.In contrast,at 0°C,the extremely weak molecule motions of both protein and ligand did not allow the increase of CBM RMSF and the conformational changes of the complex.At 45°C,the rapid dissociation of G8 from CBM hindered the modulation of the ligand direction by CBM due to the excessive flexibility.Hence,25°C was chosen as the best temperature for CBM modulating the product specificity and processivity of Sd G5 A.(8)In order to evaluate the feasibility of Sd G5 A application,we used 10%(w/v)native starch as the substrate to produce G5 at 4°C～25℃ and calculated the yield of G5.The results showed that when waxy corn starch,which contained the highest ratio of amylopectin,was hydrolyzed at 25°C,Sd G5 A could convert 72.2% of the substrate into maltooligosaccharides with the highest G5 yield being up to 48.6%.To the best of our knowledge,Sd G5 A has greater product specificity than any other known G5 As.It is also the first maltooligosaccharide-forming amylase found to be concurrently coldadapted and salt-tolerant.These properties indicated that Sd G5 A would have great potential for both basic research and industrial applications.Our study revealed the mechanism of the linkerCBM structure modulating the cold adaptation and product specificity of Sd G5 A,providing strategies to modify other amylases and optimize the process for targeted biosynthesis of G5.