Molecular Modification And High-Efficiency Expression Of Raw Starch Glucoamylase

Raw starch glucoamylase（EC3.2.1.3,RSGA）can digested starch granules into glucose at temperatures lower than the gelatinization point.RSGA is widely used in the fields of starch-sugar production,ethanol fermentation,and porous starch because of the advantages of lower reaction temperature and energy consumption.Currently,RSGA still faces problems such as limited sources,low degradation efficiency to raw starch,and low fermentation yield,which limits its large-scale production and application.Therefore,the development of RSGA with high raw starch degradation activity will strongly promote the application of in starch processing.In this study,RSGA from Aspergillus fumigatus was expressed in Komagataella phaffii（formerly known as Pichia pastoris）,firstly and its enzymatic properties and raw starch degradation efficiency were analysed.Then,the starch-binding sites and N-glycosylation sites were modified to enhance the raw starch degradation activity,respectively.Finally,an efficient synthesis of RSGA in K.phaffii was achieved using combination strategy.The main findings were as follows:（1）Expression and enzymatic properties analysis of A.fumigatus RSGAThe gene of A.fumigatus RSGA was synthesized according to the codon preference of Escherichia coli and expressed in E.coli BL21（DE3）with pel B as the signal peptide.At 48 h of fermentation,the extracellular recombinant RSGA（e RSGA）yield reached 1.2 U/m L and a clear RSGA band was observed in the supernatant（Western Blot）.Expression of the same RSGA gene in K.phaffii GS115 resulted in a maximum yield of 25 U/m L（120 h）of extracellular recombinant RSGA（p RSGA）,which was 20 times higher than that of e RSGA.The optimal temperatures of p RSGA and e RSGA were 70°C and 60°C,respectively.Furthermore,the half-life of p RSGA at 60°C reached 97 min,which is 78 min longer than that of e RSGA.Deglycosylation（Endo H）or glycosylation inhibitor（tunicamycin）reaction indicate that the glycosylation modification contributed to degradation activity of p RSGA.When adding 0.5 Uα-amylase,0.1 U pullulanase and 0.2 U p RSGA,92.2%of raw corn starch（200 g/L）can be converted into glucose after 36 h of catalytic reaction at 40°C.Our findings provide an efficient RSGA and its production strain for raw starch degradation.（2）Modification of starch-binding sites to improve the catalytic efficiency of RSGA towards raw starch.The catalytic efficiency of raw starch glucoamylase（RSGA）is critical for hydrolyzing starch granules into glucose at low temperatures.However,enzymes are deficient in that natural catalytic efficiencies,leaving room for improvement.Here,alanine scanning was used to characterize the effect of starch binding sites on the raw starch activity of p RSGA.It was found that after replacing the aromatic amino acids of Y542,W578,and W558 with Ala,the activity of p RSGA decreased by 63%,75%,and 90%,respectively.To improve the activity of p RSGA,Y542,W578,and W558 were replaced with two other aromatic amino acids,respectively.The results showed that the raw starch catalytic activity of the mutant Y542W and Y542F increased by 17.6%and 15.2%compared to p RSGA,respectively.Following this,the remaining starch-binding sites were then subjected to iterative saturation mutagenesis,which produced mutants T541D,T541E,N541E and N541P with 15%,21%,10%and 15.9%increases in starch catalytic activity,respectively.Moreover,the T541E exhibited the longest half-life,which increased by 8 minutes at 60°C,reaching 105 minutes.This study reveals the key roles of the starch-binding site in catalysis and provides a robust candidate for the efficient degradation of raw starch.（3）Engineering the N-glycosylation sites to improve the catalytic efficiency of RSGA towards raw starch.Based on prediction of Net NGlyc 1.0,N422 and N610 are N-glycosylation sites of p RSGA.Mutation of the glycosylation sites resulted in the disappearance of the smeared bands and a decrease in raw starch activity,indicating that N-glycosylation of single site has an important effect on p RSGA catalytic activity.To improve the catalytic activity of raw corn starch,N-glycosylation was introduced into the p RSGA through site-directed mutation and the recombinant expression in K.phaffii.Among them,the mutants G101S（N99L100-S101）and Q113T（N111-S112-T113）increased the specific activity of raw corn starch by 1.19-and1.21-fold,respectively.The optimal temperature of Q113T decreased from 70 to 60°C.Notably,the combined mutant G101S/Q113T increased the specific activity toward raw starch by 1.22-fold and reduced the optimal temperature from 70 to 60°C.Moreover,the mutant Q113M with a 1.5-fold increase in the catalytic activity was obtained via saturation mutation at site 113.Thus,the N-glycosylation site engineering is an efficient method to improve the activity of RSGA toward raw starch.（4）Combinatorial strategies to enhance RSGA synthesis in K.phaffii.For the efficient synthesis of RGSA in K.phaffii,theα-factor signal peptide was replaced with Pho5 signal peptide to mediates the extracellular secretion of p RSGA.The enzyme activity increased from 25 U/m L to 33.2 U/m L.On this basis,protein folding-related molecular chaperones（PDI、ERO1、Hac1 and Kar2）of K.phaffii and Saccharomyces cerevisiae origin,as well as vesicular transport pathway molecular chaperones（Sec1、Sec12、Sec16 and Sec61）,were co-expressed,respectively.The results showed that overexpression of Sec1 chaperone protein resulted in a 68%increase in RGSA activity to 42 U/m L.Furthermore,high density fermentation system were optimized in a 3 L bioreactor and the optimum conditions for protein expression were determined as follows:induction temperature of 30°C,initial induction bacterium concentration of OD₆₀₀of 250 and methanol concentration of 0.5%.Under the above conditions,the p RSGA enzyme activity reached 478 U/m L,a 19.12-fold increase compared to flask fermentation,and the enzyme production intensity reached 2.84U/m L/h,achieving efficient synthesis of p RSGA.Using signal peptide pho5,the mutant Q113M was expressed in K.phaffii overexpressing Sec1.Under the same fermentation conditions,the enzyme activity reached 688 U/m L,a 27-fold increase compared to flask level.