Recombinant Expression,Exo-hydrolysis Characteristic And Application Of Isomaltotrio-dextranase From Brevibacterium Fuscum

Isomaltooligosaccharieds（IMOs）include all glycosyl units saccharides withα-1,6glycosidic linkages such as isomaltose,panose and isomaltotriose with the degree of polymerization of 2～10.IMOs are not decomposed by human digestive enzymes so that IMOs could be applied to sugar-substitute for diabetics.Also,IMOs can be utilized by bifidobacterium to yield short-chain fatty acid so that IMOs are applied in food industry widely.It was reported that isomaltotriose（IG3）in IMOs mainly contributed to the promotion of bifidobacterium proliferation.In this study,we proposed to produce IMOs in two steps,which was combined with dextran synthesis by 4,6-α-glycosyltransferases（4,6-α-GTs）and dextran degradation by isomaltotrio-dextranase（IMTD）for the purpose of improving production and proportion of IG3.IMTD from Brevibacterium fuscum was our target enzyme which was called BfIMTD because of its product specificity.Recombinant expression and structure exploration of isomaltotriose-specific BfIMTD were performed,and the rule of endo-or exo-hydrolysis characteristic in BfIMTD could be possibly revealed.To optimize the strategy of the two-step method,4,6-α-GT from Lactobacillus fermentum（LfGT）was selected to produce dextran and improve the proportion ofα-1,6 linkages with the combination of pullulanase and isopullulanase in the dextran synthesis step.Meanwhile,the process of BfIMTD hydrolyzing dextran was optimized to obtain IMOs rich in IG3.The main conclusions are as followed:（1）Recombinant expression and enzymatic properties of BfIMTD were performed.After Escherichia coli BL21（DE3）/p ET-24a（+）-Bfimtd was constructed,the recombinant strain was induced by IPTG and fermented for 48 h.The SDS-PAGE result showed that the recombinant protein was expressed successfully with the enzyme activity of 7.8 U·m L^-1.And the specific activity was 78.7 U·mg^-1 when the purified enzyme was obtained by Ni²⁺affinity chromatography with the fold of 8.4 and the yield of 3.8%.Its optimum temperature and p H were 45℃and 8.0.IG3 was the specific product of BfIMTD,whose product proportion was91.5%when reacting with 0.1 g·m L^-1commercial dextran 20000 for 0.5 h.（2）The BfIMTD exo-hydrolysis characteristic was explored.After multiple sequence alignment and homology modeling,a characteristic loop（amino acid 392～400 HEGEKEIGV）at the non-reducing end of the active groove of BfIMTD is significantly different from other endo-dextranases in GH49 which forms a irregular coil in the structure.The activity of BfIMTD-ΔL(loop^exo truncated mutation)was 0.7 U·m L^-1.BfIMTD-ΔL yielded quite small amount of IG3.BfIMTD-ΔL no longer showed product specificity,suggesting that loop^exo plays a vital role in BfIMTD.Two of six uncharacterized dextranases with loop-like structure were mined and selected by BLAST.One was Cc Dex from Cellulosimicrobium cellulans,the other was Mr Dex from Mycetocola reblochoni.And the redundant domains of these two sequences were truncated so that Cc DexΔBC and Mr DexΔBC were formed and both showed solube expression.Also,these two both showed IG3 product specificity when reacting with0.1 g·m L^-1commercial dextran 20000 for 0.5 h with the product proportion 84.3%and 92.1%,respectively.It was revealed that both Cc DexΔBC and Mr DexΔBC were typical exo-type dextranases and loop^exocould be the determinant of exo-hydrolysis in GH49.（3）IMOs were produced by LfGT and BfIMTD in the two-step method via dextran.It could yield 24.3%（peak area ratio）when LfGT reacted with 0.2 g·m L^-1DE2 maltodextrins at37℃and p H of 6.0 with the enzyme amount of 400 U·g^-1substrate for 24 h.It could yield 73.1%dextran with the combination of LfGT(400 U·g^-1 substrate),pullulanase(40 U·g^-1 substrate)under the same condition.When combining isopullulanase(400 U·g^-1 substrate)with the above-mentioned two enzymes under the same conditon,it could yield 66.3%dextran,whoseα-1,6glycosidic linkage ratio was 99.3%.Glucose in the product was removed by yeast and the dextran was pulverized by freeze-drying.98.3 g·L^-1 IG3 was produced（65.2%from maltodextrin）when 0.1 g·m L^-1dextran was reacted with BfIMTD(150 U·g^-1substrate)at 40℃and p H of 7.5 for 10 h.