Abstract
Levan-typed fructooligosaccharide (LFOS), a β-2,6 linked oligofructose, displays the potential application as a prebiotic and therapeutic dietary supplement. In the present study, LFOS was synthesized using levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9). The wild-type LsKK9 was cloned and expressed in E. coli, and purified by cation exchanger chromatography. Additionally, Y237S variant of LsKK9 was constructed based on sequence alignment and structural analysis to enhance the LFOS production. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis indicated that Y237S variant efficiently produced a higher amount of short-chain LFOS than wild type. Also, the concentration of enzyme and sucrose in the reactions was optimized. Finally, prebiotic activity assay demonstrated that LFOS produced by Y237S variant had higher prebiotic activity than that of the wild-type enzyme, making the variant enzyme attractive for food biotechnology.
Highlights
Fructooligosaccharide (FOS) is a well-known prebiotic which is widely used in food and pharmaceutical industries
FOS was used as a sweetener and dietary supplement for low-carbohydrate consumers [1,2]
levan-typed fructooligosaccharide (LFOS) was synthesized from sucrose using both wild-type and Y237S variant levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9)
Summary
Fructooligosaccharide (FOS) is a well-known prebiotic which is widely used in food and pharmaceutical industries. FOS can be divided into 2 major categories based on the linkages between the fructosyl unit. Hydrolysis is the reactions used to produce FOS from fructan polysaccharides. This reaction can be achieved by inulinase (EC 3.2.1.7) and levanase (EC 3.2.1.65), the enzymes randomly hydrolyzed β-2,1 and β-2,6 glycosidic linkages of fructan, respectively [3,4]. The production of FOS by hydrolysis was limited because it is difficult. Bainomdolleecuvlaesn2a0s2e0,(1E0C, 6932.2.1.65), the enzymes randomly hydrolyzed β-2,1 and β-2,6 glycosidic linkag2eosf 1o3f fructan, respectively [3,4]. The production of FOS by hydrolysis was limited because it is difficult to control the molecular weight of FOS produced. CClloonniinngg ooff LLeevvaannssuuccrraassee ffrroomm BBaacciilllluuss AAmmyylloolliiqquueeffaacciieennss KKKK99 wseaqsugTeTrnhhoceeweB,nB.w.aaameasmyrgoloyrbloliioqcwluaiqnelulfyaaecefiairenconibeLsniBKcsaKblKl9ryoK, iit9snho, LlaiaBtstoe3bld7arotf◦ertCodhm.aTftrsh3oo7emil°giCnes.noTTeilhheaeininlgcaeonTnddheianaeingnladclnoeiddvdaienannngtsidulfiecevriddaansebensyutwi1cf6iraeSasdsraeRmbwNypaAls1ifias6emeSqdpurflReirnfNoicemAed, tfhroemchtrhoemchorsoommoasloDmNalADoNfAB.ofamB.yalomliyqluoelifqaucieefnacsieKnsKK9 Ku9siunsgintghethpeapratiratiladl edgeegneenrearcaycypprrimimeerrsswwhhiicchh were designed based on sequence alignment; forward primer The sequence of recombinant plasmid was verified by nucleotide sequencing and assigned to GenBank in Accession number KC477262
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