Patent Application: US-201515526402-A

Abstract:
the present invention relates to the production of the disaccharide kojibiose which is known to be a powerful prebiotic . the invention indeed discloses the generation of genetically modified sucrose phosphorylases which convert — via a transglycosylation reaction — sucrose into kojibiose in a very efficient manner . hence , the present invention relates to a cost - effective production method of kojibiose which is useful within industry .

Description:
the present invention relates to a systematic mutagenesis study of the active site of the sucrose phosphorylase of bifidobacterium adolescentis resulting in the identification of only a few mutations that surprisingly can improve the activity ratio of kojibiose over maltose formation ( k / m ratio ) to values higher than 0 . 5 . combining mutations l341i_y344a_q345n into one sequence yields the highest selectivity , corresponding to a k / m ratio of 22 . however , the double mutant l341i_q345s produces kojibiose two times faster than this triple mutant and has a k / m ratio of 15 , which is still enough to generate highly pure kojibiose . a further and simple purification procedure consists of a yeast treatment to consume the remaining glucose , fructose and sucrose . hence , the present invention relates to an isolated sucrose phosphorylase comprising an amino acid sequence given by seq id n ° 1 characterized in that it contains at least one of the following mutations : p134v , p134r , p134w , p134s , r135e , a193g , h234t , l341i , d342a , l343p , y344a , y344r , y344d , y344v , y344i , q345a , q345s and q345n and having a k / m ratio of kojibiose over maltose formation greater than 0 . 5 during a transglycosylation reaction with glucose as acceptor , or , a fragment of said sucrose phosphorylase containing at least one said mutations and having said selectivity of kojibiose . more specifically , the present invention relates to an isolated sucrose phosphorylase comprising an amino acid sequence given by seq id n ° 1 characterized in that it contains at least one of the following mutations : p134v , p134r , p134w , p134s , r135e , a193g , h234t , l341i , l343p , y344r , y344d , y344v , y344i , q345s , q345n and has an activity ratio of kojibiose over maltose formation greater than 0 . 5 during a transglycosylation reaction with glucose , or , a fragment of said sucrose phosphorylase containing at least one of said mutations and having said selectivity of kojibiose . the term ‘ sucrose phosphorylase comprising an amino acid sequence given by seq id n ° 1 ’ refers to an amino acid sequence comprising an amino acid sequence encoded by a nucleic acid having genbank accession number af543301 as described by sprogoe et al . ( 2004 ). the latter nucleic acid corresponds to a sucrose phosphorylase gene from bifidobacterium adolescentis . more specifically , the latter term refers to a sucrose phosphorylase encoded by the sucrose phosphorylase gene from bifidobacterium adolescentis lmg 10502 as described by reuter ( 1963 ) and which is synonymous to dsm20083 and attc15703 . seq id n ° 1 corresponds to the following amino acid sequence ( the amino acids of which at least one should be substituted according to the present invention are underlined ): the terms “ characterized in that it contains at least one of the following mutations : p134v , p134r , p134w , p134s , r135e , a193g , h234t , l341i , d342a , l343p , y344a , y344r , y344d , y344v , y344i , q345a , q345s and q345n ” refers to the fact that specific amino acids at specific positions of wild type seq id n ° 1 ( i . e . at amino acid positions 134 , 135 , 193 , 234 , 341 , 342 , 343 , 344 and / or 345 ) have been substituted by other specific amino acids . the latter substitutions can be obtained by any method known in the art . the latter method can be for example mutating , via for example performing site - directed mutagenesis , the sucrose phosphorylase gene from bifidobacterium adolescentis . the term “ at least one of the following mutations ” indicates that the mutant sucrose phosphorylase of the present invention must contain one of the indicated list of single mutations but may also contain 2 , 3 , 4 , . . . , 16 , 17 or all of the in total 18 single mutations , or , any combination of said list of single mutations which results in mutant sucrose phosphorylases containing multiple ( i . e . 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . . . ) mutations . preferred - but non - limiting - examples of specific combinations of 2 , 3 , 4 , 5 , 6 and 7 of said list of mutations are given in table 1 . the terms “ having an activity ratio of kojibiose over maltose formation greater than 0 . 5 during a transglycosylation reaction with glucose as acceptor ” refers to the fact that sucrose phosphorylases are capable of producing kojibiose 26 , 27 , starting from sucrose and d - glucose . it naturally catalyses the reversible phosphorolysis of sucrose into α - d - glucose 1 - phosphate and d - fructose , but it can also glycosylate alternative acceptors with yields exceeding 90 % 29 , 30 ( wo 2011 / 124538 ). in case of d - glucose as acceptor , the overall kojibiose yield is however lowered by the side - formation of maltose ( 4 - o - α - d - glucopyranosyl - d - glucopyranose ) as glucobiose product 27 . for example , the wild - type and thermostable sp from bifidobacterium adolescentis ( wo 2011 / 124538 ) displays a k / m ratio of only 0 . 5 , meaning that the velocity of maltose formation is twice as high as the velocity of kojibiose formation . the mutants of the present invention as indicated above have a k / m ratio that is greater than 0 . 5 ( i . e . 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 , 1 . 0 , . . . , 2 . 0 . . . , 3 . 0 . . . , 4 . 0 , . . . 10 . 0 , . . . 20 . 0 , . . . 30 . 0 . . . ) meaning that the mutants show a velocity of formation of kojibiose that is increased relative to the velocity of maltose formation , when compared to the wild - type enzyme . the terms “ a fragment of said sucrose phosphorylase containing at least one said mutations and having said selectivity of kojibiose ” refers to a protein ( or peptide or polypeptide ) containing fewer amino acids than the amino acid sequence as depicted by seq id n ° 1 and that retains the activity of said mutant sucrose phosphorylases of the present invention ( i . e . the velocity of kojibiose formation is increased relative to the velocity of maltose formation when compared to the wild - type enzyme ). such fragment must thus contain at least one of the indicated list of single mutations or a combination thereof as indicated above and can — for example — be a protein with a deletion of 10 % or less of the total number of amino acids at the c - and / or n - terminus . the present invention preferably relates to mutants of the present invention have a k / m ratio that is greater than 10 . 0 ( i . e . 10 . 0 , . . . 15 . 0 , . . . 20 . 0 . . . ) meaning that the mutants show a velocity of formation of kojibiose and a velocity of maltose formation which is significantly higher and lower , respectively compared to corresponding velocities of the wild - type enzyme . hence , and more specifically , the present invention relates to an isolated sucrose phosphorylase as described above containing the mutations l341i_q345s or l341i_y344a_q345n and wherein said selectivity for kojibiose is characterized by a k / m ratio of 15 and 22 , respectively . the present invention further relates to an isolated nucleic acid encoding for a sucrose phosphorylase as described above . an example of a nucleic acid encoding for mutant sucrose phosphorylase of the present invention ( seq id n ° 2 ) is as follows ( the codons of which at least one should be mutated according to the present invention are underlined ): the present invention also relates to a vector comprising a nucleic acid as described above . the present invention further relates to a host cell comprising a vector as described above . the term ‘ nucleic acid ’ as used herein corresponds to the sucrose phosphorylase gene from bifidobacterium adolescentis , and preferably from bifidobacterium adolescentis lmg 10502 . said nucleic acids can be incorporated in appropriate vectors such as plasmids and appropriate host cells such as escherichia coli can be transfected with said vectors . the present invention relates to the usage of a sucrose phosphorylase as described above to produce kojibiose . more specifically , the present invention relates to the usage of a sucrose phosphorylase to produce kojibiose wherein said sucrose phosphorylase is an isolated sucrose phosphorylase comprising an amino acid sequence given by seq id n ° 1 characterized in that it contains at least one of the following mutations : p134v , p134r , p134w , p1345 , r135e , a193g , h234t , l341i , d342a , l343p , y344a , y344r , y344d , y344v , y344i , q345a , q345s , q345n and has an activity ratio of kojibiose over maltose formation greater than 0 . 5 during a transglycosylation reaction with glucose , or , a fragment of said sucrose phosphorylase containing at least one of said mutations and having said selectivity of kojibiose . more specifically , the present invention relates to a process to produce kojibiose comprising the steps of : providing sucrose and / or alpha - glucose 1 - phosphate as donor , and , glucose as acceptor , providing a sucrose phosphorylase as described above , bringing said donor and acceptor , and , said sucrose phosphorylase in a medium wherein the synthesis of kojibiose by said sucrose phosphorylase takes place , and purifying said kojibiose from said medium . the term providing sucrose and / or alpha - glucose 1 - phosphate as donor means that sucrose or alpha - glucose 1 - phosphate or a mixture of both may be used as donor in order to glucosylate the acceptor glucose via the mutant sucrose phosphorylases of the present invention . the term providing a sucrose phosphorylase means providing a mutant sucrose phosphorylase of the present invention . a non - limiting example of the latter ‘ providing ’ encompasses : a ) mutating the sucrose phosphorylase wt gene from bifidobacterium adolescentis contained in a vector such as a plasmid , b ) transforming a host cell such as e . coli with said mutated genes , c ) checking the desired mutations by sequencing each construct , d ) growing individual colonies , e ) lysing said colonies and f ) purifying the mutant sucrose phosphorylases of interest . the term ‘ medium ’ refers to any suitable medium known in the art which allows — when the mutant enzyme of the present invention , acceptor and donor are added to said medium — the synthesis of kojibiose by a sucrose phosphorylase of the present invention . an example of such a medium is the 3 -( n - morpholino ) propanesulfonic acid ( mops ) buffer . the term “ purifying ” relates to any purification method known in the art but preferably relates to a purification consisting of a yeast treatment such as a treatment with baker &# 39 ; s yeast . the present invention thus specifically relates to a process as described above wherein said purification consists of a yeast treatment . after said treatment , the yeast can be removed via centrifugation and the supernatant can be evaporated . the kojibiose solution can then be cooled down in order to obtain pure kojibiose crystals which can be washed with ethanol . the present invention will further be illustrated by the following non - limiting example . mutagenesis was performed on the constitutive expression plasmid pcxp34h 31 containing the sucrose phosphorylase gene from bifidobacterium adolescentis lmg 10502 . saturation libraries were generated using either the quikchange tm32 protocol or the protocol described by sanchis et al . 33 , while site - directed mutagenesis was performed with the latter . in all cases , the mutated dna was transformed in e . coli cgsc 8974 ( coli genetic stock center , new haven , conn ., usa ). for each library , the constructs were subjected to nucleotide sequencing ( agowa sequence service , berlin , germany ) in order to confirm that the desired mutations were indeed introduced and to exclude the presence of undesirable mutations . individual colonies were picked , grown and lysed as described previously 34 , with the difference that the lysis buffer composition was altered ( 1 mg / ml lysozyme , 0 . 1 mm pmsf , 50 mm na 2 so 4 , 4 mm mgso 4 and 1 mm edta in 50 mm mops buffer ph 7 . 0 ). screening was performed by incubating crude cell extract with substrate solution in low well microtiter plates at 37 ° c . interesting mutants were then produced at erlenmeyer scale and purified by ni - affinity chromatography 35 for further characterization at a temperature of 58 ° c . all reactions were performed with a donor concentration of 100 mm ( sucrose or α - d - glucose 1 - phosphate ) and acceptor concentration of 200 mm ( glucose ) and were analysed by high performance anion exchange chromatography with pulsed amperometric detection ( hpaec - pad ) ( dionex ics - 3000 , thermo scientific ). the k / m ratio was defined as the ratio of initial velocity of kojibiose formation and the initial velocity of maltose formation . the production of kojibiose was performed by incubating 2 mg / ml of mutant l341i_q345s with 0 . 5m sucrose and 0 . 5m glucose in 0 . 1 l of mops buffer at ph 7 and 55 ° c . for 24 h , after which the enzyme was inactivated by heating the mixture to 95 ° c . during 10 min . next , the ph was adjusted to 5 , and 30 g / l baker &# 39 ; s yeast ( algist bruggeman ) was added . after 8 h incubation at 30 ° c . and 50 rpm on a rotary shaker , the yeast was removed by centrifugation ( 5 000 g , 4 ° c ., 20 min ), and the obtained supernatant was evaporated at 50 ° c . to a brix of 48 ( atago hand refractometer ) using a rotary evaporator ( buchi , rotavapor r - 134 ). next , the kojibiose solution was slowly cooled to 22 ° c . over a period of 4 h , and subsequently stored during 16 h at 22 ° c . the obtained crystals were washed with ethanol ( 3 times 10 ml ), and dried during 24 h in vacuo . the purity of the obtained crystals was analysed by hpaec - pad ( dionex ics - 3000 , thermo scientific ) and ion - moderated partition chromatography ( aminex hpx - 87h , bio - rad ). first , several active site residues ( y132 , p134 , r135 , y196 , v233 , l341 , d342 , l343 , y344 , q345 ) were one by one mutated to alanine to evaluate their influence on product specificity . to that end , the k / m ratio of kojibiose and maltose formation was measured for each of the single mutants and compared with that of the wild - type enzyme . in some cases the preference for maltose formation was enhanced ( like for y196a ), but for other mutants the opposite was true ( e . g . p134a , d342a , y344a and q345a ). this shows that predicting changes in product specificity is not a trivial task . interestingly , all enzymes were still active with both sucrose and glucose 1 - phosphate as glycosyl donor , meaning that their donor selectivity was not fundamentally altered . next , several active site positions were submitted to site - saturation mutagenesis to identify the best single mutants for kojibiose production ( table 1 ). the best mutant was found to be l341i , which displays a k / m ratio of 3 . 9 ( formation of kojibiose over maltose ). to further improve the enzyme &# 39 ; s specificity , the best single mutant l341i was combined with the next best mutations , i . e . p134v , q345s and q345n . surprisingly , the combination with of q345n or q345s increased the k / m ratio to 15 - 18 , whereas the combination with p134v decreased the k / m ratio . the specificity of the double mutant l341i_q345n was further optimised by introducing an alanine at position 344 , resulting in a k / m ratio of 22 . combining more than 3 mutations was also attempted but no further improvements in k / m ratio could be observed ( table 1 ). to demonstrate the practical usefulness of the obtained mutants , the production of kojibiose was performed at a larger scale ( 0 . 1 l ) and with higher substrate concentrations ( 0 . 5m sucrose and 0 . 5m glucose ), which is preferred by the industry . to that end , the double mutant l341i / q345s was employed as it displays a good balance between activity and product specificity . after incubating 2 mg / ml of enzyme with the substrates for 24 h , the reaction was terminated by heating to 95 ° c . and contaminating carbohydrates were removed by yeast treatment . finally , the solution was concentrated and then slowly cooled to induce crystallization . in that way , 12 . 4 g of crystalline kojibiose with a purity exceeding 99 % could be obtained . when the wild - 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