CN106460023A - Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes - Google Patents

Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes Download PDF

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CN106460023A
CN106460023A CN201580010439.5A CN201580010439A CN106460023A CN 106460023 A CN106460023 A CN 106460023A CN 201580010439 A CN201580010439 A CN 201580010439A CN 106460023 A CN106460023 A CN 106460023A
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alpha
glucosidase
sugar
glucosan
fraction
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K.D.纳盖
E.C.哈戈
J.K.舍蒂
S.M.亨内塞
R.迪科斯莫
L.华
R.拉米雷兹
Z.汤
Z.于
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DuPont Industrial Biosciences USA LLC
Nutrition and Biosciences USA 4 Inc
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EI Du Pont de Nemours and Co
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Abstract

A method is disclosed for hydro!yzing an alpha-1,5 giucosyl-fructose linkage in a saccharide (disaccharide or oligosaccharide) such as leucrose. This method comprises contacting the saccharide with an alpha-glucosidase enzyme such as transgiucosidase or glucoamylase under suitable conditions, during which contacting step the enzyme hydrolyzes at least one alpha-1,5 glucosyl-fructose linkage of the saccharide. This method is useful for reducing the amount of leucrose in a filtrate isolated from a glucan synthesis reaction, for example.

Description

Come enzyme hydrolysiss disaccharide and oligosaccharide using alpha-Glucosidase
This application claims U.S. Provisional Application 61/945,233 (submitting to on 2 27th, 2014), 61/945,241 (2014 2 months 27 days submit to), 62/004,290 (on May 29th, 2014 submission), 62/004,308 (on May 29th, 2014 submission), 62/ 004,312 (on May 29th, 2014 submission), 62/004,300 (on May 29th, 2014 submission), 62/004,314 (in May, 2014 Submit within 29th) and 62/004, the rights and interests of 305 (on May 29th, 2014 submissions), the full content of all these applications is with the side of quoting Formula is expressly incorporated herein.
Technical field
The invention belongs to the enzyme hydrolysiss field of less glycopolymers.In particular it relates to use alpha-Glucosidase water Solution comprises one or more α -1, the disaccharide of 5 glucityls-Fructose key and oligosaccharide.
The quoting of the sequence table electronically submitted
Electronically the document of sequence table is submitted as the sequence table of ASCII fromat by EFS-Web, this article The entitled CL6115USNP_SequenceListing_ST25.txt of part, date created is on 2 10th, 2015, file size For 266 kilobytes, and this document is submitted to this specification simultaneously.The sequence table comprising in the file of this ASCII fromat Part for described description and being incorporated by reference in its entirety herein.
Background technology
Glucoamylase (EC 3.2.1.3, α-Isosorbide-5-Nitrae-glucan glucohydralase) is two that catalyzing hydrolysis contain glucose α-the Isosorbide-5-Nitrae of non-reducing end of sugar, oligosaccharide and polysaccharide and α -1, both 6 glycosidic bonds, discharge the outer of a glucose unit every time Effect enzyme (1960, Pazur and Ando, J.Biol.Chem.235：297-302).Cracking betides the sugar connecting anomeric carbon and oxygen Glycosidic bond (1962, Fleetwood and Weigel, Nature 196：984).α-Isosorbide-5-Nitrae, α -1,6 and α -1,3 keys are only to be formed sediment by glucose The key (1957, Barker et al., J.Chem.Soc.4865-4871) that powder enzyme is hydrolyzed with notable speed.
Glucoamylase can also hydrolyze by α -1,2 (such as 2-O-alpha-D-Glucopyranosyl-D-glucose .s) or α -1, two glucose that 1 (trehalose) connects Glycosidic bond between base unit.However, with to having α-Isosorbide-5-Nitrae (maltose) or α -1, the glucose of the disaccharide of 6 (dextrinose) key Amylase activity is compared, and this kind of enzymatic activity betides lower speed and the concentration of substrate of more dilution.
Glucoamylase has been widely used for producing high glucose slurry by starch.High glucose slurry can be used as producing respectively Plant the raw material of value added compound such as alcohol fuel, high-fructose corn syrup, organic acid, aminoacid and vitamin.From multiple Isolate glucoamylase in microorganism, animal core plant, and in microorganism, many funguses are the good sources of this enzyme. The glucoamylase producing in fungal organism such as aspergillus niger (Aspergillus niger) is usually used in business application, example Starch as high glucose and produce.
Transglucosidase (EC.2.4.1.24, Isosorbide-5-Nitrae-alpha-glucanses 6- alpha-glucosyl transferring enzyme) is and α-D- glucose-oligomeric Sugar together incubate when catalyzing hydrolysis and transfer reaction D- glucosyltransferase (1951, Pazur and French, J.Amer.Chem.Soc.73：3536).Maltose is the most preferred substrate carrying out turning glucosylation reaction with this enzyme.Transfer Occur most frequently in HO-6, thus dextrinose is produced by D-Glucose, or panose (6-O- alpha-glucosyl Fructus Hordei Germinatus are produced by maltose Sugar).Transglucosidase also can make glucosyl residue be transferred to HO-2 or HO-3 of another D- glucosyl units, to form bent two Sugar or 3-O-alpha-D-Glucopyranosyl-D-glucose.This enzyme can make D- glucosyl units be transferred back to HO-4, thus improveing maltose further.
Adopt transglucosidase due to turning glucosylation reaction, so Fructus Hordei Germinatus-oligosaccharide residue is converted into comprises higher ratio α-the D-1 by non-reducing end of example, the Isomalt-oligosaccharide (IMO) of the bonded glucosyl residue of 6 glucosides.IMO sugar is in Asia For numerous food and drink formula.Brier et al. (U.S. Patent Application Publication 2003/0167929) discloses using turning Portugal Glycosidase produces IMO by barley malt juice.
Poulose et al. (U.S. Patent Application Publication 2008/0229514) discloses with transglucosidase degradation of polysaccharide example As xanthan gum and guar gum.Xanthan gum includes cellulosic backbone, wherein selects glucose 1 else, and 3- connects to comprising mannose and Portugal On the side chain of alduronic acid.The main chain of guar gum includes making galactose residue α -1 every a mannose, and the β that 6- is connected thereto - The mannose residue of Isosorbide-5-Nitrae-connection.
Lantero et al. (United States Patent (USP) 5770437) discloses and is degraded sucrose, melezitose and Sargassum ketone with transglucosidase Sugar.These sugar include being bonded, via 1,2- (sucrose), 1,3- (melezitose) or 1,1- (trehalulose), the glucose being connected to Fructose.
Although the various hydrolysing activities of glucoamylase and transglucosidase have been disclosed, these enzymes are generally viewed as Alpha-Glucosidase, gives the ability that they hydrolyze α-key between two glucosyl residue.For example, glucoamylase and turn glucose Glycosides enzyme both of which is related to having maltase activity (α-Isosorbide-5-Nitrae glycosidic bonds between two glucosyl residue of hydrolysis maltose) Connection, it is a type of alpha-glucosidase activity.
Despite aforementioned disclosure, but have been surprisingly discovered that alpha-Glucosidase such as transglucosidase (EC 2.4.1.24), α -1 of glucoamylase (EC 3.2.1.3) and other alpha-Glucosidase hydrolyzable glucityl-Fructose, 5 glucosides Key.Disclosed herein is alpha-Glucosidase comprises glucityl-α -1, the disaccharide of 5- Fructose and oligosaccharide for degraded.
Content of the invention
In one embodiment, the present invention relates to one kind makes to comprise at least one α -1, in the sugar of 5 glucityls-Fructose key α -1, the method for 5 glucityls-Fructose key hydrolysis, wherein said sugar is disaccharide or oligosaccharide, and wherein the method includes：Make Sugar is contacted under suitable conditions with alpha-Glucosidase, wherein at least one α -1 of alpha-Glucosidase hydrolysis sugar, and 5 glucityls-really Sugared key, and wherein sugared amount is compared to the amount minimizing of the sugar existing before contact procedure.
In another embodiment, the alpha-Glucosidase of method for hydrolysis is fixing.
In another embodiment, the sugar of method for hydrolysis is lucrose.In another embodiment, connecing After tactile step, the concentration of lucrose is less than the 50% of the concentration of lucrose existing before contact procedure.
In another embodiment, the suitable condition of method for hydrolysis includes：(i) glucosan synthetic reaction, or (ii) The fraction obtaining from glucosan synthetic reaction；Wherein sugar is the by-product of glucosan synthetic reaction.In another embodiment, Glucosan synthetic reaction produces at least one insoluble alpha-glucanses product.In another embodiment, level is divided into glucosan The filtrate of synthetic reaction.In another embodiment, glucosan synthetic reaction produces at least one solubility alpha-glucanses and produces Thing, it is：The product of (i) glucosyltransferase, or (ii) glucosyltransferase and alpha-glucanses hydrolytic enzyme synergism Product, this alpha-glucanses hydrolytic enzyme can hydrolyze has one or more α -1,3- glycosidic bond or one or more α -1,6- sugar The dextran polymer of glycosidic bond.In another embodiment, level is divided into the chromatograph fraction of glucosan synthetic reaction, and wherein Portugal gathers Sugared synthetic reaction produces at least one solubility alpha-glucanses product.
In another embodiment, alpha-Glucosidase is transglucosidase or glucoamylase.In another embodiment party In case, (i) transglucosidase comprises and SEQ ID NO：1 at least 90% identical aminoacid sequence；Or (ii) glucose starch Enzyme comprises and SEQ ID NO：2 at least 90% identical aminoacid sequences.
In another embodiment, the present invention relates to a kind of by making the sugar combination that contacts with alpha-Glucosidase and produce Thing, wherein sugar for disaccharide or oligosaccharide and comprise at least one α -1, and 5 glucityls-Fructose key, wherein alpha-Glucosidase hydrolyze At least one α -1 of sugar, 5 glucityls-Fructose key, and the sugar amount that wherein compositionss comprise are compared to presence before contact procedure Sugar amount reduces.
In another embodiment, the sugar of compositionss is lucrose.For example, lucrose in compositionss Concentration be less than contact before exist the concentration of lucrose 50%.
In another embodiment, the sugar of compositionss is in (i) glucosan synthetic reaction, or (ii) is anti-from glucosan synthesis In the fraction that should obtain；Wherein sugar is the by-product of glucosan synthetic reaction.In another embodiment, level is divided into glucosan The chromatograph fraction of the filtrate of synthetic reaction or glucosan synthetic reaction.
In another embodiment, the present invention relates to a kind of enrichment is present in Fructose in the fraction of glucosan synthetic reaction Method, the method includes：A () makes the fraction obtaining from glucosan synthetic reaction connect under suitable conditions with alpha-Glucosidase Touch, wherein alpha-Glucosidase hydrolyzes at least one α -1 of contained disaccharide or oligosaccharide in fraction, 5 glucityls-Fructose key；With And (b) separating levulose from step (a) is through hydrolysis fraction, to obtain the fructose concentration of the fraction that fructose concentration is than step (a) Higher compositionss.
In another 13rd embodiment, the present invention relates to a kind of fermentation process, the method includes：A () makes to gather from Portugal The fraction that sugared synthetic reaction obtains is contacted under suitable conditions with alpha-Glucosidase, wherein institute in alpha-Glucosidase hydrolysis fraction The disaccharide containing or at least one α -1 of oligosaccharide, 5 glucityls-Fructose key；B () is with the fraction of microbial fermentation step (a) to obtain Obtain product, wherein fermentation can be carried out after step (a) or with step (a) simultaneously；And (c) is optionally, separate the product of (b) Thing；Wherein compared to the product yield that the fraction of the glucosan synthetic reaction not contacted with alpha-Glucosidase is fermented, (b) Product yield increase.
Accompanying drawing and BRIEF DESCRIPTION OF THE SEQUENCES
Fig. 1：(parent material) and (treated material) glucosan reaction afterwards before being processed with NOVO 188 enzyme hydrolysiss Filtrate material1H NMR spectra (referring to embodiment 2-3).
Fig. 2：(parent material) and (treated material) Portugal afterwards before with TG L-2000 transglucosidase hydrolysis process Formose Reaction filtrate material1H NMR spectra (referring to embodiment 2-3).
Table 1 nucleic acid and the general introduction of protein sequence identification number
Specific embodiment
The disclosure of the patent of all references and non-patent literature is incorporated by reference in its entirety herein.
As used herein, term " invention " or " disclosed in this invention " are not intended to restriction but apply in general to claim Defined in or any invention as herein described.These terms are used interchangeably herein.
Except as otherwise noted, term " sugared ", " glycan molecule " and " carbohydrate " is used interchangeably herein, and refers to Disaccharide or oligosaccharide." disaccharide " refers to the carbohydrate with two monosaccharide bonded by glucosides herein." oligomeric herein Sugar " refers to by the carbohydrate for example consisting of 2 to 9 bonded monosaccharide of glucosides.Herein, oligosaccharide also can claim For " oligomer ".In disaccharide or oligosaccharide, contained monosaccharide can be described as such as " monosaccharide unit " or " monomeric unit ".Excellent herein The monosaccharide of choosing is Fructose and glucose.
Term " glycoside link " and " glycosidic bond " are used interchangeably herein, and are to instigate a carbohydrate molecule The class covalent bond being connected with another carbohydrate molecule.
This paper term " α -1,3 glucityls-glucose key ", " α -1,3 glucose-glucose keys " and " glucose-α -1,3- Glucose " refers to α -1 between two alpha-D-glucose molecules, 3- glycosidic bond.This paper term " α -1,6 glucityls-glucose Key ", " α -1,6 glucose-glucose keys " and " glucose-α -1,6- glucose " refer between two alpha-D-glucose molecules α -1,6- glycosidic bond.In certain embodiments, one or more α -1,3 glucityls-glucose key and/or α -1 herein, 6 Portugals Glycosyl-glucose key is included within disaccharide or oligosaccharide.
This paper term " α -1,5 glucityls-Fructose key ", " α -1,5 Glucose-Fructose keys " and " glucose-α -1,5- fruit Sugar " refers to α -1 between alpha-D-glucose molecule and Fructose molecule, 5- glycosidic bond.In certain embodiments, this paper α -1,5 Glucityl-Fructose key is included within disaccharide or oligosaccharide.
" alpha-D-glucose " of this paper is alternatively referred to as " glucose ".
Comprise α -1, the disaccharide of 5 glucityls-Fructose key is referred to herein as lucrose.Term " lucrose " " D- glycopyranosyl-α (1-5)-D- fructopyranose " herein used interchangeably.Lucrose has following structure：
Term " alpha-Glucosidase ", " α-Isosorbide-5-Nitrae-glucosidase " and " α-D- glucoside glucohydralase " herein exchange Use.It is oligomeric that alpha-Glucosidase (EC 3.2.1.20) (" EC " refers to enzyme identifier) had previously been accredited as catalyzing hydrolysis release The enzyme of the alpha-D-glucose residue of sugared (such as disaccharide) and the end of polysaccharide substrate non-reducing (Isosorbide-5-Nitrae)-connection.Now public herein Also to α -1,5 glucityls-Fructose key has hydrolysing activity to the alpha-Glucosidase opened, and to α -1, and 3 and α -1,6 glucityls-really Sugared key has hydrolysing activity.Transglucosidase and the example that glucoamylase is the alpha-Glucosidase with such activity.
Term " transglucosidase " (TG), " transglucosidase " and " Isosorbide-5-Nitrae-alpha-glucanses 6- alpha-glucosyl transferring enzyme " exist Used interchangeably herein.Transglucosidase (EC 2.4.1.24) had previously been accredited as together with some α-D- glucose-oligosaccharide The D- glucosyltransferase of catalyzing hydrolysis and transfer reaction during incubation.Presently disclosed transglucosidase is also to α -1 herein, 5 glucityls-Fructose key has hydrolysing activity, and to α -1, and 3 and α -1,6 glucityls-Fructose key has hydrolysing activity.
Term " glucoamylase " (GA), " glucoamylase " and " α-Isosorbide-5-Nitrae-glucan glucohydralase " are herein Used interchangeably.Glucoamylase (EC 3.2.1.3) be previously accredited as catalyzing hydrolysis contain the disaccharide of glucose, oligosaccharide and α-the Isosorbide-5-Nitrae of polysaccharide non-reducing end and α -1, the outer effect enzyme of both 6 glycosidic bonds.Herein presently disclosed glucoamylase also to α- 1,5 glucityl-Fructose key has hydrolysing activity.
Enzyme hydrolysiss are that wherein in the case of adding key element water, enzymatic enters the process of the bond fission in molecule." water herein Solution ", " hydrolysis " or " to its hydrolysing activity " α -1,5 glucityls-Fructose key refers to by alpha-Glucosidase such as glucose starch Enzyme or transglucosidase come α -1 between enzyme hydrolysiss glucose and Fructose, 5 glycosidic bonds.Such hydrolysis is comprising α -1,5 glucose Occur when the disaccharide of base-Fructose key or oligosaccharide are contacted under suitable conditions with the alpha-Glucosidase of this paper.Therefore, herein " hydrolysis " at least include：I () comprises α -1, the disaccharide of 5 glucityls-Fructose key or oligosaccharide, and (ii) alpha-Glucosidase.
" saccharifying " refers to the process of for sugared (disaccharide or oligosaccharide) to resolve into its monosaccharide component herein.Can hydrolyze anti-herein Make sugar that saccharifying occurs in answering.
For making to comprise at least one α -1, the sugar (disaccharide or oligosaccharide) of 5 glucityls-Fructose key and the α-glucose of this paper " the suitable condition " of the contact of glycosides enzyme refers to support one or more α -1 of alpha-Glucosidase hydrolysis sugar, 5 glucityls-Fructose key Those conditions (such as temperature, pH, time).Suitable condition may include " aqueous conditions ", for example, include at least 20 weight % Water.Aqueous conditions can behave as solution or mixture.Wherein make to comprise at least one α -1, the sugar of 5 glucityls-Fructose key and α - Glucosidase contact solution or mixture can be described as alpha-Glucosidase reaction (for example, transglucosidase or glucoamylase are anti- Should).
" fix " enzyme herein to refer to be bound to the enzyme of the soluble material of inertia.It is public that such as United States Patent (USP) discloses 5541097 Open the method for preparing immobilized enzyme, the disclosure of which is herein incorporated by reference.
Term " glucosan " and " dextran polymer " are in this paper used interchangeably, and refer to the Fructus Vitis viniferae bonded through glucosides The polysaccharide of sugar monomer." alpha-glucanses " refer to dextran polymer herein, it comprises at least about 80%, 81%, 82%, 83%, 84%th, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% α-glycosidic bond.
" insoluble glucan " refers to the dextran polymer insoluble in aqueous conditions herein.This paper insoluble glucan One example is poly- α -1 that DP is at least 8 or 9,3- glucosan.In certain embodiments, as presently disclosed glucityl Transfer enzyme reaction produces at least one insoluble glucan product.
Term " soluble glucan ", " solubility alpha-glucanses ", " soluble fiber ", " soluble glucan fiber ", " water soluble dietary fiber " etc., in this paper used interchangeably, dissolves in the dextran polymer of aqueous conditions with finger.This paper solubility The example of glucosan is specific oligosaccharide, poly- α -1 that such as DP is less than 8,3- glucosan, and disclosed in example presented below Specific oligosaccharide.In certain embodiments, the glucosylation enzyme reaction generation as presently disclosed is at least one solvable Property beta-glucan products.In certain embodiments, another stack features characterizing this paper solubility alpha-glucanses compound are：Its Be (i) Water-Soluble Glucose oligomer, there is the 3 or bigger degree of polymerization, (ii) digestion anti-(show extremely slowly digestible or There is no digestibility), seldom or do not absorb in people's intestinal absorption, and (iii) is at least partly fermentable in lower gastrointestinal tract. The digestibility of soluble glucan fiber composition can for example be measured with AOAC method 2009.01.
Term " poly- α -1,3- glucosan " and " α -1,3- dextran polymer " herein used interchangeably.Poly- α -1,3- Portugal Polysaccharide is the polymer of the glucose monomer unit comprising to link together through glycosidic bond, and wherein at least about 50% glycosidic bond is α -1,3- glycosidic bond.As used herein, term " α -1,3- glycosidic bond " refers to by the carbon 1 and 3 on adjacent alpha-D-glucose ring The class covalent bond that alpha-D-glucose molecule is engaged with each other.
" molecular weight " of this paper glucosan is represented by number-average molecular weight (Mn) or weight average molecular weight (Mw).Alternatively, molecule Amount be represented by dalton, gram/mol, DPw(weight average degree of polymerization) or DPn(number-average degree of polymerization).For calculating these molecule measurings Fixed various methods are well known in the art, for example with high pressure lipuid chromatography (HPLC) (HPLC), size exclusion chromatography (SEC) Or gel permeation chromatography (GPC) (SEC).
Term " glucosyltransferase ", " gtf enzyme ", " gtf enzyme catalyst ", " gtf ", " dextransucrase " etc. are herein Used interchangeably.The gtf enzyme catalysiss sucrose substrate reactions of activity are to be obtained product glucosan and Fructose herein.Other of gtf reaction Product (by-product) may include glucose (obtaining when from glucityl-gtf enzyme intermediate hydrolyzation of glucose), various solvable Property oligosaccharide (such as DP2-DP7) and lucrose (glucityl-gtf enzyme intermediate glucose and Fructose even Obtain when connecing).The glucosyltransferase of wild-type form generally comprises (N- end direction is to C- end direction) signal peptide, variable Domain, catalytic domain and glucan binding domian domain.According to CAZy (carbohydrate-organized enzyme) data base, this paper glucosyltransferase is returned Class is glycoside hydrolase Families 70 (GH70) (Cantarel et al., Nucleic Acids Res.37：D233-238,2009).
This paper term " sucrose " refers to through α -1, the bonded alpha-D-glucose molecule of 2- glucosides and β-D-Fructose group of molecules The non-reducible disaccharide becoming.Sucrose is commonly referred to sugar.
Term " glucosan synthetic reaction ", " glucosan reaction ", " gtf reaction " etc. are in this paper used interchangeably, and refer to The reaction being carried out by glucosyltransferase.As used herein, glucosan synthetic reaction is usually directed to such a solution：In bag At least one activity glucosyltransferase is comprised in solution containing sucrose and water and optional other components.Can be in this paper Portugal Other components in polysaccharide synthetic reaction include such as Fructose, glucose, lucrose, soluble oligosaccharide (for example ) and one or more soluble glucan product DP2-DP7.In addition, in some respects, glucosan synthetic reaction may include One or more alpha-glucanses hydrolytic enzyme.It should be appreciated that some beta-glucan products such as degree of polymerization (DP) be at least 8 or 9 poly- α -1,3- glucosan is water-insoluble and insoluble therefore in glucosan synthetic reaction, but can separate out solution.
Term " alpha-glucanses hydrolytic enzyme " and " glucan hydrolase " in this paper used interchangeably, and be refer to hydrolyzing alpha- The enzyme of glucan oligomer.Alpha-glucanses hydrolytic enzyme can be defined to the endo hydrolysis activity of specific α-D- glycosidic bond by it. The example of this paper alpha-glucanses hydrolytic enzyme includes glucanase (EC 3.2.1.11；Can endo hydrolysis α -1,6- connect glucosides Key), mutant enzyme (EC 3.2.1.59；Can endo hydrolysis α -1, the glycosidic bond that 3- connects) and alternan enzyme (alternanases)(EC 3.2.1.-；Can endo hydrolysis cracking alternan (alternan)).Various factors include but It is not limited to degree of branching in some alpha-glucanses, branched type and opposed branch length, alpha-glucanses water can be negatively affected The ability of solution enzyme endo hydrolysis some glucoside key.
" the dry solid percentage ratio " of glucosan synthetic reaction refers to weight % of all sugar in glucosan synthetic reaction.Can example As calculated the dry solid percentage ratio of gtf reaction based on the sucrose amount for preparing product.
" fraction " of the glucosan synthetic reaction of this paper refers to the liquid solution part of glucosan synthetic reaction.Fraction can be Derive from the part or all of liquid solution of glucosan synthetic reaction, and separate the solubility of synthesis or insoluble Portugal in autoreaction Polysaccharide product.In some embodiments, fraction is optional referred to as " mother solution ", and wherein product is insoluble (solid) glucosan Product.One example of fraction is the filtrate of glucosan synthetic reaction.Because fraction can comprise the sugar such as sucrose, really dissolving Sugar, glucose, lucrose, soluble oligosaccharide (such as DP2-DP7), so fraction can also referred to as come from glucosan and close Become " sugar juice of mixing " of reaction." fraction through hydrolysis " refers to process through the alpha-Glucosidase of this paper to hydrolyze herein It is present in the fraction of lucrose in fraction and/or oligosaccharide.
Term " filtrate ", " glucosan reacts filtrate ", " glucosan filtrate " etc. are in this paper used interchangeably, and refer to from Portugal The fraction filtering out in the solid beta-glucan products of synthesis in polysaccharide synthetic reaction." filtrate through hydrolysis " refers to herein Cross this paper alpha-Glucosidase to process to hydrolyze the filtrate of the lucrose being present in filtrate and/or oligosaccharide.
Term " percentage ratio by volume ", " percent by volume ", " volume % ", " v/v% " etc. are in this paper used interchangeably.Molten In liquid, the percent by volume of solute can be measured with following formula：[(solute volume)/(v liquor capacity)] × 100%.
Term " weight % ", " percentage by weight (weight %) ", " weight-weight percentages (w/w %) " etc. exist This paper used interchangeably.Weight % refers to material percentage when it is comprised in compositionss, mixture or solution in mass Than.Except as otherwise noted, all percentage ratios of this paper are all weight percentage.
As used herein, " polydispersity index ", " PDI ", " heterogeneity index ", " dispersibility " etc. refer to that measure gives Polymer (such as glucose oligomer, such as solubility alpha-glucanses) sample middle-molecular-weihydroxyethyl is distributed, and can pass through weight average Molecular weight to calculate (PDI=M divided by number-average molecular weightw/Mn).
Term " increase ", " enhanced " and " improvement " is in this paper used interchangeably.These terms refer to bigger amount or Activity, for example, be slightly larger than the amount of primary quantity or activity or activity or compared to primary quantity or active large excess of amount or activity, And including intervenient all amounts or activity.Alternatively, these terms can refer to such as this quantity or activity with and it compared with Quantity relatively or activity are compared, height at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%th, 14%, 15%, 16%, 17%, 18%, 19% or 20%.
As used herein, for polynucleotide or peptide sequence, term " sequence iden " or " homogeneity " refer to referring to Identical nucleic acid base or amino acid residue in two sequences when fixed comparison window scope compares for obtaining maximum correspondence.Cause This, " Percentage of sequence identity " or " homogeneity percentage " refers to the sequence by comparing two best alignment in comparison window And the value recording, wherein when being compared with reference sequences (it does not comprise to add or lacks), the many nucleoside in comparison window The part of acid or peptide sequence can comprise to add or lack (i.e. breach) to realize the optimal comparison of two sequences.By with lower section Formula calculates this percentage ratio：The number determining the position identical nucleic acid base or amino acid residue in the two sequences is to obtain To the number of the position of coupling, by the number of the position of coupling divided by the total number of position in comparison window, then result is taken advantage of With 100 to obtain Percentage of sequence identity.
Available online in National Center for Biotechnology Information (NCBI) website Basic Local Alignment Search Tool (BLAST) algorithm can for example be used for calculating two disclosed herein or Percentage identities between more polynucleotide sequences (BLASTN algorithm) or peptide sequence (BLASTP algorithm).Alternative Ground, the percentage identities between sequence can be calculated using Clustal algorithm (such as ClustalW or ClustalV).For Using the multiple alignment of Clustal comparison method, default value can be equivalent to GAP PENALTY=10 and GAP LENGTH PENALTY=10.With Clustal method carry out in contrast with to and protein sequence percentage identities calculate default parameterss Can be KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.For nucleic acid, these ginsengs Number can be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.Still alternatively, sequence Between homogeneity percentage can be carried out using EMBOSS algorithm (such as pin (needle)), parameter be such as GAP OPEN= 10, GAPEXTEND=0.5, END GAP PENALTY=false, END GAP OPEN=10, END GAP EXTEND= 0.5, using BLOSUM matrix (such as BLOSUM62).
Disclosed herein is multiple polypeptides aminoacid sequence is as the feature of some embodiments.Can use with disclosed herein The variant of sequence at least about these sequences of 70-85%, 85-90% or 90%-95% identical.Alternatively, variant amino acids sequence Row and sequence disclosed herein can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%th, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%th, 95%, 96%, 97%, 98% or 99% homogeneity.This paper Variant amino acid sequences have identical with open sequence Function/activity, or have open sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%th, 95%, 96%, 97%, 98% or 99% function/activity.
As term " detached " used in certain embodiments refer to be kept completely separate with its natural origin any thin Born of the same parents' component (for example detached polynucleotide or peptide molecule).In some cases, detached polynucleotide or peptide molecule are A part for larger compositionss, buffer system or reagent mixture.For example, detached polynucleotide or peptide molecule can be heterologous Mode is included in cell or biological interior.Another example is that detached alpha-Glucosidase (such as glucoamylase, turns glucoside Enzyme) or glucosyltransferase.Enzyme reaction disclosed herein (such as alpha-Glucosidase reaction, glucosylation enzyme reaction) is Synthesis, non-naturally occurring process.
Some embodiments disclosed in this invention are related to one kind and make to comprise at least one α -1,5 glucityls-Fructose key α -1 of sugar, the method for 5 glucityls-Fructose key hydrolysis.Sugar is disaccharide or oligosaccharide.The method includes making sugar and alpha-Glucosidase Contact under suitable conditions.In contact procedure, at least one α -1 of alpha-Glucosidase hydrolysis sugar, 5 glucityls-Fructose key. Due to this kind of hydrolysis, sugar amount reduces compared to the sugar amount existing before contact procedure.Therefore, this method for hydrolysis or alternatively The method referred to as reducing sugar amount in compositionss.
Significantly it is believed that alpha-Glucosidase hydrolyzable α -1,5 glucityls-Fructose key is previously unknown.According to this hydrolysis Method alpha-Glucosidase reaction can thus be accordingly used in from glucosan synthetic reaction and/or from the fraction that it obtains remove comprise α- The lucrose of 1,5 glucityl-Fructose key and other oligosaccharide by-product.Such removal illustrates poly- with respect to may result in Portugal The by-product of sugared product degradation removes chemical process (such as acid hydrolysis) and improves to some extent.Finally, processed according to above method for hydrolysis Glucosan reaction fraction be more suitable for for example downstream application as ferment because the content of glucose and Fructose monosaccharide increases in fraction Plus.For downstream process, monosaccharide is generally more disposable compared to lucrose and oligosaccharide by-product.
Alpha-Glucosidase used in embodiments herein (EC 3.2.1.20) hydrolyzes and comprises at least one α -1, and 5 α -1 of the sugar of glucityl-Fructose key, 5 glucityls-Fructose key.Alpha-Glucosidase previously identified for catalyzing hydrolysis release Oligosaccharide (such as disaccharide) and the alpha-D-glucose residue of the end of polysaccharide substrate non-reducing (Isosorbide-5-Nitrae)-connection.Now public herein Also to such as α -1,5 glucityls-Fructose key has hydrolysing activity to these enzymes opened.
Alpha-Glucosidase is available from for example any source (such as plant, animal, microorganism, such as antibacterial or funguses/yeast), Transglucosidase for example disclosed below and/or glucoamylase may originate from its those sources.For example, alpha-Glucosidase can be The alpha-Glucosidase of funguses.Herein the other examples of suitable alpha-Glucosidase include United States Patent (USP) 6355467,5922580, 5795766th, those disclosed in 5763252 and 8633006, above-mentioned document is all herein incorporated by reference.
In certain embodiments, alpha-Glucosidase can include SEQ ID NO：5、6、8、9、11、12、14、15、17、 18th, 20,22,24,26,28,30,32,34,36,38 aminoacid sequence, or DIAZYME RDF ULTRA (DuPont Industrial Biosciences) aminoacid sequence.Alternatively, alpha-Glucosidase may include and SEQ ID NO：5、6、8、 9th, the aminoacid of 11,12,14,15,17,18,20,22,24,26,28,30,32,34,36,38 or DIAZYME RDF ULTRA Sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical aminoacid sequence, And to sugared α -1,5 glucityls-Fructose key has hydrolysing activity.For example several foregoing sequences are to lack N- terminal signal peptide Ripe alpha-Glucosidase.If for such sequence it will be appreciated that being expressed to it and (for example not adopted using signal peptide Expressed in the cell with wherein enzyme and the expression system available from cell lysate), then it is usually added into N- end initial methionine (if necessary) (directly or via insertion heterologous amino acid sequence such as epi-position).
Transglucosidase (EC 2.4.1.24；Isosorbide-5-Nitrae-alpha-glucanses 6- alpha-glucosyl transferring enzyme) can be in some realities of this paper Apply and in scheme, be used as alpha-Glucosidase, comprise at least one α -1 to hydrolyze, α -1 of the sugar of 5 glucityls-Fructose key, 5 glucityls - Fructose key.It is anti-that this fermentoid had previously been accredited as catalyzing hydrolysis and transfer when incubating together with specific α-D- glucose-oligosaccharide Should both D- glucosyltransferases.Also to α -1,5 glucityls-Fructose key has water to transglucosidase as presently disclosed in this paper Solution activity.
The transglucosidase of this paper can derive from any microbial source, such as antibacterial or funguses.The transglucosidase of funguses Example include but is not limited to trichoderma (Trichoderma) strain (such as trichoderma reesei (T.reesei)), aspergillus (Aspergillus) those of strain and Neosartorya (Neosartorya) strain (such as N.fischeri).Turn glucoside Enzyme may originate from its aspergillus bacterium example include but is not limited to aspergillus niger (A.niger), aspergillus awamori (A.awamori), Aspergillus oryzae (A.oryzae), aspergillus terreus (A.terreus), Aspergillusclavatus (A.clavatus), Aspergillus fumigatus (A.fumigatus) and structure Nest aspergillosis (A.nidulans).Can be used for this paper transglucosidase other examples Barker et al. (1953, J.Chem.Soc.3588-3593)；Pazur et al. (1986, Carbohydr.Res.149：137-147), Nakamura et al. (1997, J.Biotechnol.53：It is described 75-84) and in U.S. Patent Application Publication 2008/0229514, these patents All it is herein incorporated by reference.Can be used for the transglucosidase of this paper other examples be heat-staple those；United States Patent (USP) 4689296 disclose the method for preparing heat stability transglucosidase, and the disclosure of which is herein incorporated by reference.Can For the transglucosidase of this paper more examples can be in GENBANK data base (NCBI) any those, for example log in Number：D45356(GID：2645159, aspergillus niger), BAD06006.1 (GID：4031328, aspergillus awamori), BAA08125.1 (GID：1054565, aspergillus oryzae), XP_001210809.1 (GID：115492363, aspergillus terreus), XP_001216899.1 (GID： 115433524, aspergillus terreus), XP_001271891.1 (GID：121707620, Aspergillusclavatus), XP_751811.1 (GID： 70993928, Aspergillus fumigatus), XP_659621.1 (GID：67523121, aspergillus nidulanses), XP_001266999.1 (GID： 119500484, N.fischeri) and XP_001258585.1 (GID：119473371, N.fischeri), it is all with the side of quoting Formula is expressly incorporated herein.Alternatively, the transglucosidase of this paper can have the amino with any aforementioned disclosed transglucosidase sequence Acid sequence has the aminoacid sequence of at least 90% or 95% homogeneity, and to sugared α -1,5 glucityls-Fructose key has hydrolysis Activity.When all aforementioned transglucosidases are used for the hydrolysis of this paper, preferably lack the ripe shape of N- terminal signal peptide Formula.
In some embodiments of this paper, transglucosidase can comprise SEQ ID NO：1 (transglucosidase L-2000) Aminoacid sequence, its be aspergillus niger transglucosidase (U.S. Patent Application Publication 2008/0229514).Alternatively, turn glucose Glycosides enzyme can comprise and SEQ ID NO：1 at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical aminoacid sequence, and to sugared α -1,5 glucityls-Fructose key has hydrolysing activity.Any SEQ ID NO：1 Or its variant can the disclosure for example according to U.S. Patent Application Publication 2008/0229514 be obtained, this application is with way of reference It is expressly incorporated herein.SEQ ID NO：1 is the ripe transglucosidase lacking N- terminal signal peptide.Because SEQ is ID NO：1 with first If methyllanthionine residue starts it will be appreciated that being expressed to it and not using signal peptide (for example with wherein enzyme in cell Interior expression the expression system available from cell lysate), then generally N- end initial methionine is added to SEQ ID NO：1 (directly or via insertion heterologous amino acid sequence such as epi-position).
Glucoamylase (EC 3.2.1.3；α-Isosorbide-5-Nitrae-glucan glucohydralase) can be in some embodiments of this paper As alpha-Glucosidase, comprise at least one α -1, α -1 of the sugar of 5 glucityls-Fructose key, 5 glucityls-Fructose key to hydrolyze. This fermentoid had previously been accredited as catalyzing hydrolysis and had contained the α-Isosorbide-5-Nitrae of disaccharide, oligosaccharide and polysaccharide non-reducing end of glucose and α -1, and 6 The outer effect enzyme of both glycosidic bonds.Also to α -1,5 glucityls-Fructose key has hydrolysis to glucoamylase as presently disclosed in this paper Activity.In certain embodiments, alpha-Glucosidase is not glucoamylase.
The glucoamylase of this paper can derive from any microbial source, such as antibacterial or funguses.The glucoamylase of antibacterial Example include but is not limited to bacillus (Bacillus) strain (such as Alkaliphilic bacillus (B.alkalophilus), Bacillus amyloliquefaciens (B.amyloliquefaciens), bacillus lentus (B.lentus), Bacillus licheniformis (B.licheniformis), bacstearothermophilus (B.stearothermophilus), bacillus subtilises (B.subtilis), bacillus thuringiensiss (B.thuringiensis)) and streptomyces (Streptomyces) strain (example As (S.lividans)).The example of the glucoamylase of funguses includes but is not limited to trichoderma strain (such as Richter scale wood Mould, long shoot trichoderma (T.longibrachiatum), T.strictipilis, Trichoderma asperellum (T.asperellum), the long Trichoderma spp. of health (T.konilangbra), Trichoderma harzianum (T.hazianum)), aspergillus bacterium (such as aspergillus niger, aspergillus oryzae, aspergillus terreus, rod Aspergillosis, aspergillus nidulanses, Aspergillus candidus, aspergillus awamori), Rhizopus (Rhizopus) strain (such as Rhizopus oryzae (R.oryzae), snow Rhizopus (R.niveus)), Talaromyces (Talaromyces) strain (such as Ai Mosen indigo plant shape bacterium (T.emersonii), thermophilic Actinomycetes (T.thermophilus), T.duponti), Mucor (Mucor) strain, Hypocrea (Hypocrea) strain (such as H.gelatinosa, H.orientalis, H.vinosa, H.citrina), Fusarium (Fusarium) strain are (for example Fusarium oxysporum (F.oxysporum), rose-colored fusarium (F.roseum), F.venenatum), neurospora (Neurospora) strain (such as Neurospora crassa (N.crassa)), Humicola (Humicola) strain (for example grey detritus Mould (H.grisea), H.insolens, Humicola lanuginosa (H.lanuginose)), Penicillium (Penicillium) strain (example As Penicllium notatum (P.notatum), Penicllium chrysogenum (P.chrysogenum)) and active yeast genus (Saccharomycopsis) Those of strain (such as button capsule laminating adhesive yeast (S.fibuligera)).These antibacterials for this paper and Fungal Glucoamylases Study Example be disclosed in U.S. Patent Application Publication 2013/0102035, the document is hereby incorporated herein by.Can be used for The other examples of the glucoamylase of this paper are in Svensson et al. (1983, Carlsberg Res.Commun.48：529- 544), (1984, EMBO J.3 for Boel et al.：1097-1102), Havashida et al. (1989, Agric.Biol.Chem.53： 923-929)；United States Patent (USP) 5024941, United States Patent (USP) 4794175, United States Patent (USP) 4247637, United States Patent (USP) 6255084, the U.S. Patent 6620924, Ashikari et al. (1986, Agric.Biol.Chem.50：957-964), Ashikari et al. (1989, Appl.Microbiol.Biotechnol.32：129-133), United States Patent (USP) 4863864；United States Patent (USP) 4618579, Houghton-Larsen et al. (2003, Appl.Microbiol.Biotechnol.62：210-217) and United States Patent (USP) It is described in 7413887, these patents are all herein incorporated by reference.Alternatively, the glucoamylase of this paper can have There is the aminoacid sequence of at least 90% or 95% homogeneity with the aminoacid sequence of any aforementioned disclosed glucose starch enzyme sequence, And to sugared α -1,5 glucityls-Fructose key has hydrolysing activity.When all aforementioned glucoamylases are anti-for the hydrolysis of this paper At once, preferably lack the mature form of N- terminal signal peptide.The commercially available glucoamylase that can be used for this paper includes Such as OPTIDEX L-400, GC 147, GC 321, G ZYME G990 4X, OPTIMAX 7525, DEXTROZYME, DISTILLASE and GLUCZYME.
In some embodiments of this paper, glucoamylase can comprise SEQ ID NO：The aminoacid sequence of 2 (GC 321) Row, it is trichoderma reesei glucoamylase.Alternatively, glucoamylase can comprise and SEQ ID NO：2 at least about 90%, 91%th, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical aminoacid sequence, and to sugared α -1,5 Glucityl-Fructose key has hydrolysing activity.Any SEQ ID NO：2 or its variant can for example according to United States Patent (USP) 7413887 or The disclosure of U.S. Patent Application Publication 2013/0102035 is obtained, and these applications are herein incorporated by reference.SEQ ID NO：2 is the ripe glucoamylase lacking N- terminal signal peptide.Because SEQ is ID NO：2 are not started with methionine residues, should Work as understanding, if being expressed to it and (not expressing in the cell and available from cell for example with wherein enzyme using signal peptide The expression system of lysate), then generally N- end initial methionine is added to SEQ ID NO：2 (directly or via insertion Heterologous amino acid sequence such as epi-position).
The alpha-Glucosidase of this paper such as transglucosidase or glucoamylase are available from commercial source (such as DuPont Industrial Biosciences/Genencor, USA；Megazyme International, Ireland；Amano Enzyme Inc., Japan).Alternatively, this fermentoid can be obtained by any method known in the art, such as in United States Patent (USP) Application discloses described in 2008/0229514, United States Patent (USP) 7413887 or U.S. Patent Application Publication 2013/0102035, These applications are hereby incorporated herein by.For example, alpha-Glucosidase can be recombinated generation in heterologous expression system, for example micro- Biology or funguses heterologous expression system.The example of heterologous expression system includes antibacterial (such as escherichia coli (E.coli), spore bar Pseudomonas (Bacillus sp.)) and eukaryotic system.Eukaryotic system can adopt such as yeast (for example, pichia (Pichia Sp.), Saccharomyces (Saccharomyces sp.)) or funguses (for example, trichoderma, such as trichoderma reesei；Aspergillus bacterium, example As aspergillus niger) expression system.SEQ ID NO：1 transglucosidase and SEQ ID NO：2 glucoamylase and they Variant can be expressed for example in trichoderma reesei host.
When alpha-Glucosidase is used for the hydrolysis of this paper, preferably lack the mature form of N- terminal signal peptide.With Expression system in the ripe alpha-Glucosidase producing this paper can be using the polynucleotide of codase, the polynucleotide of this codase The sequence also comprising to encode N- terminal signal peptide is instructing exocytosiss.In such embodiment, believe during secretion process Number peptide cuts down from enzyme.Signal peptide can be natural or heterologous for transglucosidase or glucoamylase.Separately Selection of land, the alpha-Glucosidase of mature form can be by expressing and the table available from cell pyrolysis liquid for example with wherein enzyme in the cell The system that reaches is expressed (not using signal peptide) and is provided to it.In either case (secretion or cell inner expression), heterologous Aminoacid sequence such as epi-position may be optionally contained in the N- end of alpha-Glucosidase.
In certain embodiments, the directly cell using one or more enzyme of expression can be passed through in this paper hydrolysis Alpha-Glucosidase to be provided.In other words, the alpha-Glucosidase contacting with sugar can be because it be by the appropraite condition being placed in for hydrolysis In cell expression and exist.Such cell therefore can be used for the detached alpha-Glucosidase system replacing adding to hydrolysis Agent.Cell for the purpose can be such as antibacterial, yeast or fungal cell.The example of yeast includes deriving from following those： Saccharomyces (such as saccharomyces cerevisiae (S.cerevisiae)), Kluyveromyceses (Kluyveromyces), candida mycoderma (Candida), pichia (Pichia), fission yeast (Schizosaccharomyces), Hansenula (Hansenula), Kloeckera (Kloeckera), perhaps prosperous Saccharomyces (Schwanniomyces).Can be used for this paper Other expression systems are disclosed in U.S. Patent Application Publication 2013/0323822, and this application is hereby incorporated herein by.
The sugar of this paper comprises at least one α -1,5 glucityls-Fructose key.Therefore, according to sugared length, it can comprise for example 1st, 2,3,4,5,6,7 or 8 α -1,5 glucityls-Fructose key.Sugar preferably comprises 1,2 or 3 this generic keys.
Because the sugar of this paper comprises at least one α -1,5 glucityls-Fructose key, sugar comprises at least one glucose list Unit and at least one fructose units.In certain embodiments, the sugar of this paper only comprises glucose and fructose units.Such combination Thing can behave as disaccharide and the oligosaccharide by-product of glucosan synthetic reaction.Alternatively, in addition to glucose and Fructose, herein Sugar also can comprise other monosaccharide, such as galactose, ribose and xylose.
The sugar hydrolyzing in certain embodiments disclosed by the invention can be oligosaccharide.The oligosaccharide of this paper can have for example 2nd, 3,4,5,6,7,8 or 9 monosaccharide units.As understood in the art, the oligosaccharide of this paper can refer to its degree of polymerization (DP) number this specify the number of monomeric unit in oligosaccharide.For example, DP3 oligosaccharide has 3 monomeric units.Cause This, oligosaccharide may be, for example, DP3, DP4, DP5, DP6, DP7, DP8 or DP9 oligosaccharide.In certain embodiments, the DP of sugar is 3 to 7 (that is, DP 3-7).
Except at least one α -1, (it may be noted that having the oligosaccharide of 2 monosaccharide units outside 5 glucityls-Fructose key I.e. disaccharide is lucrose, and the sugar being given in the method for hydrolysis of this paper has at least one α -1,5 glucityls-really Sugared key), this paper there are 3 or the oligosaccharide of more monosaccharide units also can comprise other keys.For example, also may be used in oligosaccharide There is α -1,3, α -1,6 and/or α-Isosorbide-5-Nitrae key, it is also easy to be hydrolyzed by alpha-Glucosidase as illustrated herein.
In certain embodiments, sugar only comprises through α -1, and 3 and/or α -1, the bonded glucose monomer of 6 glucosides.Cause This, such oligosaccharide only comprises α -1,3 glucityls-glucose key and/or α -1,6 glucityls-glucose key.Such oligosaccharide Example only comprises α -1,3 keys or α -1,6 keys.In certain embodiments, oligosaccharide can comprise at least 85%, 86%, 87%, 88%th, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% glucityl-glucose key. In other embodiments, there may be α -1 of about 75-85%, 3 glucityls-glucose key and about 15- in the oligosaccharide of this paper 25% α -1,6 glucityls-glucose key.Alternatively, the oligosaccharide of this paper can comprise any percentage ratio (between 1% to 99% Between any integer value) α -1,3 glucityls-glucose key and any percentage ratio (arbitrarily whole between 1% to 99% Numerical value) α -1,6 glucityls-glucose key, as long as these percentage ratios be not more than 100%.These oligosaccharide any can be Derive among the fraction of glucosan synthetic reaction, it is (for example poly- that this glucosan synthetic reaction produces such as (i) insoluble alpha-glucanses α -1,3- glucosan), or (ii) solubility alpha-glucanses product.This linkage content can characterize：(i) individually each oligosaccharide, or (ii) one group of oligosaccharide (i.e. average linkage content).Only comprise via α -1,3 and/or α -1, the bonded glucose monomer of 6 glucosides Oligosaccharide may be, for example, DP2-DP7 or DP3-DP7.It should be appreciated that concrete distribution in oligosaccharide for the key can be low according to producing The condition (such as gtf enzyme) of the glucosan synthetic reaction of polysaccharide by-product and change.It is also understood that the concrete distribution of key for Presently disclosed method is not vital.
The example of this paper illustrate alpha-Glucosidase (such as transglucosidase and glucoamylase) hydrolyzable (i) comprise α- The lucrose of 1,5 glucityl-Fructose key, and (ii) only comprise α -1,3 glucityls-glucose and/or α -1,6 glucose Both oligosaccharide of base-glucose key.Therefore, can be for example for hydrolyzing alpha -1,5 glucityls-Fructose key, α -1,3 glucityls - Glucose key and/or α -1, using alpha-Glucosidase in the reaction of 6 glucityls-glucose key.
At least one α -1 of the sugar of this paper, 5 glucityls-Fructose key can be hydrolyzed by the alpha-Glucosidase of this paper.Alternatively, It is believed that the 2 of sugar, 3,4,5 or more α -1,5 glucityls-Fructose key can for example be hydrolyzed by alpha-Glucosidase.In some embodiment party In case, at least one α -1, the hydrolysis of 5 glucityls-Fructose key can occur in sugared non-reducing end.For example, wherein sugar for disaccharide is Lucrose, non-reducing end glucose is cleaved from Fructose, obtains free glucose and Fructose.And for example, wherein Sugar for having α -1,5 connect the non-reducing end glucose to Fructose oligosaccharide it is believed that this glucose can cleaved fall, oligomeric Residue of fructose is left at the non-reducing end of sugar.
In disclosed method for hydrolysis, the amount of sugar reduces compared to the amount of the sugar existing before contact procedure.This minimizing results from At least one α -1 of sugar, the hydrolytic cleavage of 5 glucityls-Fructose key.Sugar amount in this paper method for hydrolysis, after contact procedure (before making this paper alpha-Glucosidase contact under suitable conditions with sugar) institute before (such as concentration) is smaller than contact procedure There is about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of sugar amount (or any integer value between 1% to 90%).
The sugar hydrolyzing in certain embodiments disclosed by the invention is lucrose, and it is to have α -1,5 glucose The disaccharide of base-Fructose key.In this paper method for hydrolysis, the concentration of the lucrose after contact procedure is smaller than contact step The leukonid that before rapid, (before making this paper alpha-Glucosidase be contacted under suitable conditions with lucrose) is existed About the 1% of the concentration of disaccharide, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (or any integer value between 1% to 90%).At some aspects of this paper, method for hydrolysis is alternatively referred to as dropping The method of the amount of lucrose in low compositionss.
In the method for hydrolysis of this paper, lucrose and transglucosidase can be for example made for example to comprise SEQ ID NO： 1 transglucosidase (transglucosidase L-2000) contact.In certain embodiments, complete the leukonid after the method The concentration of disaccharide is smaller than initial lucrose concentration about 1-3%.
In disclosed method for hydrolysis, the amount of sugar reduces compared to the amount of the sugar existing before contact procedure.It should be understood that this ratio Relatively can carry out in any manner.For example, both sugared concentration can be measured before and after the method for being hydrolyzed.Alternatively, remove Outside such as presently disclosed alpha-Glucosidase not being added to control reaction, can with respect to the control reaction with the same terms It is compared.
In certain embodiments, alpha-Glucosidase can be fixing.Can using any method known in the art and/ Or mode comes immobilized enzyme, such as those disclosed in United States Patent (USP) 5541097 and 4713333, this two patents are all to quote Mode is expressly incorporated herein.For example, can be added by making one or more enzyme contact with amine reactive explosive (such as glutaraldehyde) to be formed Compound (such as enzyme-glutaraldehyde adduct), thereafter this adduct is attached to through polyamine (such as polyethyleneimine, such as EPOMIN One or more enzyme to be fixed on the solid carrier P-1050) processing.
In certain embodiments, the solid carrier (solid support) that alpha-Glucosidase is fixed to the upper can be made can be nothing Machine or organic material.Such material include for example gama-alumina, titanium dioxide, granular active carbon, granular kieselguhr, bead, Cellular glass, foam, silica gel, metal-oxide and aluminium oxide.
Polyamine can be used for processing solid carrier, so that solid carrier is subsequently exposed to including enzyme and amine reactive explosive Adduct, lead to enzyme to be bound to solid carrier.The example that can be used for the polyamine of this paper includes：Polyethylenediamine, polyethyleneimine Amine (for example poly- diethylenetriamines, poly- trien, poly- penten, polyhexamethylene diamidogen), polymethylene Dicyclohexylamine, polymethylene diphenylamines, poly- tetren, polyphenylene diamidogen and two or more these polyamine The blend of compound.Preferably polyamine is molecular weight that is water miscible and/or having about 500 to 100,000 dalton. Can be in certain embodiments using polyethyleneimine such as EPOMIN P-1050.
For prepare comprise this paper enzyme adduct amine reactive explosive may be, for example, aldehyde, organohalogen compounds, anhydride, Azo-compound, isothiocyanate and/or isocyanates.The example of these amine reactive explosives includes：Glutaraldehyde, succinaldehyde, Terephthalaldehyde, two-diazo benzidine -2,2 '-disulfonic acid, 4,4 '-two fluoro- 3,3 '-diphenylsulfone dinitro, diphenyl -4,4 ' - Two sulfocyanic ester -2,2 '-disulfonic acid, 3- methoxyl group diphenyl methane -4,4 '-diisocyanate, Toluene-2,4-diisocyanate-isocyanates -4- Isothiocyanate, Toluene-2,4-diisocyanate, -4- diisocyanate resin, diazo benzidine, diazo benzidine -3,3 '-dianisidine, N, N '-six Methylene iodide acetamide, hexamethylene diisocyanate, Cyanuric Chloride and/or fluoro- 2, the 4- dinitro benzene of 1,5- bis-.Preferably Ground, amine reactive explosive is aldehyde, such as glutaraldehyde.
Can make to contact with the solid carrier through polyamine treatment with the alpha-Glucosidase of amine reactive compound adduction, so that Enzyme is fixing on a solid support.The enzyme fixed herein can be used in various reactor assemblies, for example post (such as packed column) or stir Mix groove reactor, to carry out hydrolysis as disclosed herein.
For the conjunction making the sugar of this paper contact with the alpha-Glucosidase (such as transglucosidase or glucoamylase) of this paper Suitable condition is to support one or more α -1 of sugar, those conditions that 5 glucityls-Fructose key is hydrolyzed by alpha-Glucosidase.Properly The example of condition be disclosed in following examples.For making condition that the alpha-Glucosidase of this paper contacted with sugared substrate (for example Temperature, pH, time) it is also disclosed in U.S. Patent Application Publication 2008/0229514, United States Patent (USP) 7413887 and United States Patent (USP) Shen Please disclose (these patents are all herein incorporated by reference) in 2013/0102035, and be equally applicable to disclosed hydrolysis Method.
In disclosed method for hydrolysis, disaccharide and oligosaccharide usually can be dissolved in water or aqueous solution.Therefore, make this paper's Sugar is contacted with alpha-Glucosidase and preferably to carry out under the suitable aqueous conditions of dissolved sugar wherein.Aqueous conditions can behave as comprising The solution of at least about 20 weight % water or mixture.Alternatively, aqueous conditions of this paper for example, at least about 20,30,40,50, 60th, 70,80,85,90 or 95 weight % water (or any integer value between 20 to 95 weight %).Aqueous conditions also can be wrapped Include the buffer of such as suitable concn, for example acid, neutral or alkaline buffer, and based on the pH scope being provided by buffer To select.The example of buffer/buffer agent includes citrate, acetate (such as sodium acetate), KH2PO4, MOPS, CHES, boron Hydrochlorate, sodium carbonate and sodium bicarbonate.
The pH of the hydrolysis of this paper for example can be about 3.0 to 9.0.Hydrolysis pH for example can be about 3.0,3.5,4.0, 4.5th, 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5 or 9.0.Alternatively, pH can be about 4-5.For setting the technology of pH Including use such as buffer, alkali and/or acid, and it is well known in the present art.
The temperature of the hydrolysis of this paper for example can be about 20 DEG C to about 80 DEG C.Hydrolysising reacting temperature for example can be about 20, 30th, 40,50,60,70 or 80 DEG C (or any integer value between 20 to 80 DEG C).In certain embodiments, about 60 DEG C, 65 DEG C or 60-65 DEG C of hydrolysis temperature is preferred.
The hydrolysis of this paper can carry out the period of for example, at least about 10 minutes to about 90 hours.The time example of hydrolysis As being at least about 0.5,1,2,3,4,8,12,16,20,24,30,36,42,48,54,60,66,72,78,84 or 90 hours (or any integer value between 0.5 to 72 hour).In some embodiment party for example lucrose being hydrolyzed In case, can for example the carrying out less than 4 hours (such as 0.5-4 hour) of hydrolysis.Realize needed for desired hydrolysis level when Between section will be changed according to actual conditions used, and it will be appreciated by those skilled in the art that.For example, make interpolation to reaction or solid The fixed enzyme amount increase on the solid carrier for reaction will reduce time of contact.
In certain embodiments, one or more alpha-Glucosidase of this paper can be used for hydrolysis.For example, turn glucose Glycosides enzyme and glucoamylase can be used for reacting.In the hydrolysis of this paper, the amount of alpha-Glucosidase can be for example than any Amount add drop 10% to 20% (or 5% to 10%) for following examples (such as embodiment 2).Alternatively, about 0.1-0.5 The alpha-Glucosidase of volume % or 0.1-1.0 volume % can be used for hydrolysis.Still alternatively, the alpha-Glucosidase of this paper with About or at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15ppm be used for hydrolysis.Transglucosidase list Position (TGU) can for example be defined as the amount of the transglucosidase producing micromole's panose per minute under conditions of following mensure. Transglucosidase activity can for example measure as follows：By transglucosidase introduce comprise 4mM p- nitrobenzophenone-α-glucoside and In the 100mM sodium acetate buffer (pH 4.5) of 1mg/ml bovine serum albumin (BSA).Incubate 30 minutes afterwards at 30 DEG C, By adding isopyknic 1M sodium carbonate terminating reaction, and record OD405.Glucoamylase unit (XU) can for example be defined as by Produce the amount of the glucoamylase of 1g reducing sugar, be calculated as at pH 4.2 and 60 DEG C being derived from per hour soluble starch substrate The glucose of (4%DS [substitution value]).
In some embodiments disclosed by the invention, in hydrolysis, the initial concentration of sugar may be, for example, about 1 weight % To 50 weight %.For example, the concentration of lucrose can be about 5,10,15,20,25,30,35 or 40 weight % (or between 5 Any integer value to 40 weight %).And for example, in the hydrolysis of this paper, one or more oligosaccharide (such as DP2, DP3, DP4, DP2-DP7, DP3-DP7) concentration can be about 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15 weights Amount %.Those skilled in the art recognizes, the concentration of total sugar (including disaccharide and oligosaccharide) can be to the activity of alpha-Glucosidase Have an impact；In some respects, make the maximized preferred total sugar concentration of enzymatic activity be smaller than 50 weight % in hydrolysis to do Solid (DS), most preferred concentration is 20-35 weight %DS.
In certain embodiments, the suitable condition for making sugar be contacted with the alpha-Glucosidase of this paper be may include：(i) Glucosan synthetic reaction, or the fraction that (ii) obtains from glucosan synthetic reaction；Wherein sugar is the by-product of glucosan synthetic reaction Thing.In other words, the hydrolysis of this paper can in the scope of glucosan synthetic reaction or glucosan synthetic reaction a part In carry out although it is carried out generally among the latter.The glucosan synthetic reaction of this paper can for example produce one or more insoluble Property and/or solubility alpha-glucanses product.Therefore, in some embodiments of this paper, glucosan synthetic reaction can characterization For " alpha-glucanses synthetic reaction ".
Glucosan synthetic reaction is usually directed to such a solution：It comprises at least one sucrose, water and a kind of activity Portugal Glycosyl transferase and optional other components.Can the other components in glucosan synthetic reaction include Fructose, glucose, Lucrose, soluble oligosaccharide (such as DP2-DP7) and one or more soluble glucan product.In addition, Some aspects, glucosan synthetic reaction may include one or more alpha-glucanses hydrolytic enzyme.It should be appreciated that some beta-glucan products Such as DP is at least 8 or 9 poly- α -1, and 3- glucosan can be water-insoluble and therefore insoluble in glucosan synthetic reaction Solution, but solution can be separated out.Therefore, the glucosan being produced by the glucosan synthetic reaction of this paper can be insoluble.Can The alpha-Glucosidase of this paper was added to wherein in any stage of glucosan synthetic reaction, such as during reaction initial preparation Or when reaction is nearly completed (for example, completing 80 to 90%) or completes, wherein latter two time point is preferred.
The glucosan synthetic reaction of this paper, in addition to producing beta-glucan products, also can produce by-product such as leukonid Disaccharide and/or soluble oligosaccharide.In some respects, glucosan is poly- alpha-glucanses.Therefore, the glucosan synthetic reaction of this paper Can for example be used for producing poly- α -1,3- glucosan or mutan, it is bright with least one generally in glucosan synthetic reaction Beading bacterium disaccharide and/or oligosaccharide by-product produce jointly.
In certain embodiments, glucosan synthetic reaction includes producing poly- alpha-glucanses such as α -1, the Portugal of 3- glucosan Glycosyl transferase.The example that can be used for such glucosyltransferase of this paper is disclosed in United States Patent (USP) 7000000 and United States Patent (USP) Application discloses 2013/0244288,2013/0244287 and 2014/0087431, and these patents are all herein incorporated by reference.
The glucosyltransferase of this paper can derive from any microbial source, such as antibacterial or funguses.Antibacterial glucosylation The example of enzyme is from Streptococcus (Streptococcus) strain, Leuconostoc (Leuconostoc) strain or newborn bar Those of Pseudomonas (Lactobacillus) strain.The example of Streptococcus species include Lactobacillus salivarius (S.salivarius), Streptococcus sobrinus (S.sobrinus), S.dentirousetti, sobrinus (S.downei), Streptococcus mutans (S.mutans), Streptococcus oralis (S.oralis), solution gallic acid streptococcus (S.gallolyticus) and Streptococcus sanguiss (S.sanguinis).The example of Leuconostoc strain includes (L.mesenteroides), Leuconostoc mesenteroides (L.amelibiosum), Argentinian leukonid (L.argentinum), meat leukonid (L.carnosum), thermophilic citric acid Leukonid (L.citreum), Streptococcus cremoriss (L.cremoris), leuconostoc dextranicum bacteria (L.dextranicum) and L.fructosum.The example of Lactobacillus species includes bacillus acidophilus (L.acidophilus), Deshi Lactobacilluss (L.delbrueckii), (L.helveticus), lactobacillus helveticuss (L.salivarius), Lactobacillus casei (L.casei), lactobacillus curvatuses (L.curvatus), Lactobacillus plantarum (L.plantarum), Lactobacillus saki (L.sakei), Lactobacillus brevis (L.brevis), (L.buchneri), Lactobacillus buchneri (L.fermentum) and Lactobacillus reuteri (L.reuteri).
The glucosyltransferase of this paper can be independent of primer or rely on primer.The glucosyltransferase being independent of primer is not required to There is the primer carrying out glucosan synthesis.During Macroscopic single crystal, the glucosyltransferase relying on primer needs to react molten There is the starting molecule serving as enzyme primer in liquid.As used herein, term " primer " refers to any can act as glucosylation The molecule of the initiator of enzyme.The primer that can be used for some embodiments includes such as glucosan and other carbohydrate-based and draws The glucosan of thing, such as hydrolysis.U.S. Patent Application Publication 2013/0244287 (it is herein incorporated by reference) discloses By poly- α -1,3- glucosan is used as parent material to prepare the glucosan of hydrolysis.Glucosan as primer can be such as glucosan T10 (has the glucosan of 10kD molecular weight).
Glucosyltransferase for the glucosan synthetic reaction of this paper can be produced by any method being known in the art Raw.For example, glucosyltransferase can be in heterologous expression system, such as restructuring in microorganism heterologous expression system produces.Heterologous table The example reaching system includes antibacterial (such as escherichia coli, such as TOP10 or MG1655；Bacillus) and eucaryon (such as yeast, As pichia and Saccharomyces) expression system.
Glucosyltransferase described herein can be with any purification state (for example pure or impure) use.For example, glucityl Transferring enzyme can be purification and/or detached at it before use.The example of impure glucosyltransferase includes cell pyrolysis liquid Those of form.Cell pyrolysis liquid or extract can obtain from the antibacterial (such as escherichia coli) for heterogenous expression enzyme.For example, Using French crusher, antibacterial can be destroyed.In the embodiment of alternative, available homogenizer (such as APV, Rannie, Gaulin) make antibacterial homogenization.Glucosyltransferase usually can be dissolved in the preparation of these types.This paper's is thin Bacterium cell pyrolysis liquid, extract or homogenate for example can be used for reaction solution with about 0.15-0.3% (v/v), thus by sucrose Produce poly- alpha-glucanses, for example poly- α -1,3- glucosan.
If so desired, the temperature of the glucosan synthetic reaction of this paper can be controlled.In certain embodiments, react Temperature be about 5 DEG C to about 50 DEG C.In some other embodiments, temperature is about 20 DEG C to about 40 DEG C.
In the glucosan synthetic reaction of this paper, the initial concentration of sucrose can be e.g., from about 20g/L to about 400g/L.Alternative Ground, the initial concentration of sucrose can be about 75g/L to about 175g/L or about 50g/L to about 150g/L.Still alternatively, sucrose is first Beginning concentration can for e.g., from about 40,50,60,70,80,90,100,110,120,130,140,150 or 160g/L (or between 40 to Any integer value between 160g/L)." initial concentration of sucrose " refer to only add all reaction solution components (at least water, Sucrose, gtf enzyme) after sucrose concentration in gtf reaction solution.
Sucrose for the glucosan synthetic reaction of this paper can be highly purified (>=99.5%) or for any other pure Degree or grade.For example, sucrose can have at least 99.0% purity or can be reagent grade cane sugar.And for example, can use incomplete Refined sucrose.The not completely refined sucrose of this paper refers to the undressed sucrose for refining white sucrose.Therefore, completely not refined Sucrose can for completely unpurified or partly refine.The example of unpurified sucrose is " melada " (" raw sugar ") and its molten Liquid.The example of the sucrose partly refining does not experience one, two, three or more crystallisation step.This paper's is completely not refined Sucrose ICUMSA (International Commission for Uniform Methods of Sugar Analysis 150 can) be greater than.This paper sucrose can derive from any renewable sugar source, for example Caulis Sacchari sinensis, sugar beet, Maninot esculenta crantz., Sugar grass or Semen Maydiss.The sucrose that can be used for the suitable form of this paper is, for example, crystal form or non-crystalline forms (such as syrup, sugarcane Juice, beet juice).The other suitable form of completely refined sucrose is not disclosed in U. S. application 61/969,958.
The method measuring sucrose ICUMSA value is well known in the present art, and for example by International Commission for Uniform Methods of Sugar Analysis is disclosed asICUMSA Methods of Sugar Analysis：Official and Tentative Methods Recommended by the International Commission for Uniform Methods of SugarAnalysis(ICUMSA)(H.C.S.de Whalley compiles Volume, Elsevier Pub.Co., 1964), document way of reference is expressly incorporated herein.ICUMSA can for example as by R.J.McCowage, R.M.Urquhart know M.L.Burge(Determination of the Solution Colour of Raw Sugars, Brown Sugars and Coloured Syrups at pH 7.0-Official, Verlag Dr Albert Bartens, 2011 revised editions) described in ICUMSA method GS1/3-7 measure, the document is incorporated by reference this Literary composition.
In certain embodiments, the pH of glucosan synthetic reaction can be about 4.0 to about 8.0.Alternatively, pH can be about 4.0th, 4.5,5.0,5.5,6.0,6.5,7.0,7.5 or 8.0.Can be adjusted by interpolation or the suitable buffer of incorporation or control PH processed, this buffer includes but is not limited to：Phosphate, tris, citrate or combinations thereof.Glucosan synthetic reaction Buffer concentration may be, for example, 0mM to about 100mM or about 10,20 or 50mM.
Poly- α -1 producing in the glucosan synthetic reaction of this paper, 3- glucosan can have at least about 50%, 60%, 70%th, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or arbitrarily whole between 50% to 100% Numerical value) α -1,3 glycosidic bonds.In such embodiment, poly- α -1,3- glucosan has less than about 50%, 40%, 30%, 20%th, non-alpha -1 of 10%, 5%, 4%, 3%, 2%, 1% or 0% (or any integer value between 0% to 50%), 3 Glycosidic bond.
Poly- α -1 of this paper, 3- glucosan preferably have straight chain/main chain of non-branched.In certain embodiments, poly- α- 1,3- glucosan does not have branching-point or has less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% point Scolus (as the percentage ratio of glycosidic bond in polymer).The example of branching-point includes α -1,6 branching-points.
Poly- α -1 producing in the glucosan synthetic reaction of this paper, the molecular weight of 3- glucosan can be determined as number-average molecular weight (Mn) or weight average molecular weight (Mw).Alternatively, molecular weight can by dalton or gram/mol based on mensure.It also can be used to refer to poly- α -1, The DP of 3- dextran polymerw(weight average degree of polymerization) or DPn(number-average degree of polymerization).
Poly- α -1 of this paper, the M of 3- glucosannOr MwCan be at least about 1000.Alternatively, MnOr MwMay be, for example, at least about 1000 to about 600000 (or any integer value between 1000 to 600000).Still alternatively, poly- α -1,3- glucosan can There is such molecular weight：At least about 100 or at least about 100 to 1000 (or any integer value between 100 to 1000) MnOr Mw.
The fraction of glucosan synthetic reaction is suitable configurable for make sugared and such as presently disclosed alpha-Glucosidase contact Condition.Fraction can be the part or all of liquid solution deriving from glucosan synthetic reaction.Generally, make to synthesize in fraction and reaction One or more solubility or insoluble glucan product separate.For example, fraction and one or more can be made in its synthesis phase Between from solution separate out water-fast beta-glucan products (for example poly- α -1,3 glucosans) separate.Some preferred in the disclosure Embodiment in fraction obtain autohemagglutination α -1,3- glucosan synthetic reaction.
In certain embodiments, the volume (before optionally dilution or concentration stage divide, see below) of fraction can be Therefrom obtain this fraction the volume of glucosan synthetic reaction at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%th, 80% or 90% (or any integer value between 10% to 90%).Generally, producing insoluble glucan (example As poly- α -1,3 glucosans) glucosan synthetic reaction in, fraction will for reaction liquid solution component a part (not complete Portion).Fraction can be obtained in any stage of glucosan synthetic reaction, but preferably be nearly completed in reaction and (be greater than completing 80% or 90%) or obtain after completing.
In certain embodiments, the example of the fraction of glucosan synthetic reaction includes filtrate and supernatant.Therefore, at it Middle synthesis insoluble glucan product those embodiments in, can using funnel, filter (such as filter press), centrifuge, Or known in the art allow to remove any other method of some or all liquid from solid or equipment closes from glucosan Reaction is become to obtain the fraction of (separation) this paper.Filtration can for example be carried out by gravity, vacuum or filter pressing.Filter and preferably remove Wholly or largely insoluble glucan；Can be enough to remove from liquid admittedly using average cell size (e.g., from about 40-50 micron) Any filtering material (such as filter paper) of body.Fraction generally retains the component of wholly or largely its dissolving, and such as glucosan closes Become the by-product of reaction.In the filtrate of this paper, lucrose is preferred sugar.
If so desired, the fraction of this paper optionally being diluted or being concentrated.The concentration of fraction can adopt and be suitable to concentrate Any other method known in the art of solution or equipment are carried out.For example, Rotary Evaporators (example can for example be used by evaporation As being set as about 40-50 DEG C of temperature) concentrating fraction.At some aspects of this paper, fraction can be made to be concentrated into such body Long-pending：About initial level partial volume 75%, 80%, 85%, 90% or 95%.Concentrated fraction (for example concentrated filtrate) can be appointed Selection of land is referred to as syrup.
Fraction can comprise water in some respects, and this water substitutes water present in the compositionss therefrom obtaining fraction.For example, may be used Separate one or more from glucosan synthetic reaction in some chromatographic processes that initial solvent is substituted by another kind of solvent wherein Sugared by-product (the sugared by-product [thus removing from initial solvent] being for example bound to post can be eluted in novel solvent).
In some respects, fraction can be processed by this way：Have disclosed above for make sugar with α-glucoside Any appropraite condition (for example, temperature, pH and reagent) of enzyme contact.For example, before alpha-Glucosidase adds to fraction, can make Fraction changes into the pH with about 4 to 5.And for example, the temperature with regard to the hydrolysis of fraction can be about 55-65 DEG C (e.g., from about 60 ℃).And for example, concentrate the fraction for syrup and can be used for hydrolysis.
Fraction obtains autohemagglutination α -1,3- glucosan synthetic reaction in some preferred embodiments of this paper；For example fraction is excellent Selection of land is filtrate.Poly- α -1 of this paper, the fraction of 3- glucosan synthetic reaction at least includes water, Fructose and one or more class The sugar (lucrose and/or oligosaccharide, such as DP2-DP7) of type.Can the other components in such fraction for example include Sucrose (sucrose of remnants not consumed in gtf reaction), one or more gtf enzyme, glucose, buffer, salt,Borate, sodium hydroxide, hydrochloric acid, cell pyrolysis liquid component, protein and/or nucleic acid.Minimally, Autohemagglutination α -1, the component of the fraction of 3- glucosan synthetic reaction includes such as water, Fructose, glucose, one or more type Sugared (lucrose and/or oligosaccharide, such as DP2-DP7) and optional sucrose.It should be appreciated that the composition portion of fraction Divide depending on the condition therefrom obtaining the glucosan synthetic reaction of fraction.In those fraction comprising one or more gtf enzyme, Preferably, in the hydrolysis of this paper using fraction before, such one or more gtf enzyme inactivates (such as heat inactivation).
It should be appreciated that the concrete distribution of the sugared by-product producing via polymerising sucrose in glucosan synthetic reaction can be based on Reaction condition used and gtf enzyme, especially temperature and sucrose concentration and change.It is also understood that sugar is in glucosan synthetic reaction Fraction in concrete composition not vital for disclosed method for hydrolysis.In general, the amount with sucrose increases, Reaction will increase to the selectivity of lucrose and oligosaccharide.On the contrary, increasing with temperature, reaction is to bright beading The selectivity of bacterium disaccharide tends to reducing, and the selectivity for oligosaccharide is generally not affected by affecting.It should be appreciated that passing through sugar The sugar that calculates divided by total solution weight of quality be that weight % dry solid (DS) can be by evaporating the water (preferably with the ratio of water Under vacuo with less than at a temperature of 50 DEG C) or add water and to adjust, and substantially without impact sugar in glucosan synthetic reaction Fraction in Relative distribution.Can also react to increase by termination gtf before realizing converting (being converted into glucosan) completely The percentage ratio of sucrose in big fraction, this termination by falling below the field of activity of gtf enzyme or by making gtf enzyme heat inactivation by pH Realize.
In certain embodiments, the glucosan synthetic reaction of this paper can produce one or more solubility alpha-glucans and produce Thing.Solubility alpha-glucanses product (or " soluble fiber ") can be：The direct product of (i) glucosyltransferase, or (ii) Glucosyltransferase and the synergistic product of alpha-glucanses hydrolytic enzyme, described alpha-glucanses hydrolytic enzyme can hydrolyze to be had One or more α -1,3- glycosidic bond or one or more α -1, the dextran polymer of 6- glycosidic bond.
The solubility alpha-glucanses of this paper may include, for example：
A) at least 75% α -1,3- glycosidic bond；
B) it is less than 25% α -1,6- glycosidic bond；
C) it is less than 10% α -1,3,6- glycosidic bonds；
D) it is less than the M of 5000 daltonw；
E) at 20 DEG C, under 12 weight % in water, less than the viscosity of 0.25 pascal second (Pa s)；
F) scope is 4 to 40 dextrose equivalent (DE)；
G) it is less than 10% digestibility, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) method 2009.01 surveyed；
H) at 25 DEG C, the dissolubility of at least 20% (w/w) in pH 7 water；With
I) it is less than 5 polydispersity index (PDI).
Such solubility alpha-glucanses can be prepared as disclosed in U. S. application 62/004,290.
For example, solubility alpha-glucanses fiber composition can comprise at least 75%, preferably at least 80%, more preferably at least 85%th, even more desirably at least 90% and most preferably at least 95% α-(1,3) glycosidic bond.
And for example, outside above-mentioned α-(1,3) glycosidic bond embodiment, solubility alpha-glucanses fiber composition also can wrap Contain less than 25%, preferably less than 10%, more preferably 5% or less and be even more preferably less than 1% α-(1,6) glycosidic bond.
And for example, outside above-mentioned α-(1,3) and α-(1,6) glucosides linkage content embodiment, solubility alpha-glucanses are fine Dimension compositionss also can comprise less than 10%, preferably less than 5% and be most preferably in less than 2.5% α-(1,3,6) glycosidic bond.
And for example, solubility alpha-glucanses fiber composition can comprise 93 to 97% α-(1,3) glycosidic bond and less than 3% α- (1,6) glycosidic bond, and there is the weight average molecular weight corresponding to 3 to 7 mixing DP.In another embodiment, solubility α- Glucosan fiber composition can include about 95% α-(1,3) glycosidic bond and about 1% α-(1,6) glycosidic bond, and has corresponding to 3 Weight average molecular weight to 7 mixing DP.In the another aspect of embodiments above, solubility alpha-glucanses fiber composition can comprise About 1 to 3% α-(1,3,6) key or preferably from about 2% α-(1,3,6) key.
And for example, in addition to above-mentioned glucosides linkage content embodiment, solubility alpha-glucanses fiber composition is also Can comprise less than 5%, preferably less than 1% and be most preferably in less than 0.5% α-(Isosorbide-5-Nitrae) glycosidic bond.
And for example, in addition to above-mentioned glucosides linkage content embodiment, solubility alpha-glucanses fiber composition is also May include less than 5000 dalton, preferably smaller than 2500 dalton, more preferably 500 to 2500 dalton and most preferably from about 500 Weight average molecular weight (M to about 2000 daltonw).
And for example, in addition to any features above, at 20 DEG C and under 12 weight % in water, solubility alpha-glucanses fiber Compositionss may also include less than 250 centipoises (0.25Pa s), preferably smaller than 10cP (0.01Pa s), preferably smaller than 7cP (0.007Pa s), more preferably less than 5cP (0.005Pa s), more preferably less than 4cP (0.004Pa s) and most preferably little Viscosity in 3cP (0.003Pa s).
In certain embodiments, as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) method 2009.01 surveyed, and solubility alpha-glucanses fiber composition can have less than 10%, preferably Digestibility less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% digestibility.On the other hand, the phase of digestibility To level or AOAC 2011.25 may also be employed (comprehensive total dietary fiber detection is fixed, Integrated Total Dietary Fiber Assay) (McCleary et al., 2012, J.AOAC Int., 95 (3), 824-844) mensure.
In addition to any embodiments above, solubility alpha-glucanses fiber composition can have in pH 7 water at 25 DEG C The dissolubility of at least 20% (w/w), preferably at least 30%, 40%, 50%, 60% or 70%.
In one embodiment, the content of the reducing sugar that solubility alpha-glucanses fiber composition can comprise is less than 10 weights Measure %, preferably smaller than 5 weight % and be more preferably less than weight 1% or less.
In one embodiment, solubility alpha-glucanses fiber composition may include less than 4 kilocalories/g, is preferably less than 3 Kilocalorie/g, more preferably less than 2.5 kilocalories/g and most preferably from about 2 kilocalories/g or less heat content.
And for example, the solubility alpha-glucanses of this paper may include：
A) 10% to 30% α -1,3- glycosidic bond；
B) 65% to 87% α -1,6- glycosidic bond；
C) it is less than 5% α -1,3,6- glycosidic bonds；
D) it is less than the weight average molecular weight (Mw) of 5000 dalton；
E) at 20 DEG C, under 12 weight % in water, less than the viscosity of 0.25 pascal second (Pa s)；
F) scope be 4 to 40, preferably 10 to 40 dextrose equivalent (DE)；
G) it is less than 10% digestibility, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) method 2009.01 surveyed；
H) at 25 DEG C, the dissolubility of at least 20% (w/w) in pH 7 water；With
I) it is less than 5 polydispersity index (PDI).
Such solubility alpha-glucanses can be prepared as disclosed in U. S. application 62/004,308.
And for example, the solubility alpha-glucanses of this paper may include：
A) 25-35 α -1,3- glycosidic bond；
B) 55-75% α -1,6- glycosidic bond；
C) 5-15% α -1,3,6- glycosidic bonds；
D) it is less than the weight average molecular weight of 5000 dalton；
E) at 20 DEG C, under 12 weight % in water, less than the viscosity of 0.25 pascal second (Pa s)；
F) scope is 4 to 40 dextrose equivalent (DE)；
G) it is less than 10% digestibility, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) method 2009.01 surveyed；
H) at 25 DEG C, the dissolubility of at least 20% (w/w) in water；With
I) it is less than 5 polydispersity index.
Such solubility alpha-glucanses can be prepared as disclosed in U. S. application 62/004,312.
And for example, the solubility alpha-glucanses of this paper may include：
A) at least 95% α -1,6- glycosidic bond；
B) 1% or less α -1,3- glycosidic bond；
C) it is less than 2% α -1,3,6- glycosidic bonds；
D) it is less than 1.5% α-Isosorbide-5-Nitrae-glycosidic bond；
E) it is less than the weight average molecular weight of 20000 dalton；
F) at 20 DEG C, under 12 weight % in water, less than the viscosity of 0.25 pascal second (Pa s)：
G) scope is 1 to 30 dextrose equivalent (DE)；
H) it is less than 10% digestibility, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) method 2009.01 surveyed；
I) at 25 DEG C, the dissolubility of at least 20% (w/w) in pH 7 water；With
J) it is less than 5 polydispersity index.
Such solubility alpha-glucanses can be prepared as disclosed in U. S. application 62/004,314.
And for example, the solubility alpha-glucanses of this paper may include：
A) scope is：
I) 1% to 50% α -1,3- glycosidic bond；Or
Ii) it is more than 10% list but the α-Isosorbide-5-Nitrae-glycosidic bond less than 40%；Or
Iii) i) and ii) any combinations；
B) 1 to 50% α -1,2- glycosidic bond；
C) 0-25% α -1,3,6- glycosidic bonds；
D) it is less than 98% α -1,6- glycosidic bond；
E) it is less than the weight average molecular weight of 300kDa；
F) at 20 DEG C, under 12 weight % in water, less than the viscosity of 0.25 pascal second (Pa s)；
G) it is less than 20% digestibility, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) method 2009.01 surveyed；
H) at 25 DEG C, the dissolubility of at least 20% (w/w) in pH 7 water；With
I) it is less than 26, preferably smaller than 5 polydispersity index.
Such solubility alpha-glucanses can be prepared as disclosed in U. S. application 62/004,305.
In certain embodiments, solubility alpha-glucanses are the direct product of glucosyltransferase.Gather in suitable Portugal In sugared synthetic reaction, such glucosyltransferase and can be as disclosed herein for its condition, or as special in any U.S. Disclosed in profit application such as 62/004,290,62/004,308,62/004,312,62/004,314 and/or 62/004,305.
Solubility alpha-glucanses or alternatively such as glucosyltransferase and alpha-glucanses hydrolytic enzyme synergism Product, described alpha-glucanses hydrolytic enzyme can hydrolyze has one or more α -1,3- glycosidic bond or one or more α -1,6- The dextran polymer of glycosidic bond.In some respects, the glucosan synthetic reaction for producing solubility alpha-glucanses product can Including at least one glucosyltransferase and at least one alpha-glucanses hydrolytic enzyme.In other side, glucosan synthesis is anti- One or more glucosyltransferase should initially be comprised as unique enzyme component.Such reaction generation not yet passes α-Portugal and gathers The first alpha-glucanses that glycosylhydrolase is modified.Then, at least one alpha-glucanses hydrolytic enzyme is added to reaction suitably Time period, to allow for the first product to be modified to solubility alpha-glucanses product.Accordingly, there exist by it via glucosylation Enzyme and the different modes of alpha-glucanses hydrolytic enzyme synergism synthesizing soluble alpha-glucanses product.Anti- in glucosan synthesis Should during and/or glucosan synthesis after, be used for carrying out glucosan synthetic reaction (wherein comprising one or more alpha-glucanses water Solution enzyme) condition can as disclosed herein, or as in any U.S. Patent application such as 62/004,290,62/004,308,62/ 004,312nd, disclosed in 62/004,314 and/or 62/004,305.
The alpha-glucanses hydrolytic enzyme of this paper can for such as glucanase (can hydrolyzing alpha -1, the glycosidic bond that 6- connects； E.C.3.2.1.11), mutant enzyme (can hydrolyzing alpha -1,3- connect glycosidic bond；E.C.3.2.1.59), mycodextranase (energy Enough endo hydrolysis comprise (1-4)-α-D- glycosidic bond of the α-D- glucosan of (1-3)-and (1-4)-key；EC 3.2.1.61), glucosan 1,6-a- glucosidase (EC 3.2.1.70) and alternan enzyme (EC 3.2.1.-；Being capable of inscribe water Solution cracking alternan；E.C.3.2.1.-；Referring to United States Patent (USP) 5786196).
Can be in certain aspects using inclusion SEQ ID NO：47 mutant enzyme.Alternatively, mutant enzyme can for example comprise with SEQ ID NO：47 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino Acid sequence, and there is mutation enzymatic activity.
As the presently disclosed glucosan synthetic reaction for producing one or more solubility alpha-glucanses product can Be directly used as wherein carrying out the suitable condition of the hydrolysis of this paper, wherein alpha-Glucosidase be used for hydrolyzing alpha -1,5 glucityls - Fructose key.Can be according to regard to producing for example poly- α -1, any above public affairs of the hydrolysis process of glucosan synthetic reaction of 3- glucosan The condition opened carries out such hydrolysis.Alternatively, the glucosan for producing one or more solubility alpha-glucanses product synthesizes The fraction (such as chromatograph fraction) of reaction can be used as wherein to α -1, and 5 glucityls-Fructose key carries out the water of alpha-Glucosidase mediation The suitable condition of solution.
In some embodiments of this paper, fraction can be the chromatograph fraction of glucosan synthetic reaction.For example, fraction can be Produce the chromatograph fraction of the glucosan synthetic reaction of one or more solubility alpha-glucanses product as disclosed herein.Such anti- Should during glucosan synthesis and/or glucosan synthesis complete after optionally include one or more alpha-glucans and hydrolyze Enzyme.Obtain fraction generally in the embodiment of these types any, to make wholly or largely (for example, at least about 60%, 70%th, 80%, 90%, 95%) solubility alpha-glucanses product separates with producing its response composite.Once with whole or big Part solubility alpha-glucanses product separates so that it may so that fraction is stood using one or more alpha-glucanase disclosed herein Any α -1,5 glucityls-Fructose hydrolytic process.
The liquid chromatography that the chromatograph fraction of this paper can be usually used proper types obtains.Liquid chromatograph can be for example using chi Very little exclusion chromatography (SEC), column chromatography, high performance liquid chromatography (HPLC), ion exchange chromatography, affinity chromatography, ultrafiltration, Microfiltration or dialysis are carried out.
The disclosure further relate to a kind of by making sugar contact with alpha-Glucosidase (such as transglucosidase or glucosidase) The compositionss producing, wherein (i) sugar is for comprising at least one α -1, the disaccharide of 5 glucityls-Fructose key or oligosaccharide, and (ii) At least one α -1 of alpha-Glucosidase hydrolysis sugar, 5 glucityls-Fructose key.The sugar amount phase that the compositionss producing in like fashion comprise Less compared with the sugar amount existing before contact.The example of compositionss includes those disclosed herein, for example, derive from glucosan synthesis The filtrate through hydrolysis of reaction or the fraction through hydrolysis for producing the glucosan synthetic reaction of solubility alpha-glucanses.With In upper disclosed and embodiment, any feature with regard to method for hydrolysis and its product can characterize compositionss.The following spy of compositionss Levy as example.
In some embodiments of compositionss, alpha-Glucosidase can comprise and SEQ ID NO：5、6、8、9、11、12、 14th, 15,17,18,20,22,24,26,28,30,32,34,36,38, or DIAZYME RDF ULTRA (DuPont Industrial Biosciences) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Identical aminoacid sequence.In some embodiments of compositionss, transglucosidase can comprise and SEQ ID NO：1 at least 90% identical aminoacid sequence.In some embodiments of compositionss, glucoamylase can comprise and SEQ ID NO：2 to Few 90% identical aminoacid sequence.Alternatively, any alpha-Glucosidase disclosed herein can be used for producing disclosed compositionss.
By this paper method for hydrolysis produce compositionss sugar such as lucrose concentration can for example be less than sugar with The 50% of the concentration of lucrose existing before alpha-Glucosidase contact.
In some embodiments of this paper, the compositionss that produced by method for hydrolysis can for glucosan synthetic reaction thing or Its fraction, the wherein by-product of glucosan synthetic reaction are contacted with alpha-Glucosidase.In this embodiment, fraction can example As the filtrate for glucosan synthetic reaction or the fraction for producing the synthetic reaction of the glucosan of solubility alpha-glucanses.? In this embodiment, sugar may be, for example, lucrose.
It will be appreciated by the skilled person that presently disclosed embodiments part is used for saccharifying being otherwise likely difficult to decompose Disaccharide and oligosaccharide.For example, it is possible to implement enhanced method using this feature：The enrichment of (i) Fructose and (ii) fermentation.
Example 6 below shows, compared to using unhydrolysed filtrate, when using by alpha-Glucosidase (transglucosidase) During the glucosan filtrate of hydrolysis, Fructose enrichment is enhanced by chromatography.
Therefore, invention disclosed further relates to a kind of method that enrichment is present in Fructose in the fraction of glucosan synthetic reaction. The method includes：A () makes fraction and alpha-Glucosidase (the such as transglucosidase or glucose shallow lake obtaining from glucosan synthetic reaction Powder enzyme) contact under suitable conditions, at least one α -1 of contained disaccharide or oligosaccharide, 5 glucose wherein in enzyme hydrolysiss fraction Base-Fructose key；And (b) separating levulose from step (a) is through hydrolysis fraction, to obtain the level that fructose concentration is than step (a) Point the higher compositionss of fructose concentration.
Disclosed for example with regard to alpha-Glucosidase (such as transglucosidase or glucoamylase) and glucosan synthetic reaction The feature of the Fructose enrichment method of fraction can be according to any disclosure being related to these features every kind of provided herein.
The step (b) of separating levulose can be carried out by any method known to those skilled in the art.For example, can as with Disclosed in lower embodiment, or chromatography is used according to European Patent Publication EP2292803B1, this patent is with way of reference simultaneously Enter herein.
Derive from the compositionss (such as fructose soln or fructose syrup) with higher concentration Fructose of disclosed enrichment method Can have at least about 90,91,92,93,94,95,96,97,98 or 99 weight % Fructose.
The Fructose enrichment method of this paper is than the method using the filtrate without such as presently disclosed alpha-Glucosidase hydrolysis Expressively more preferable.The performance of such increase can measure according to the Fructose recovery percentage of at least 40%, 45% or 50%.
The disclosure further relates to a kind of fermentation process, and the method includes：(a) make the fraction that obtains from glucosan synthetic reaction with Alpha-Glucosidase (for example, transglucosidase or glucoamylase) contacts under suitable conditions, wherein alpha-Glucosidase hydrolysis At least one α -1 of contained disaccharide or oligosaccharide in fraction, 5 glucityls-Fructose key；B () uses microbial fermentation step (a) Fraction is to obtain product；And (c) is optionally separated the product of (b).The fermentation step of step (b) can after step (a) or Carry out with step (a) simultaneously.Significantly, the filtrate through hydrolysis of glucosan synthetic reaction of fermenting can for example be passed through, using the party Method produces ethanol.Derive from this process ethanol yield be higher than fermentation still unhydrolysed glucosan filtrate when the ethanol yield that obtains.
Synthesize instead with regard to alpha-Glucosidase (for example, transglucosidase or glucoamylase), disaccharide and oligosaccharide, glucosan The feature of the disclosed fermentation process of the fraction answered and suitable contact conditions for example can according to provided herein be related to every kind of Any disclosure of these features.
Microorganism for the fermentation process of this paper can be such as antibacterial, yeast or funguses.Can be used for the antibacterial of this paper Example includes Lactobacillus species, Streptococcus species, Bifidobacterium strain, Leuconostoc strain, Escherichia (Escherichia) strain (such as escherichia coli) and Bacillus sp.The example that can be used for the yeast of this paper includes ferment Female genus strain, such as saccharomyces cerevisiae (S.cerevisiae) and saccharomyces bayanuses (S.bayanus).
The fermentation process of this paper can obtain product such as ethanol or acid (such as lactic acid).However, it is believed that if so desired, can produce Raw other product.It will be appreciated by those skilled in the art that will depend on respectively using as disclosed fermentation process produces specific product The condition of kind, for example, be used for one or more microorganism of fermentation.For example, the condition for fermentation of this paper can be as following examples Disclosed, or such as E1-Mansi et al. (2006,Fermentation Microbiology and Biotechnology, the Two editions, CRC Press) and Stanbury et al. (1999,Principles of Fermentation Technology, second Version, Butterworth-Heinemann) disclosed, the two is all herein incorporated by reference.
In some embodiments of the fermentation process of this paper, product yield is higher than the alpha-Glucosidase without this paper for the fermentation The product yield obtaining during the glucosan filtrate of hydrolysis.This relatively can refer to the non-hydrolysed grade for example using glucosan synthetic reaction The control fermentation divided.The product yield fermenting herein can for example increase at least about 10%, 20%, 40%, 60%, 80% or 100% (or any integer value between 10% to 100%).Additionally, can be increased by the speed that the fermentation of this paper forms product Greatly.
Example 7 below shows, lucrose can be provided that the charging comprising unhydrolysed glucosan filtrate Yeast ferment for ethanol.Therefore, there is further disclosed herein it is product (example that lucrose is fermented by one kind microorganism As ethanol) method.The method may include fermentation (i) or (ii) Portugal of hydrolyzing without alpha-Glucosidase as disclosed herein Polysaccharide filtrate.No matter whether lucrose is provided in glucosan filtrate or provides (such as half purification in another form Or enriched form), the method for the beading bacterium disaccharide that ferments may include and makes microorganism (such as yeast, such as saccharomyces cerevisiae) be suitable to profit Use lucrose.Such adaptation may include makes microorganism grow in the presence of lucrose and optional other sugar Few 2 or 3 growth cycles, microorganism thereafter is using more lucroses come tunning.In certain embodiments, micro- Biology (i) can grow (1 complete cycle) in the first charging comprising lucrose, and (ii) moves from the first charging Remove, (iii) comprise lucrose second charging in growth (two complete cycles, (iv) from second charging optionally Remove, and (v) optionally grows (three complete cycles) in the 3rd charging.In certain embodiments, fit in like fashion The microorganism answered can increase the ability of fermentation lucrose.
Example 9 below shows, when glucosan filtrate is fermented by yeast while being hydrolyzed by transglucosidase, deposits Nearly all (the such as ＞ 98% or ＞ 99%) lucrose being in glucosan filtrate can be used for being fermented by yeast.Cause This, enhanced beading bacterium disaccharide fermentation process may include with alpha-Glucosidase (for example, transglucosidase or glucose starch herein Enzyme) hydrolysis beading bacterium disaccharide, use fermentable beading bacterium disaccharide simultaneously.
The non-limiting example of the compositions disclosed herein and method includes：
1. one kind makes to comprise at least one α -1, α -1 in the sugar of 5 glucityls-Fructose key, 5 glucityls-Fructose key hydrolysis Method, wherein sugar is disaccharide or oligosaccharide, and wherein said method includes：
Sugar is made to contact under suitable conditions with alpha-Glucosidase, wherein at least one α -1 of alpha-Glucosidase hydrolysis sugar, 5 glucityls-Fructose key,
And wherein sugar amount reduces compared to the sugar amount existing before contact.
2. the method according to embodiment 1, wherein alpha-Glucosidase are fixing.
3. the method according to embodiment 1 or 2, wherein sugar are lucrose.
4. the method according to embodiment 3, the concentration of lucrose wherein after the contacting step is less than The 50% of the concentration of the lucrose existing before contact.
5. the method according to embodiment 1,2,3 or 4, wherein suitable condition includes：
(i) glucosan synthetic reaction, or
(ii) fraction obtaining from glucosan synthetic reaction；
Wherein sugar is the by-product of glucosan synthetic reaction.
6. the method according to embodiment 5, wherein glucosan synthetic reaction produce at least one insoluble α-Portugal and gather Sugared product.
7. the method according to embodiment 6, wherein level are divided into the filtrate of glucosan synthetic reaction.
8. the method according to embodiment 5, wherein glucosan synthetic reaction produce at least one solubility α-Portugal and gather Sugared product, it is：
The product of (i) glucosyltransferase, or
(ii) glucosyltransferase and the synergistic product of alpha-glucanses hydrolytic enzyme, described alpha-glucanses hydrolytic enzyme Can hydrolyze and there are one or more α -1,3- glycosidic bond or one or more α -1, the dextran polymer of 6- glycosidic bond.
9. the method according to embodiment 8, wherein level are divided into the chromatograph fraction of glucosan synthetic reaction.
10. the method according to any one of embodiment 1-9, wherein alpha-Glucosidase are transglucosidase or glucose Amylase.
A kind of 11. compositionss by making sugar contact with alpha-Glucosidase and produce,
Wherein sugar for disaccharide or oligosaccharide and comprises at least one α -1,5 glucityls-Fructose key,
Wherein at least one sugared α -1 of enzyme hydrolysiss, 5 glucityls-Fructose key,
And the sugar amount that wherein compositionss comprise reduces compared to the sugar amount existing before contact.
12. compositionss according to embodiment 11, wherein sugar are lucrose.
13. compositionss according to embodiment 11 or 12, wherein sugar in (i) glucosan synthetic reaction, or (ii) from In the fraction that glucosan synthetic reaction obtains；
Wherein sugar is the by-product of glucosan synthetic reaction.
The method that a kind of 14. enrichments are present in Fructose in the fraction of glucosan synthetic reaction, the method includes：
A () makes the fraction obtaining from glucosan synthetic reaction contact under suitable conditions with alpha-Glucosidase, and wherein α- Glucosidase hydrolyzes at least one α -1 of contained disaccharide or oligosaccharide in fraction, 5 glucityls-Fructose key；And
B () separating levulose from step (a) is through hydrolysis fraction, to obtain the fruit of the fraction that fructose concentration is than step (a) The higher compositionss of sugared concentration.
A kind of 15. fermentation process, the method includes：
A () makes the fraction obtaining from glucosan synthetic reaction contact under suitable conditions with alpha-Glucosidase, and wherein α- Glucosidase hydrolyzes at least one α -1 of contained disaccharide or oligosaccharide in fraction, 5 glucityls-Fructose key；
B () is wherein fermented after step (a) or and step with the fraction of microbial fermentation step (a) to obtain product A () is carried out simultaneously；And
C () optionally, separates the product of (b)；
Wherein receive compared to the product that the fraction of the glucosan synthetic reaction not contacted with alpha-Glucosidase is fermented Rate, the product yield of (b) increases.
Embodiment
Disclosed invention will be further elaborated in the following embodiments.Although it should be understood that these embodiment explanations Some preferred aspects of the present invention, but be only given in an exemplary manner.By above-mentioned discussion and these embodiments, ability The technical staff in domain can determine that the essential feature of the present invention, and in without departing from the spirit and scope of the invention on the premise of, Variations and modifications can be carried out to adapt to multiple use and condition to the present invention.
Abbreviation
Used herein some abbreviation implication as follows：" g " refers to gram, and " h " refers to hour, and " mL " refers to milliliter, " psi " Refer to pound per square inch, " wt% " refers to percentage by weight, and " μm " refers to micron, " % " refers to percentage ratio, " DEG C " refers to take the photograph Family name's degree, " mg " milligram, " mm " refers to millimeter, and " mL/min " refers to that millimeter is per minute, and " m " refers to rice, and " uL " refers to microlitre, " mmol " refers to mM, and " min " refers to minute, and " mol% " refers to a mole %, and " M " refers to mole, and " mg/g " refers to that milligram is every Gram, " rpm " refers to rpm, and " MPa " refers to MPa.
Conventional method
Unless otherwise noted, all reagent are all purchased from Sigma-Aldrich (St.Louis, MO).Sucrose is purchased from VWR (Radnor, PA).
The preparation of the crude extract of glucosyltransferase (gtf)
Using isopropyl ss-D-1- thiogalactoside (IPTG)-induction expression system in coli strain DH10B Middle expression streptococcus salivariuss gtfJ enzyme (SEQ ID NO：3).Compared to streptococcus salivariuss gtfJ aminoacid sequence, (GENBANK knows Alias 47527), SEQ ID NO：3 have N- end 42 residue deletions, but include initial methionine.In brief, greatly Enterobacteria DH10B cell is converted to express SEQ ID NO by the DNA sequence through codon optimization：3, thus in escherichia coli Middle expression gtfJ enzyme.This DNA sequence is included in expression vectorIn (DNA 2.0, Menlo Park CA). The cell of conversion is inoculated in initial optical density, and (OD, 600nmPlace) be 0.025 LB culture medium (10g/L tryptone；5g/L Yeast extract, 10g/L NaCl) in, and so that it is grown in incubator at 37 DEG C, it is stirred under 250rpm simultaneously. When it reaches the OD of 0.8-1.0600When, add 1mM IPTG and carry out Induced cultures.The culture of induction is placed on shaking machine simultaneously Harvest after inducing 3 hours.
Existed by making the cell of cultureIt is centrifuged (25 DEG C, 16000rpm) results in centrifuge and obtain enzyme GtfJ(SEQ ID NO：3) cell, is made to be resuspended in 5.0mM phosphate buffer (pH7.0) and in cooled on ice to 4 DEG C.Adopt Making cell breakage with 0.1-mm silicon dioxide bead using grain beater, being then centrifuged so that not breaking under 4 DEG C and 16000rpm Broken cell and pellet cell debris.Crude extract is made (to comprise soluble g tfJ enzyme, SEQ ID NO：And precipitate and separate, and pass through 3) Bradford protein determination is analyzed and to be measured protein concentration (mg/mL).
Following preparation Streptococcus C150gtf-S enzyme (SEQ ID NO：40).SG1184 is bacillus subtilises (Bacillus subtilis) expression strain, its expression truncate pattern Streptococcus C150 (GI： 321278321) glycosyl transferase Gtf-S (" GTF0459 ").Will be from escherichia coli expression matter under the effect of aprE promoter Grain pMP79 (SEQ ID NO：41) protein G TF0459 (the SEQ ID NO of coding N- end truncate：42) gene cloning With bacillus subtilises in NheI the and HindIII site of bacillus subtilises integrated expression plasmid p4JH and on carrier AprE signal peptide merges.First construct is transformed in escherichia coli DH10B and containing ampicillin (100 μ g/mL) Selected on LB flat board.Then construct pDCQ984 of attested expression GTF0459 is transformed into and comprises nine protease (amyE in the bacillus subtilises BG6006 of disappearance：：XylRPxylAcomK-ermC, degUHy32, oppA, Δ SpoIIE3501, Δ aprE, Δ nprE, Δ epr, Δ ispA, Δ bpr, Δ vpr, Δ wprA, Δ mpr-ybfJ, Δ nprB) and LB flat board containing chloromycetin (5 μ g/mL) selects.The bacterium colony of growth on the LB flat board containing 5 μ g/mL chloromycetin is made to contain On the LB flat board of 25 μ g/mL chloromycetin, streak inoculation is several times.The bacillus subtilises expression strain SG1184 of gained is made to exist first In LB culture medium containing 25 μ g/mL chloromycetin growth and and then in the GrantsII culture medium containing 25 μ g/mL chloromycetin subculture Culture, grows 2-3 days at 30 DEG C.Culture is made to be centrifuged 30 minutes and so that supernatant liquid filtering is passed through at 15,000g and 4 DEG C 0.22- μm of wave filter.Supernatant after filtering is divided into equal portions and in -80 DEG C of freezings.
Expression GTF0459 (SEQ ID NO is made by conventional fed-batch fermentation：42) bacillus subtilises SG1184 Bacterial strain grows under deep water aerobic conditions.Using containing 0-0.25% corn steep solids (Roquette), 5-25g/L sodium phosphate and Potassium phosphate, the solution of 0.3-0.6M ferrous sulfate, manganese chloride and calcium chloride, 0.5-4g/L magnesium sulfate and 0.01-3.7g/L sulphuric acid The Nutrient medium of the solution of zinc, cuprous sulfate, boric acid and citric acid.Defoamer FOAMBLAST 882 is added with 2-4mL/L Control foaming.When can't detect the initial glucose sugar in batch, add 10-L fermentation with the charging of 50% (w/w) glucose.Portugal Grape sugar feed rate rose within a few houres.Make ferment control in 30 DEG C and 20%DO, and initially stir as 750rpm.With PH is controlled in 7.2 by 50% (v/v) ammonium hydroxide.In the fermentation running through 2- days runs, monitor fermentation parameter such as pH, temperature Degree, air mass flow and DO%.Harvest in end of run and obtain culture fermentation broth and it is centrifuged to obtain supernatant. Then make to comprise GTF0459 (SEQ ID NO：42) supernatant stored frozen at -80 DEG C.
Prepare Streptococcus mutans MT-4239gtf-C enzyme (SEQ ID NO as follows：43).Using being optimized in hay bud In spore bacillus expression codon composite coding truncate pattern glucosyltransferase (gtf) (It is identified as GI：3130088, SEQ ID NO：43；Derive from the gtf-C of Streptococcus mutans MT-4239) gene and its by GenScript Synthesis.Be there is GTF0088BsT1 (the SEQ ID of the truncate of N- end and the T1 truncate of C- end by GENSCRIPT plasmid amplification coding NO：45) gene (SEQ ID NO：44), and it is cloned into the integrated expression of bacillus subtilises under the effect of aprE promoter Merge with bacillus subtilises AprE signal peptide in NheI the and HindIII site of plasmid p4JH and on carrier.First by structure Build body to be transformed in escherichia coli DH10B and selected on the LB flat board containing ampicillin (100 μ g/mL).Then will Construct pDCQ1021 of attested expression GTF0088BsT1 is transformed into the bacillus subtilises comprising nine protease deficiencies (amyE in BG6006：：XylRPxylAcomK-ermC, degUHy32, oppA, Δ spoIIE3501, Δ aprE, Δ nprE, Δ Epr, Δ ispA, Δ bpr, Δ vpr, Δ wprA, Δ mpr-ybfJ, Δ nprB) and in the LB flat board containing chloromycetin (5 μ g/mL) Upper selection.The bacterium colony of growth on the LB flat board containing 5 μ g/mL chloromycetin is made to draw on the LB flat board containing 25 μ g/mL chloromycetin Line is inoculated several times.The bacillus subtilises expression strain SG1221 making gained is first in the LB culture medium containing 25 μ g/mL chloromycetin Upper growth then successive transfer culture in the GrantsII culture medium containing 25 μ g/mL chloromycetin, grow 2-3 days at 30 DEG C.? Culture is made to be centrifuged 30 minutes and make supernatant liquid filtering pass through 0.22- μm of wave filter at 15,000g and 4 DEG C.Upper after filtering Clear liquid is divided into equal portions and freezes at -80 DEG C.
Expression GTF0088BsT1 (SEQ ID NO is made by conventional fed-batch fermentation：45) bacillus subtilises SG1221 bacterial strain grows under deep water aerobic conditions.Using containing 0-0.25% corn steep solids (Roquette), 5-25g/L phosphorus Sour sodium and potassium phosphate, the solution of 0.3-0.6M ferrous sulfate, manganese chloride and calcium chloride, 0.5-4g/L magnesium sulfate and 0.01-3.7g/ The Nutrient medium of the solution of L zinc sulfate, cuprous sulfate, boric acid and citric acid.Defoamer FOAMBLAST is added with 2-4mL/L 882 controlling foaming.When can't detect the initial glucose sugar in batch, adding 2-L with the charging of 50% (w/w) glucose and sending out Ferment.Glucose feed rate rose within a few houres.Make ferment control in 30 DEG C and 20%DO, and initially stir and be 400rpm.With 50% (v/v) ammonium hydroxide, pH is controlled in 7.2.In the fermentation running through 2- days runs, monitor fermentation parameter example As pH, temperature, air mass flow and DO%.Harvest in end of run and obtain culture fermentation broth and it is centrifuged to obtain Supernatant.Then make to comprise GTF088BsT1 (SEQ ID NO：45) supernatant stored frozen at -80 DEG C.
GlucosyltransferaseGTF0459Mensure with GTF0088BsT1 activity
Glucosyl transferase activity is measured and is carried out by procedure below：Presence or absence of 25g/L glucosan (MW～ 1500, Sigma-Aldrich, Cat.#31394) in the case of, under 37 DEG C and the shake of 125rpm track, with sugarcane containing 200g/L 25mM the or 50mM sodium acetate buffer (under pH 5.5) of sugar incubates 1-10% (v/v) crude protein extract comprising GTF enzyme.? The aliquot of a reactant mixture is taken out and in 90 DEG C of heating 5min so that GTF inactivates when 1h, 2h and 3h.By 13, Under 000xg, centrifugation makes it be filtered through 0.2um RC (regenerated cellulose) film to remove insoluble material in 5 minutes afterwards.85 Two series connection AMINEX HPX-87C posts are adopted to analyze gained filtrate (Bio-Rad, Hercules, CA) to determine by HPLC at DEG C Amount sucrose concentration.Draw the sucrose concentration of Each point in time with respect to the response time and determined by the slope of linearity curve initially anti- Answer speed.One GTF active unit is defined as the enzyme amount consuming needed for a micromolar sucrose per minute under condition determination.
The preparation of the crude extract of α-(1,3)-glucan hydrolase (mutant enzyme)
Coding Ma Erneifei penicillium sp (Penicillium marneffei)18224TMMutant enzyme (It is identified as GI：212533325) gene is synthesized by GenScript (Piscataway, NJ).Open in CBHI Under the control of mover and terminator, by coded protein sequence (MUT3325；SEQ ID NO：47) nucleotide sequence (SEQ ID NO：46) it is subcloned in SacII the and AscI restriction site of plasmid pTrex3, this plasmid is designed for expressing Richter scale The carrier of the target gene in Trichoderma spp. (Trichoderma reesei), is used for selecting using aspergillus niger acetamidase.By base Because the plasmid of gained is transformed in trichoderma reesei for rifle injection.The concrete grammar of via Particle Bombardment Transformation is described in international PCT patent Shen WO2009/126773 A1 please be disclose, and the disclosure of which is herein incorporated by reference.Using the spore having from stable clone The 1-cm of son2Agar rod TRM05-3 prepares culture medium (described below) to inoculate.Under 28 DEG C and 220rpm, culture is made to exist Grow 4-5 days in shaking flask.In order to harvest the protein obtaining secreting, first pass through centrifugation under 4000g and remove cell in 10 minutes Group, and make supernatant liquid filtering pass through 0.2- μm of sterilizing filter.Mutant enzyme MUT3325 (SEQ ID NO：47) expression is passed through SDS-PAGE is confirmed.
It is listed below preparing nutrient media components.
NREL-Trich Lactose Defined
Trichoderma reesei trace element
By using the freezing spore suspension of the MUT3325 expression strain TRM05-3 of 1.0mL in the flask with baffle plate for the 2-L (minimal medium is by 5g/L ammonium sulfate, 4.5g/L di(2-ethylhexyl)phosphate to prepare fermentation seed culture for the minimal medium of inoculation 0.5L Hydrogen potassium, 1.0g/L Magnesium sulfate heptahydrate, 14.4g/L anhydrous citric acid, 1g/L calcium chloride dihydrate, 25g/L glucose, and trace unit Element includes 0.4375g/L citric acid, 0.5g/L ferrous sulfate heptahydrate, 0.04g/L zinc sulphate heptahydrate, 0.008g/L five water sulphuric acid Copper, 0.0035g/L Manganous sulfate monohydrate and 0.002g/L boric acid composition, pH is 5.5).8L in being transformed into 14-L fermentation tank Before preparation culture medium, culture is made to grow 48 hours under 32 DEG C and 170rpm.Preparation culture medium by 75g/L glucose, 4.5g/L potassium dihydrogen phosphate, 0.6g/L CALCIUM CHLORIDE DIHYDRATE, 1.0g/L Magnesium sulfate heptahydrate, 7.0g/L ammonium sulfate, 0.5g/L are anhydrous Citric acid, 0.5g/L ferrous sulfate heptahydrate, 0.04g/L zinc sulphate heptahydrate, 0.00175g/L copper sulphate pentahydrate, 0.0035g/L mono- Anhydrous manganese, 0.002g/L boric acid and 0.3mL/L FOAMBLAST 882 form.
In the batch of grown on glucose 24h, run fermentation under 34 DEG C and 500rpm first.At the end of 24h, Temperature is made to be reduced to 28 DEG C and make mixing speed increase to 1000rpm.Then biological in 0.030g glucose-sophorose solid/g Under the specific feed rate of matter/hour, feed fermentation tank with glucose and sophorose mixture (62%w/w).In end of run, Biomass are removed by centrifugation, and retains ultrafiltration core (UFP-10-E-35 using 10-kD molecular weight；GE Healthcare, Little Chalfont, Buckinghamshire, UK) will be containing MUT3325 mutant enzyme (SEQ ID NO by filtration：47) About 10 times of supernatant concentration.Condensing protein is made to store in -80 DEG C.
The mensure of alpha-glucanses hydrolytic enzyme (mutant enzyme) activity
Using by Streptococcus sobrinus (Streptococcus sobrinus)33478TMThe enzyme of the secretion producing Carry out formation determination and be mutated the insoluble mutan polymer needed for enzymatic activity.Specifically, (Brain on BHI agar plate Heart Infusion agar, Teknova, Hollister, CA) streak inoculation one ring Streptococcus sobrinus33478TM Glycerol stock, and so that flat board is incubated 2 days at 37 DEG C.Choose several bacterium colonies with ring (to derive from initial medium bottle Teknova inoculation 2X 100mL BHI fluid medium in), and it is incubated culture at 37 DEG C, static holding 24h.By from The heart removes gained cell and makes gained supernatant liquid filtering pass through 0.2- μm of sterilizing filter；Collect the filtrate of 2X 101mL.To filter Add the 200g/L sucrose (final sucrose 20g/L) of 2X 11.2mL in liquid.Under non-stirring state, make to react on 37 DEG C of incubations 67h.Gained polysaccharide polymer is collected by centrifugation 10min under 5000xg.Carefully drain supernatant.Aseptic with 40mL Insoluble polymer is washed 4 times by water.Make the polymer lyophilized 48h of mutan of gained.Make mutan polymer (390mg) it is suspended in the sterilized water of 39mL to prepare 10mg/mL suspension.By supersound process, (40% amplitude is up to compared with agglomerate Block disappears, total～10min) homogenize mutan suspension.Homogenized suspension is divided into equal portions and in 4 DEG C of storages.
By at pH 5.5 and 37 DEG C with containing 0.5mg/mL's mutan polymer (prepared as described above) 25mM KOAc buffer incubates proper amount of enzyme, starts to be mutated enzymatic determination.In Each point in time, take out the reaction of aliquot Mixture is simultaneously quenched with isopyknic 100mM glycine buffer (pH 10).Removed respectively by centrifugation 5min under 14,000xg The individual insoluble material through being quenched in sample.By P-hydroxybenzoic acid hydrazides solution (PAHBAH) (Lever M., Anal.Biochem., (1972) 47：273-279) measure the reduction of the oligosaccharide that Each point in time is produced and polysaccharide polymer End carries out quantitation, and initial rate is come really by the slope of the junior three in time course or the linear diagram of four time points Fixed.By the response sample supernatant of 10 μ L is added carry out to the PAHBAH working solution of 100 μ L PAHBAH mensure and 95 DEG C of heating 5min.By a reagent A (concentrated hydrochloric acid of 0.05g/mL P-hydroxybenzoic acid hydrazides and 5 volumes %) of mixing and four Part reagent B (0.05g/mL NaOH, 0.2g/mL sodium potassium tartrate tetrahydrate) carrys out preparation work solution.Absorbance at record 410nm, and And by deducting suitable background absorption and being used the standard curve that the glucose (as reference material) of various concentration produces to count Calculate the concentration of reducing end.
Response feature ana lysis are carried out by HPLC
From reaction periodic sample and use equipped with RI-detector1260HPLC is analyzed. At the flow velocity and 85 DEG C of 0.6mL/min, using having deionized waterHP-87C post (BioRad, Hercules, CA) come to measure gtf reaction in sucrose, glucose, lucrose and Fructose content.0.6mL/min's At flow velocity and 85 DEG C, using having deionized waterHP-42A post (BioRad) come to quantitative determine gtf reaction Middle soluble oligosaccharide by-product (DP2-DP7).
Equipped with RI-detectorUltiMateTM3000HPLC (Thermo Scientific) is used for Sample (embodiment 4) including immobilized enzyme.At the flow velocity and 85 DEG C of 0.3mL/min, using having deionized waterRezexTMCalcium monosaccharide post is analyzing sugar.
Oligosaccharide is analyzed by NMR
Allied the communists using 5-mm low temperature three shake that pulse field gradient (PFG) pops one's head in 500MHz (for1H work under) NMR data is gathered on Agilent DD2 spectrogrph.It is real by observation transmitter frequency is carefully placed in " Pu Shi (presat) " At the resonance of residual water signal tested, and and then using the incorporation time with complete phase circulation (more than 32 time) and 10ms First data slice (slice) of NOESY experiment obtains water suppression.One-dimensional1H spectra collection is in the spectral width of 6410Hz, 5.1s Acquisition time, the 90- degree pulse of 65536 data points, 4s presaturation and 5.85 μ s.Sample temperature is made to be maintained at 25 DEG C. By by the D of 50 μ L and 450 μ L2(4, the 4- dimethyl -4- silicyl pentane -1- sodium sulfonates of DSS containing 12.4mM of O and 60 μ L Salt) interior target D2O adds to prepare sample to 5-mm NMR pipe together, and methyl resonance is set as 0ppm.Different different head keys Chemical shift positioning derives from：Goffin et al. (2009, Bull Korean Chem.Soc.30：2535-2541.For α (1,3) For key, peak is distributed as 5.35ppm, and lucrose is 5.1ppm, and α (1,6) key is 4.95.For α RE reducing end (RE) it is assigned as 5.2, be 4.65 for β RE.
Embodiment 1
Syrup is prepared by polymerising sucrose
This embodiment disclose by making sucrose be polymerized to produce soluble sugar using gtf enzyme in glucosan synthetic reaction Mixture general fashion.Specifically, it is prepared for the filtrate of glucosan synthetic reaction, then concentrated as syrup.
Sucrose (3000g) is added to the polyethylene bucket of the 5- gallon of cleaning.By water (18.1L) and FermasureTM (10mL) add to bucket, and by adding 5 volumes %NaOH and 5 volumes %H2SO4By pH regulator to 7.0.Final volume is About 20L, and as surveyed by HPLC, the initial concentration of sucrose is 152.5g/L.By adding as in conventional method partly middle institute State rough gtf enzyme (the SEQ ID NO of 0.3 volume % of preparation：3) extract is causing glucosan polyreaction.This extract Comprise the protein of about 2.9mg/mL.There is provided to reaction solution using the overhead mechanical motor equipped with glass axle and PTFE blade Stirring.
After 48 hours, HPLC analysis display, has consumed 96% sucrose and has thought that reaction completes.Using 325- mesh Steel wire and 40- urn filter filter insoluble poly- α -1 removing reaction, 3- beta-glucan products using Bu Shi filter funnel. Then using Rotary Evaporators (40-50 DEG C of bath temperature), mother solution (filtrate) is concentrated into the total sugar concentration of about 320g/L sugar.Concentrate The composition of filtrate is provided in table 2.
Table 2
The composition of the concentration filtrate of glucosan synthetic reaction
Sucrose Lucrose Glucose Fructose DP2 DP3+ Total amount
g/L 13.5 130.6 25.5 103.8 18.3 28.3 320.1
Wt% 4.2 40.8 8 32.4 5.7 8.9 100
Table 2 points out that the concentration filtrate of glucosan synthetic reaction comprises sucrose, Fructose, glucose, lucrose and low Polysaccharide DP2-DP7.
Embodiment 2
The impact to sucrose solution solution in the filtrate of glucosan synthetic reaction for the enzyme
This embodiment determines various glucoamylases (EC 3.2.1.3), transglucosidase (EC 2.4.1.24), β-Portugal The activity of glycosidase (EC 3.2.1.21), α-amylase (EC 3.2.1.1) and glucosidase (EC 3.2.1) is it is therefore intended that subtract The concentration concentrating lucrose and/or oligosaccharide by-product in filtrate of little glucosan synthetic reaction.Some enzymes are for example DIAZYME RDF ULTRA, transglucosidase (EC 2.4.1.24) and glucoamylase (EC 3.2.1.3) are α-glucoside Enzyme, is found particularly effectively to reduce the amount of these by-products, leads to monosaccharide (the glucose and Fructose) phase in treated filtrate Should increase.
Brief outline of procedure according to embodiment 1 is obtained the filtrate of glucosan synthetic reaction first and is concentrated as syrup.Should The composition concentrating filtrate is provided in table 3.NMR analysis shows, the α (1,3) being present in syrup with the ratio of (1,6) key is 78：22.
Table 3
The composition of the concentration filtrate of glucosan synthetic reaction
The syrup of table 3 is used for testing various enzymes to the lucrose of glucosan synthetic reaction and oligosaccharide by-product Hydrolysing activity.It is assumed that lucrose comprise special keys [α (1,5)-glucityl Fructose] and oligosaccharide mainly comprise α (1, 3) and α (1,6) glucityl-glucose key, when these experiments start which kind of enzyme can be used for hydrolyzing both by-products this not It is obvious.For should analyzing, select the enzyme (table 4) with different activities.
Table 4
Enzyme assessment for lucrose and oligosaccharide hydrolysis
a DuPont Industrial Biosciences
It is provided in table 5 (carrying enzyme amount, time, temperature, pH, sugared concentration) with the condition of the syrup that each enzyme processes table 3 above In.Dilute with water syrup is to reach the sugared concentration for each hydrolysis.Table 5 additionally provides by bright beading for every kind of enzyme hydrolysiss Bacterium disaccharide and the percentage ratio of DP3+ (at least DP3-DP7) oligosaccharide.DP3+ percent hydrolysis are calculated as (1- (DP3 in final syrup Weight % of+oligosaccharide)/(weight % of DP3+ oligosaccharide in initial syrup)).Similarly, lucrose hydrolysis percentage Ratio is calculated as (1- (lucrose weight % in final syrup)/(lucrose weight % in initial syrup)).
Table 5
Concentrate lucrose and oligosaccharide in filtrate by various enzyme hydrolysiss
The sugared concentration (sucrose, glucose, Fructose, lucrose and oligosaccharide) that a HPLC records；The value quilt of record It is rounded up to closest to 10g/L increment.
B DP3+ comprises DP3-DP7, but also can comprise larger soluble oligosaccharide, and it is when being produced with some gtf enzymes There is higher α -1,6 keys and α -1, the ratio of 3 keys.
Table 5 shows that Isosorbide-5-Nitrae-alpha-Glucosidase and 1,6- alpha-Glucosidase show part (embodiment 2.1) or few (enforcement Example 2.2) hydrolysis lucrose, but discharge some glucoses from oligosaccharide.α-amylase (embodiment 2.3 and reality Apply example 2.4) use show that the activity to target compound is minimum.Similarly, the use of amylopectase (embodiment 2.5) Minimum activity is shown.
Cellulase (embodiment 2.14 and 2.15) is largely invalid when hydrolyzing lucrose, but hydrolysis Some oligosaccharide.
Although oligosaccharide comprises β key, astoundingly, β-glucosyl enzym also show that extremely low (ACCELERASE BG, Embodiment 2.9) to high (NOVO 188, embodiment 2.10 and 2.11) hydrolysis scope.The relative potency of these enzymes is big Amplitude of variation.In some cases, the amount of the oligosaccharide of hydrolysis substantially exceeds (embodiment 2.11) or close to (embodiment 2.12) percentage ratio of the lucrose hydrolyzing.In other cases, lucrose is by β-glucosyl enzym height water Solution, oligosaccharide is by moderate hydrolysis (embodiment 2.13) simultaneously.Relatively High Defferential between the result of observed β-glucosyl enzym Show, present in tested β-glucosyl enzym preparation, other enzyme such as glucoamylases or another kind of alpha-Glucosidase are to cause Observe active the reason.
On the contrary, the result in table 5 shows, transglucosidase (TG L-2000, embodiment 2.6) illustrates to hydrolyze oligosaccharide High activity with lucrose.The lucrose being hydrolyzed by transglucosidase seems in some cases It is quantitative, and the DP3+ material more than 95% is hydrolyzed to glucose and DP2 (embodiment 2.7) under higher load enzyme amount. Show similar activity (embodiment 2.8) using the transglucosidase of purified pattern, show observed hydrolysis owing to Transglucosidase rather than background activity.
Glucoamylase (embodiment 2.16-2.18) illustrates a range of work for lucrose and oligosaccharide Property.Only a kind of tested glucoamylase (embodiment 2.18) gives the water that lucrose and oligosaccharide are less than 30% Solution.
Result in table 5 shows, alpha-Glucosidase such as DIAZYME RDF ULTRA, glucoamylase and turn glucoside Enzyme hydrolyzable is present in glucosan and reacts the lucrose by-product in filtrate.Alpha-Glucosidase hydrolyzes lucrose Ability show these enzyme hydrolyzable α -1,5 glucityls-Fructose key.By lucrose be used as substrate and illustrate above this Plant during activity it is believed that this activity also extends to comprise α -1, the oligosaccharide of 5 glucityls-Fructose key.
The result of table 5 further demonstrates that, alpha-Glucosidase such as glucoamylase and transglucosidase hydrolyzable are present in Glucosan reacts the oligosaccharide by-product in filtrate.Because these oligosaccharide are mainly by through α -1,3 and/or α -1,6 is bonded Glucose monomer unit forms (embodiment 3), so as shown by data alpha-Glucosidase hydrolyzable α -1 of table 5,3 glucityls-Fructus Vitis viniferae Sugar and/or α -1,6 glucityls-Fructose key.
Because the alpha-Glucosidase usually effective lucrose of hydroglucan synthetic reaction and/or oligosaccharide pair Product, so can be used alone or in combination these enzymes to reduce the Portugal of the sugared by-product from the monosaccharide comprising incrementss and reduction amount Formose Reaction filtrate produces the process time needed for high-purity syrup.Effectively the example of enzyme combination can be for hydrolyzing bright beading The transglucosidase of bacterium disaccharide such as TG L-2000, and effectively hydrolyze glucoamylase (the such as GC of oligosaccharide by-product 321).
Therefore, α-glucoamylase can independently hydrolyze (i) α -1 in specific sugar, 5 glucityls-Fructose key, and (ii) α - 1,3 and α -1,6 glucityls-glucose key.
Embodiment 3
Before and after enzyme hydrolysiss, glucosan reacts the comparison of the key distribution of filtrate component
This embodiment measures transglucosidase (EC 2.4.1.24) and β-glucosyl enzym (EC 3.2.1.21) to being present in The lucrose concentrating in filtrate of glucosan synthetic reaction and the hydrolysing activity of oligosaccharide by-product.Discovery turns glucoside Enzyme decreases the amount of these by-products, leads to the corresponding increase of the monosaccharide (glucose and Fructose) in treated filtrate.
The oligosaccharide by-product being present in the filtrate of above glucosan synthetic reaction comprises the glucose-Fructus Vitis viniferae of ＞ 90% Sugared key, as (conventional method) measured by NMR.In glucose-glucose key, about 78% represents α -1,3 keys and about 22% represents α -1,6 keys.
NMR is used for measuring the key feature of the material producing after hydrolyzing in above example 2.11.As shown in figure 1, it is corresponding In α -1, the peak of 3 keys reduces by 86%, and corresponding to α -1, the peak of 6 keys only reduces by 2.3%, and the peak corresponding to lucrose Peak reduce by 21%.Although almost quantitatively by this enzyme hydrolysis, Novo 188 seems to be unable to hydrolyzing alpha -1 sucrose, 6 keys.
NMR is similarly used for mensure TG L-2000 (SEQ ID NO：1) the key feature of the material that transglucosidase produces (Fig. 2).D by the concentration filtrate (deriving from the material of table 3) of 210 μ L, 300 μ L2O and 90 μ L contains 12.4mM DSS (as interior Mark) D2O mixes in NMR pipe, to provide the total sugar concentration of 300g/L and to be heated to 60 DEG C.Obtain after 60 DEG C of thermal balances Time zero spectrum (parent material in Fig. 2), and then add the enzyme of 0.5 volume %.At 60 DEG C, make sample in probe Reequilibrate simultaneously adds pad, and measure within a few minutes of analysis.With TG L-2000 enzyme (treated material in Fig. 2 Material) process 10 hours afterwards, corresponding to α -1, the peak of 3 keys reduces by 41%, and corresponding to α -1, the peak of 6 keys reduces by 36%, and right ＞ 95% (Fig. 2) should be reduced in the peak of lucrose.Observe that α-reducing end and β-reducing end peak both of which increase, this is right Should be in the increase (Fig. 2) of Fructose and glucose.
These results show that transglucosidase can will comprise α -1,3 and α -1, the oligosaccharide of 6 keys be converted into glucose and Lucrose can be converted into Fructose and glucose.Therefore, (i) α -1 in transglucosidase hydrolyzable specific sugar, 5 Portugals Glycosyl-Fructose key and (ii) α -1,3 and α -1,6 glucityls-glucose key.
Embodiment 4
React lucrose and the oligosaccharide in filtrate using immobilized enzyme hydrolysis glucosan
This embodiment describes using fixing glucoamylase (EC 3.2.1.3) and transglucosidase (EC 2.4.1.24) hydrolysis is present in available from the lucrose in the filtrate of glucosan synthetic reaction and other oligosaccharide.Specifically Ground, have studied fixing transglucosidase TG L-2000 (SEQ ID NO：1, available from Genencor/DuPont Industrial ) and fixing glucoamylase GC-147 is (available from Genencor/DuPont Industrial Biosciences Biosciences) to lucrose in the filtrate of glucosan synthetic reaction and oligosaccharide DP2, DP3 and HS (high sugar, DP4 +) impact that hydrolyzes.
Method according to described in United States Patent (USP) 5541097 is fixed to glucoamylase and transglucosidase, and it is public Open content to be herein incorporated by reference.
For fixing in glucoamylase and the typical method of transglucosidase, make the two batches porous that about 8.0g/ criticizes Granular kieselguhr (EP Minerals, Reno, NV) with distillation water hydratable and is then transferred to diameter 1.5-cm, height 30-cm In glass column type reactor.Pumping water is flowed up with about 6-7mL/min, thus removing particulate from whole three posts.Generally, exist In one hour, water effluent does not contain particulate.Water is drained into the top of granular bed of diatomaceous earth and with the poly- nitrogen of 0.1%w/v from post Third pyridine (PEI, EPOMIN P-1050) aqueous solution is replaced.Flowing pumps the PEI solution of 3500mL and so that effluent is followed then up Ring passes through bed 2 hours.Then, at room temperature, use the distilled water wash graininess bed of diatomaceous earth flowing up 2 hours, to remove Free PEI.So, obtain particulate Si diatomaceous earth-PEI.
Meanwhile, the 3.5mL glucoamylase GC-147 with the activity that table 4 limits is added to the 0.02M acetic acid of 315ml In salt buffer (pH 4.5).Then, by the 50%w/w glutaraldehyde of 1.575g (GA-50) it is added slowly to Portugal In saccharogenic amylase aqueous solution, it is gently mixed, and make glutaraldehyde and glucose starch enzyme aqueous solution under being gently mixed at 20-25 DEG C At a temperature of react 4 hours, it results in the treated enzyme-glutaraldehyde adduct comprising treated glucoamylase.Using tool Have table 4 limit activity transglucosidase TG L-2000 rather than glucoamylase repeat these steps respectively, thus result in Comprise the treated enzyme-glutaraldehyde adduct of treated transglucosidase.
Then, make every kind of treated enzyme-glutaraldehyde adduct its comprise particulate Si diatomaceous earth-PEI carrier from Shenzhu 4 hours (20-25 DEG C) of circulation in (produced above).With water, excessive treated adduct is washed out from carrier.Thus prepare There is the post of fixing glucoamylase or transglucosidase.
By have table 3 limited composition glucosan filtrate be diluted to 180g/L, adjust to pH 4.5, and be passed to wrap Post containing immobilized enzyme.Column temperature is controlled in 60 DEG C.In column equilibration 16 hours afterwards, timing sampling under different in flow rate.Pass through HPLC measures sugar composition (table 6) of hydrolysis product.When each change flow rate set when, reequilibrate is extremely before sampling to make post Few 1-2 bed volume.Calculate the degree of hydrolysis of lucrose and oligosaccharide with the method described in embodiment 2.Three kinds of posts of test Configuration：1) fixing glucoamylase, 2) fixing transglucosidase, and 3) fixing after fixing transglucosidase Glucoamylase.
Table 6
Fixing glucoamylase and the application of transglucosidase hydrolysis oligosaccharide and lucrose
Table 6 shows, increases with average contact time (being defined as nominal column volume divided by mean flow rate), leukonid two The degree of hydrolysis of sugar and oligosaccharide generally increases.It is particularly preferred with fixing transglucosidase hydrolysis lucrose, because Even if by not observing significant difference using being surveyed the fastest flow velocity yet.Although each post is shown respectively suitable conversion, Portugal The combination of saccharogenic amylase and transglucosidase gives the highest hydrolysis of oligosaccharide.
Therefore, the use of the enzyme that fixing glucoamylase or transglucosidase or this two class are fixed shows hydrolysis Comprise α -1,3 and α -1, the oligosaccharide of 6 glucityls-glucose key and the effective technology of lucrose.These results Meet embodiment 2 those.The fixation of other alpha-Glucosidases should provide similar result.
Embodiment 5
It is enriched with Fructose with chromatography from glucosan reaction filtrate
This embodiment disclose and how glucosan can be enriched with further by chromatography and react the Fructose in filtrate.
Generally, when by chromatography separation glycan molecule, component eluting is negatively correlated with molecular dimension, therefore elutes first Maximum molecule.Therefore, for the filtrate of glucosan synthetic reaction, elute oligosaccharide first, be disaccharide afterwards, followed by singly Sugar.Using sodium cation resin separate cannot be sufficiently separated Fructose and glucose, and all of lucrose, sucrose and DP2 is eluted out jointly.Preferably use the ion exchange resin separating glucose that cation is calcium and Fructose.
Brief outline of procedure according to embodiment 1 is obtained the filtrate of glucosan synthetic reaction first and is concentrated as syrup.Should The composition concentrating filtrate is provided in table 7.
Table 7
The composition of the concentration filtrate of glucosan synthetic reaction
The syrup of table 7 is filtered and is diluted to 25g dry solid/100g solution with nonionic exchanged water, and feed to comprising In the crosslinked post of strong-acid ion exchange resin (calcium form).The physical parameter of this post shows in table 8.Syrup by dilution (15.8L) send in the post being maintained at 65 DEG C, the water elution post being hereafter 30L/ hour with flow velocity.
Table 8
The physical parameter of post
Resinous type FINEX CS11GC
Ionic speciess Ca2+
Crosslinking, divinylbenzene % 5.5
Granularity (mm) 0.34
Bed length (m) 5.0
Column diameter (m) 0.225
This separate in, the lucrose remaining in post is longer than sucrose, this be likely due to beading bacterium disaccharide with Calcium cation complex, and be in fact jointly eluted out with glucose.Two fraction comprising Fructose are made to separate.47 to Elute fraction 5.1 between 120 minutes, and elute fraction 5.2 between 120 to 172 minutes.In feed in chromatographic isolation Fructose in, 95.7% Fructose is separated with ＞ 90% purity.As HPLC surveys, the product in each fraction (5.1 and 5.2) divides Cloth is shown in table 9.
Table 9
Comprise the products distribution of the chromatograph fraction of significant quantity Fructose
Because comprising 36.0% Fructose for this detached feed composition, 34.5% total stream is to have altogether The fructose syrup of ＞ 90 weight %DS Fructose reclaims.If ignoring the sucrose in charging, 40.7% sugar is to have ＞ 90 weight The fructose syrup of amount %DS Fructose reclaims.
Therefore, the Fructose that glucosan reacts in filtrate can be enriched with further by chromatography.Example 6 below shows, This process can be strengthened using the glucosan filtrate of transglucosidase hydrolysis.
Embodiment 6
Reacted from the glucosan of hydrolysis by chromatography and filtrate, be enriched with Fructose
This enforcement exemplifies, compared with separating levulose from unhydrolysed glucosan filtrate, from oligosaccharide and leukonid In the glucosan filtrate that disaccharide has been hydrolyzed, separating levulose leads to the yield of high-purity fructose syrup to increase.
By under 60 DEG C and pH 4.5 to 1 volume % transglucosidase TG L-2000 (SEQ ID NO：1) process The glucosan filtrate of 24 hours is concentrated (vacuum at 50 DEG C) to prepare syrup.Observe that some are low during concentration process Polysaccharide formation, it can be desired because it is known that transglucosidase produces oligosaccharide under the monosaccharide of high concentration.Syrup has The final product described in Table A is had to be distributed.
Table A
The composition of the concentration glucosan filtrate hydrolyzing before it is concentrated
Syrup described in Table A is filtered and is diluted to 25.4g DS/100g solution with nonionic exchanged water, and feed to bag Containing in the crosslinked post of storng-acid cation exchange resin (calcium form).The physical parameter of post is shown in table B.Then by dilution Syrup (169g) is sent in the post being maintained at 65 DEG C, the water elution post being hereafter 50mL/min with flow velocity.
Table B
The physical parameter of post
Resinous type FINEX CS11GC
Ionic speciess Ca2+
Crosslinking, divinylbenzene % 5.5
Granularity (mm) 0.34
Bed length (m) 1.69
Column diameter (m) 0.093
Two fraction comprising Fructose are made to separate.Elute fraction 6.1 between 73 to 103 minutes, and 103 to Fraction 6.2 is eluted between 120 minutes.In feed in the Fructose of chromatographic isolation, feed to post 93.0% Fructose in level Divide in 6.2 and separated with ＞ 90% purity.As HPLC surveys, the products distribution in each fraction (6.1 and 6.2) is shown in table C.
Table C
Comprise to derive from the products distribution of the chromatograph fraction of the Fructose of hydroglucan filtrate
The separation efficiency of this embodiment is attributable in the difference of post scale and sample more compared to the reduction of embodiment 5 High glucose fraction.Even so, prepared by the glucosan filtrate hydrolyzing without transglucosidase compared to by chromatography The embodiment 5 of syrup is obtained, and the chromatogram purification of this material leads to the yield of high-purity fructose syrup to increase.Because for being somebody's turn to do Detached feed composition comprises 47% Fructose (Table A), so 43.7% total stream is to have ＞ 90 weight %DS Fructose Fructose syrup reclaims.43.7% response rate is significantly better than 34.5% response rate of embodiment 5 herein.
Therefore, compared with from unhydrolysed glucosan filtrate during separating levulose, from the Portugal having been hydrolyzed by transglucosidase In polysaccharide filtrate, separating levulose leads to the yield of Fructose to increase.
Embodiment 7
Ethanol is prepared by the filtrate of glucosan synthetic reaction of fermenting
This embodiment disclose and glucosan filtrate yeast ferments for ethanol.
It is suspended in tap water (2.4L, optical density is 65 at 600nm) and and then adopts LEGEND XTR by making cream Yeast extract centrifugation to be washed yeast (saccharomyces cerevisiae) cream for 5 minutes under 4500g by centrifuge (Thermo Scientific) (Tonon mill, Brazil).After decantation supernatant, make yeast cells resuspended by two times centrifugal again and concentrate.? After three washings, by adding 5 weight % sulphuric acid by pH regulator to 2.Using GENESYS 204001 spectrophotometer (Thermo Scientific) measures optical density and is adjusted to 100 (at 600nm) by adding tap water.Will be through adjusting Section yeast extract (1.5L) adds to 7.5-L BIOFLO310 fermentor vessel (New Brunswick).Fermentation tank is set as It is maintained at 30 DEG C of temperature, and stir at 100 rpm.Although determining pH during fermentation, its do not pass through add acid or Aqueous slkali is controlling.
Preparation comprises yeast extract (10g/L), peptone (20g/L) and derives from entering of the 200g/L sugar of glucosan filtrate Material solution is simultaneously sterilized 15 minutes at 121 DEG C with PHOENIX AV-250 PLUS autoclave.Before fermentation starts, make charging Solution is cooled to 25 DEG C (room temperatures).Under 684mL/ hour speed, sterilized feedstock solution (3.5L) is added to fermentation tank about 5 Hour, and make fermentation carry out 22 hours.
Timing sampling with GENESYS 20 4001 spectrophotometric analysis optical density during fermentation, is reflected with PAL-3 Meter (Atago) analysis Brix Scale, analyzes sugar and concentration of alcohol with HPLC (conventional method).These results are summarized in table 10.
Table 10
The charging of the first alcohol fermentation and time-histories fermenting characteristic
List the dense of in charging and each fermentation time point (0-22 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
When fermentation ends, it is centrifuged 5 minutes by using LEGEND XTR centrifuge that to separate yeast thin under 4500g Born of the same parents.After decantation supernatant, make yeast resuspended twice and concentration by being centrifuged again.After third time is washed, by adding 5 Weight % sulphuric acid is by pH regulator to 2.Using GENESYS 20 4001 spectrophotometric determination optical density and by adding tap water Adjusted to 100 (at 600nm).According to above-mentioned the same terms, carried out again using the yeast cells reclaiming from previous fermentation Fermenting twice circulates, every time all using fresh feed.The fermentation results that the yeast being reclaimed with for the first time and second obtains are respectively It is provided in table 11 and 12.
Table 11
Charging and time-histories fermenting characteristic using the first recovery yeast cells
List the dense of in charging and each fermentation time point (0-21 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
Table 12
Charging and time-histories fermenting characteristic using the second recovery yeast cells
List the dense of in charging and each fermentation time point (0-21 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
Consume few lucrose in first time ferments, but yeast cells reclaim via second and start to fit And lucrose should be consumed.After using three fermentation cycle reclaiming yeast, alcohol fermentation titre is by 33g/L (table 10,22 hours) increase to 54g/L (table 12,21 hours), but even if or exist notable in last circulation wild Oryza species The lucrose of amount.
Therefore, glucosan filtrate can be used for sweat to produce ethanol.
Embodiment 8
Ethanol is prepared by the glucosan filtrate of hydrolysis fermentation
This enforcement exemplifies makes wherein lucrose and oligosaccharide side components previously by the glucosan of saccharifying Filtrate fermentation leads to ethanol yield to increase.
Fermentation is carried out according to the method for embodiment 7 description, but used here as previous transglucosidase (TG L- 2000, SEQ ID NO：1) the glucosan filtrate processing.The glucosan filtrate of following preparation hydrolysis.By glucosan filtrate adjust to 300g sugar/L, then using 1.0M sodium hydroxide and 5 weight % sulphuric acid by pH regulator to 4.0.The final volume of said preparation is 6.75L.Then using PHOENIX AV-250PLUS autoclave, filtrate is sterilized 15 minutes at 121 DEG C, then temperature is adjusted Save to 60 DEG C.So that the TG L-2000 enzyme extract as described in table 4 (135mL) is mixed with sterilized filtrate, and at 60 DEG C and Solution is made to incubate 72 hours in culture shaking table (IKA KS4000) under 100rpm.Thus it is prepared for the glucosan filtrate hydrolyzing.
By make cream be suspended in tap water (2.4L, at 600nm optical density be 65) and and then adopt LEGEND XTR from Yeast extract centrifugation to be washed yeast (saccharomyces cerevisiae) cream (Bom Retiro mill, Brazil) for 5 minutes under 4500g by scheming. After decantation supernatant, make yeast resuspended by two times centrifugal again and concentrate.After third time is washed, by adding 5 weights Amount % sulphuric acid is by pH regulator to 4.5, and using GENESYS204001 spectrophotometric determination optical density and passes through to add from the beginning Water is adjusted to 100 (at 600nm).Adjusted yeast extract (1.5L) is added and holds to 7.5-L BIOFLO310 fermentation tank In device.Fermentation tank is set as keeping 30 DEG C of temperature, stirs at 100 rpm, and use 4M ammonium hydroxide aqueous solution or 5 weights Amount % aqueous sulfuric acid makes pH be maintained at 4.5.
Preparation comprises the 200g/L sugar of the filtrate of yeast extract (10g/L), peptone (20g/L) and hydrolysis of must hanging oneself Feedstock solution is simultaneously sterilized 15 minutes at 121 DEG C with PHOENIX AV-250 Plus autoclave.Before fermentation starts, make charging Solution is cooled to 25 DEG C (room temperatures).Under 684mL/ hour speed, sterilized feedstock solution (3.5L) is added to fermentation tank about 5 Hour, and make fermentation carry out 22 hours.
Timing sampling with GENESYS 20 4001 spectrophotometric analysis optical density during fermentation, is reflected with PAL-3 Meter analysis Brix Scale, analyzes sugar and concentration of alcohol with HPLC (conventional method).These results are summarized in table 13.
Table 13
Charging and time-histories fermenting characteristic using the first alcohol fermentation through hydroglucan filtrate
List the dense of in charging and each fermentation time point (0-22 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
When fermentation ends, it is centrifuged 5 minutes by using LEGEND XTR centrifuge that to separate yeast thin under 4500g Born of the same parents.After decantation supernatant, make yeast cells resuspended twice and concentration by being centrifuged again.After third time is washed, pass through Add 5 weight % sulphuric acid by pH regulator to 2.Using GENESYS 20 4001 spectrophotometric determination optical density and by adding Tap water is adjusted to 100 (at 600nm).According to above-mentioned the same terms, using the yeast cells reclaiming from previous fermentation Carry out fermenting twice circulation again, every time using fresh feed.What the yeast cells reclaiming using for the first time and second obtained sends out Ferment result is respectively provided in table 14 and 15.
Table 14
Reclaim yeast cells and the charging through hydroglucan filtrate and time-histories fermenting characteristic using first
List the dense of in charging and each fermentation time point (0-21 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
Table 15
Reclaim yeast cells and the charging through hydroglucan filtrate and time-histories fermenting characteristic using second
List the dense of in charging and each fermentation time point (0-21 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
Start fermentation about six hours in, all fermentations are basically completed, and lead to the ethanol titre of 57-60.0g/L.Will These fermentation with embodiment 7 those be compared show than with using unhydrolysed glucosan filtrate fermentation obtain those Compare, glucosan filtrate hydrolyzed before it is fermented and leads to faster and bigger ethanol yield.
Therefore so as to middle lucrose and oligosaccharide side components are led to by the glucosan filtrate fermentation of saccharifying Ethanol yield is increased with faster speed.This saccharifying can be carried out using such as transglucosidase.
Embodiment 9
The synchronous saccharification of glucosan filtrate and fermentation
This embodiment disclose the synchronous saccharification of the charging comprising glucosan filtrate and fermentation may result in enhanced fermentation special Property.
By make cream be suspended in tap water (2.4L, at 600nm optical density be 65) and and then adopt LEGEND XTR from Yeast extract centrifugation to be washed yeast (saccharomyces cerevisiae) cream (Bom Retiro mill, Brazil) for 5 minutes under 4500g by scheming. After decantation supernatant, make yeast cells resuspended by two times centrifugal again and concentrate.After third time is washed, by adding 5 weight % sulphuric acid are by pH regulator to 4.5, and using GENESYS 20 4001 spectrophotometric determination optical density and pass through to add Plus tap water is adjusted to 100 (at 600nm).Adjusted yeast extract (1.5L) is added and sends out to 7.5-L BIOFLO310 In fermentation tank container.Fermentation tank is set as keeping 30 DEG C of temperature, stirs at 100 rpm, and use 4M ammonium hydroxide aqueous solution Or 5 weight % aqueous sulfuric acid make pH be maintained at 4.5.
Preparation comprises yeast extract (10g/L), peptone (20g/L) and derives from entering of the 200g/L sugar of glucosan filtrate Material solution is simultaneously sterilized 15 minutes at 121 DEG C with PHOENIX AV-250 PLUS autoclave.Before fermentation starts, make charging molten Liquid is cooled to 25 DEG C (room temperatures).Before adding the solution to fermentation tank, TG L-2000 as described in Table 4 is turned glucose Glycosides enzyme (1%v/v) adds to sterilized feedstock solution.684mL/ is little at present will be molten for the charging comprising TG L-2000 enzyme Liquid (3.5L) adds to fermentation tank about 5 hours, and makes fermentation carry out 48 hours.
Timing sampling with GENESYS 20 4001 spectrophotometric analysis optical density during fermentation, is reflected with PAL-3 Meter (Atago) analysis Brix Scale, analyzes sugar and concentration of alcohol with HPLC (conventional method).These results are summarized in table 16.
Table 16
The charging of saccharifying and alcohol fermentation and time-histories fermenting characteristic while glucosan filtrate
List the dense of in charging and each fermentation time point (0-48 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
Fermentation nominally completed in 6 hours, the fermentation (embodiment being hydrolyzed similar to filtrate before fermentation step , and be given compared to using not hydrolyzing the slightly more excellent ethanol titre (62g/L) of filtrate (embodiment 7) 8).Additionally, 6 is little When consume almost all of lucrose (table 16 is compared) with table 13-15.If directly saccharifying enzyme is added to sending out Ferment thing, then except face fermentation before saccharifying enzyme such as TG L-2000 is added to the charging comprising glucosan filtrate in addition to it should Obtain similar result.
Therefore, comprise the synchronous saccharification of the charging of glucosan filtrate and fermentation may result in enhanced fermentation character, for example, increase The consumption of (i) glucosan filtrate component (such as lucrose) and (ii) ethanol yield and generation speed greatly.
Embodiment 10
The preparation of various alpha-Glucosidases
This embodiment disclose prepare except for some previous embodiment those alpha-Glucosidases (transglucosidase, Glucoamylase, DIAZYME RDF ULTRA) outside various alpha-Glucosidases.Test these other alpha-Glucosidases pair Comprise α -1,5 glucityls-Fructose key or α -1,3 and/or α -1, the hydrolysing activity of the oligosaccharide of 6 glucityls-glucose key (with In the embodiment 11,12,15 and 16 of lower offer).
The discovery of Aspergillusclavatus alpha-Glucosidase (Aclglu1)
The bacterial strain of Aspergillusclavatus is chosen as can be used for the possible source of other enzymes of various commercial Application.Identify in Aspergillusclavatus A kind of gene code alpha-Glucosidase (referred to as " Aclglu1 "), and the sequence of this gene is provided in SEQ ID NO：In 4. By SEQ ID NO：The corresponding protein of 4 codings is provided in SEQ ID NO：5.Aclglu1 belongs to based on PFAM search The glycosyl hydrolase family 31 of (pfam.sanger.ac.uk web link).In N- end, protein (SEQ ID NO：5) have There is the signal peptide that length is 19 aminoacid, predict as SignalP edition 4 .0 (Nordahl Petersen et al. 2011, Nature Methods, 8：785-786).The presence of signal peptide shows that Aclglu1 is secreted enzyme.The mature form of prediction The aminoacid sequence of Aclglu1 be shown as SEQ ID NO：6.
The expression of Aspergillusclavatus alpha-Glucosidase Aclglu1
Aclglu1 gene cloning will be synthesized in pTrex3gM expression vector, (be described in U.S. Patent Application Publication 2011/ 0136197, it is herein incorporated by reference) and gained plasmid is named as pJG294.The sequence of Aclglu1 gene is passed through DNA sequencing is confirmed.
Using ballistic methods (Te ' o VS et al., J Microbiol Methods, 51：393-9,2002) by plasmid PJG294 is transformed in the Li's Trichoderma strains of quadruple disappearance and (is described in WO05/001036).Prediction comprises SEQ ID NO：6 protein secreting is in extracellular medium, and the culture medium after filtering is used for carrying out SDS-PAGE and α-glucoside Enzyme assay is confirming expression of enzymes.
Fei Shi Xin Satuo bacterium (Neosartorya Fischeri) the discovery of alpha-Glucosidase Nfiglu1
The bacterial strain of Fei Shi Xin Satuo bacterium is chosen as can be used for the possible source of other enzymes of various commercial Application.Identify in expense One of family name Xin Satuo bacterium gene code alpha-Glucosidase (referred to as " Nfiglu1 "), and the sequence of this gene is provided in SEQ ID NO：In 7.By SEQ ID NO：The corresponding protein of 7 codings is provided in SEQ ID NO：8.Nfiglu1 belongs to based on PFAM The glycosyl hydrolase family 31 of search (pfam.sanger.ac.uk web link).In N- end, protein (SEQ ID NO： 8) there is the signal peptide that length is 19 aminoacid, as SignalP edition 4 .0 predicts (Nordahl Petersen et al. 2011, Nature Methods, 8：785-786).The presence of signal peptide shows that Nfiglu1 is secreted enzyme.The ripe shape of prediction The aminoacid sequence of the Nfiglu1 of formula is shown as SEQ ID NO：9.
The expression of Fei Shi Xin Satuo bacterium alpha-Glucosidase Nfiglu1
Nfiglu1 gene cloning will be synthesized in pTrex3gM expression vector, (be described in U.S. Patent Application Publication 2011/ 0136197) and by gained plasmid it is named as pJG295.The sequence of Nfiglu1 gene is confirmed by DNA sequencing.
Using ballistic methods (Te ' o VS et al., J Microbiol Methods, 51：393-9,2002) by plasmid PJG295 is transformed in the Li's Trichoderma strains of quadruple disappearance and (is described in WO05/001036).Prediction comprises SEQ ID NO：9 protein secreting is in extracellular medium, and the culture medium after filtering is used for carrying out SDS-PAGE and α-glucoside Enzyme assay is confirming expression of enzymes.
The discovery of Neuraspora crassa (Neurospora crassa) alpha-Glucosidase Ncrglu1
The bacterial strain of Neuraspora crassa is chosen as can be used for the possible source of other enzymes of various commercial Application.Identify in coarse One of neurospora gene code alpha-Glucosidase (referred to as " Ncrglu1 "), and the sequence of this gene is provided in SEQ ID NO：In 10.By SEQ ID NO：The corresponding protein of 10 codings is provided in SEQ ID NO：In 11.Ncrglu1 belongs to and is based on The glycosyl hydrolase family 31 of PFAM search (pfam.sanger.ac.uk web link).In N- end, protein (SEQ ID NO：11) there is the signal peptide that length is 22 aminoacid, as SignalP edition 4 .0 predicts (Nordahl Petersen etc. People 2011, Nature Methods, and 8：785-786).The presence of signal peptide shows that Ncrglu1 is secreted enzyme.The maturation of prediction The aminoacid sequence of the Ncrglu1 of form is shown as SEQ ID NO：12.
The expression of Neuraspora crassa alpha-Glucosidase Ncrglu1
Ncrglu1 gene cloning will be synthesized in pTrex3gM expression vector, (be described in U.S. Patent Application Publication 2011/ 0136197) and by gained plasmid it is named as pJG296.The sequence of Ncrglu1 gene is confirmed by DNA sequencing.
Using ballistic methods (Te ' o VS et al., J Microbiol Methods, 51：393-399,2002) by plasmid PJG296 is transformed in the Li's Trichoderma strains of quadruple disappearance and (is described in WO05/001036).Prediction comprises SEQ ID NO：12 protein secreting is in extracellular medium, and the culture medium after filtering is used for carrying out SDS-PAGE and α-glucose Glycosides enzyme assay is confirming expression of enzymes.
The discovery of Rasamsonia composticola alpha-Glucosidase TauSec098
The bacterial strain of Rasamsonia composticola is chosen as can be used for may coming of other enzymes of various commercial Application Source.Identify in one of Rasamsonia composticola gene code alpha-Glucosidase (referred to as " TauSec098 "), And the sequence of this gene is provided in SEQ ID NO：In 13.By SEQ ID NO：The corresponding protein of 13 codings is provided in SEQ ID NO：14.TauSec098 belongs to the glycosyl hydrolase man based on PFAM search (pfam.sanger.ac.uk web link) Race 31 and comprise N- end CBM 20 domain.In N- end, protein (SEQ ID NO：14) having length is 22 aminoacid Signal peptide, predict as SignalP edition 4 .0 (Nordahl Petersen et al. 2011, Nature Methods, 8： 785-786).The presence of signal peptide shows that TauSec098 is secreted enzyme.The amino of the TauSec098 of mature form of prediction Acid sequence is shown as SEQ ID NO：15.
The expression of Rasamsonia composticola alpha-Glucosidase TauSec098
Synthesis TauSec098 gene is cloned into trichoderma reesei table by Generay Biotech Co (Shanghai, China) Reach in carrier pGXT (pTTT- plasmid) and gained plasmid is named as pGX256-TauSec098.TauSec098 gene Sequence confirmed by DNA sequencing.
Using protoplast transformation (Te ' o et al., J.Microbiol.Methods 51：393-9,2002) by plasmid PGX256-TauSec098 is transformed in the Li's Trichoderma strains of quadruple disappearance and (is described in WO05/001036).In bag Culture medium (acetamide 0.6g/L containing the acetamide as only nitrogen source；Cesium chloride 1.68g/L；Glucose 20g/L；Di(2-ethylhexyl)phosphate Hydrogen potassium 15g/L；Magnesium sulfate heptahydrate 0.6g/L；Calcium chloride dihydrate 0.6g/L；Ferrous sulfate (II) 5mg/L；Zinc sulfate 1.4mg/L； Cobaltous chloride (II) 1mg/L；Manganese sulfate (II) 1.6mg/L；Agar 20g/L；PH 4.25) upper selection transformant.Went out in about 1 week The bacterium colony (about 50-100) now converting.After acetamide plates grow, collect the spore of transformant and transfer to new second In amide agar plate.After acetamide plates grow 5 days, by 1 × 108The 30ml Portugal in 250-mL shaking flask for the spore inoculating In grape sugar/sophorose defined medium.Shaking flask is made to shake 5 days at 28 DEG C.The supernatant deriving from these cultures is used for demonstrate,proving Real mature T auSec098 enzyme (SEQ ID NO：15) expression (SDS PAGE) and activity.
The discovery of Rasamsonia composticola alpha-Glucosidase TauSec099
The bacterial strain of Rasamsonia composticola is chosen as can be used for may coming of other enzymes of various commercial Application Source.Identify in one of Rasamsonia composticola gene code alpha-Glucosidase (referred to as " TauSec099 "), And the sequence of this gene is provided in SEQ ID NO：In 16.By SEQ ID NO：The corresponding protein of 16 codings is provided in SEQ ID NO：In 17.TauSec099 belongs to the glycosyl hydrolase based on PFAM search (pfam.sanger.ac.uk web link) Family 31.In N- end, protein (SEQ ID NO：17) there is the signal peptide that length is 17 aminoacid, such as SignalP version This 4.0 is predicted (Nordahl Petersen et al. 2011, Nature Methods, 8：785-786).The presence table of signal peptide Bright TauSec099 is secreted enzyme.The aminoacid sequence of the TauSec099 of mature form of prediction is shown as SEQ ID NO： 18.
The expression of Rasamsonia composticola alpha-Glucosidase TauSec099
Synthesis TauSec099 gene is cloned into trichoderma reesei table by Generay Biotech Co (Shanghai, China) Reach in carrier pGXT (pTTT- plasmid) and gained plasmid is named as pGX256-TauSec099.TauSec0998 base The sequence of cause is confirmed by DNA sequencing.
Using protoplast transformation (Te ' o et al., J.Microbiol.Methods 51：393-9,2002) by plasmid PGX256-TauSec099 is transformed in the Li's Trichoderma strains of quadruple disappearance and (is described in WO05/001036).In bag Culture medium (acetamide 0.6g/L containing the acetamide as only nitrogen source；Cesium chloride 1.68g/L；Glucose 20g/L；Di(2-ethylhexyl)phosphate Hydrogen potassium 15g/L；Magnesium sulfate heptahydrate 0.6g/L；Calcium chloride dihydrate 0.6g/L；Ferrous sulfate (II) 5mg/L；Zinc sulfate 1.4mg/L； Cobaltous chloride (II) 1mg/L；Manganese sulfate (II) 1.6mg/L；Agar 20g/L；PH 4.25) upper selection transformant.Went out in about 1 week The bacterium colony (about 50-100) now converting.After acetamide plates grow, collect the spore of transformant and transfer to new second In amide agar plate.After acetamide plates grow 5 days, by 1 × 108The 30ml Portugal in 250-mL shaking flask for the spore inoculating In grape sugar/sophorose defined medium.Shaking flask is made to shake 5 days at 28 DEG C.The supernatant deriving from these cultures is used for demonstrate,proving Real mature T auSec099 enzyme (SEQ ID NO：18) expression (SDS PAGE) and activity.
The sequence of bifidobacterium longum alpha-Glucosidase BloGlu1
Identify alpha-Glucosidase gene " BloGlu1 " from bifidobacterium longum subspecies-bifidobacterium longum JDM301. BloGlu1 gene (SEQ ID NO：19, GENBANK accession number NC014169.1, the complementary seriess of 140600 to 142414) Nucleotide sequence and by SEQ ID NO：Putative protein (the SEQ ID NO of 19 codings：20) aminoacid sequence is present in In GENBANK accession number YP_003660432.1.
The expression of bifidobacterium longum alpha-Glucosidase BloGlu1
To coding whole BloGlu1 protein (SEQ ID NO：20) DNA sequence is optimized with bacillus subtilis Express in bacterium, then synthesized by Generay Biotech Co. (Shanghai, China) and (obtain SEQ ID NO：21) and insert Enter in p3JM plasmid, lead to p3JM-BloGlu1.It is optimized to drive that p3JM-BloGlu1 plasmid comprises aprE promoter BloGlu1 sequence (SEQ ID NO：21) express.
Plasmid p3JM-BloGlu1 be used for converting B. subtilis cell (degUHy32, Δ nprB, Δ vpr, Δ epr, Δ scoC, Δ wprA, Δ mpr, Δ ispA, Δ bpr), and the cell of conversion is coated on is supplemented with 5ppm chloromycetin On Luria agar plate.Select to be correctly inserted into the bacterium colony of fragment and make it have with tool as confirm by PCR and sequencing (MOPS- base defined medium is supplemented with extra 5mM CaCl to MBD culture medium2) 250-mL shaking flask in fermented, with Expression BloGlu1 protein (SEQ ID NO：20).
The sequence of bifidobacterium longum alpha-Glucosidase BloGlu2
Identify alpha-Glucosidase gene BloGlu2 from bifidobacterium longum.Aminoacid sequence (the SEQ ID of BloGlu2 NO：22) it is present in ncbi database (the GENBANK number of logging in WP_007054665.10).
The expression of bifidobacterium longum alpha-Glucosidase BloGlu2
Coding BloGlu2 protein DNA sequence is optimized to express in bacillus subtilises, Ran Houyou Generay Biotech Co. synthesis (obtains SEQ ID NO：23) and be inserted in p3JM plasmid, lead to p3JM-BloGlu2. SEQ ID NO：23 coding SEQ ID NO：24 aminoacid sequence.P3JM-BloGlu2 plasmid comprises aprE promoter to drive Optimized BloGlu2 sequence (SEQ ID NO：23) express.
Plasmid p3JM-BloGlu2 be used for converting B. subtilis cell (degUHy32, Δ nprB, Δ vpr, Δ epr, Δ scoC, Δ wprA, Δ mpr, Δ ispA, Δ bpr), and the cell of conversion is coated on is supplemented with 5ppm chloromycetin On Luria agar plate.Select to be correctly inserted into the bacterium colony of fragment and make it have with tool as confirm by PCR and sequencing (MOPS- base defined medium is supplemented with extra 5mM CaCl to MBD culture medium2) 250-mL shaking flask in fermented, with Expression BloGlu2 protein (SEQ ID NO：24).
The sequence of bifidobacterium longum alpha-Glucosidase BloGlu3
Identify alpha-Glucosidase gene " BloGlu3 " from bifidobacterium longum subspecies-bifidobacterium longum F8.BloGlu3 base Because of (SEQ ID NO：25, GENBANK accession number NC_021008.1,2130627 to 2132441) nucleotide sequence and by SEQ ID NO：Putative protein (the SEQ ID NO of 25 codings：26) aminoacid sequence is present in GENBANK accession number YP_ In 007768249.1.
The expression of bifidobacterium longum alpha-Glucosidase BloGlu3
To coding whole BloGlu3 protein (SEQ ID NO：26) DNA sequence is optimized with bacillus subtilis Express in bacterium, then synthesized by Generay Biotech Co. and (obtain SEQ ID NO：27) and be inserted in p3JM plasmid, lead Cause p3JM-BloGlu3.P3JM-BloGlu3 plasmid comprises aprE promoter to drive optimized BloGlu3 sequence (SEQ ID NO：27) express.
Plasmid p3JM-BloGlu3 be used for converting B. subtilis cell (degUHy32, Δ nprB, Δ vpr, Δ epr, Δ scoC, Δ wprA, Δ mpr, Δ ispA, Δ bpr), and the cell of conversion is coated on is supplemented with 5ppm chloromycetin On Luria agar plate.Select to be correctly inserted into the bacterium colony of fragment and make it have with tool as confirm by PCR and sequencing (MOPS- base defined medium is supplemented with extra 5mM CaCl to MBD culture medium2) 250-mL shaking flask in fermented, with Expression BloGlu3 protein (SEQ ID NO：26).
The sequence of bifidobacterium pseudolongum alpha-Glucosidase BpsGlul
Identify alpha-Glucosidase gene BpsGlu1 from bifidobacterium pseudolongum.Aminoacid sequence (the SEQ ID of BpsGlu1 NO：28) it is present in ncbi database (the GENBANK number of logging in WP_022858408.1).
The expression of bifidobacterium pseudolongum alpha-Glucosidase BpsGlu1
Coding BloGlu1 protein DNA sequence is optimized to express in bacillus subtilises, Ran Houyou Generay Biotech Co. synthesis (obtains SEQ ID NO：29) and be inserted in p3JM plasmid, lead to p3JM-BpsGlul. SEQ ID NO：29 coding SEQ ID NO：30 aminoacid sequence.P3JM-BpsGlu1 plasmid comprises aprE promoter to drive Optimized BpsGlu1 sequence (SEQ ID NO：29) express.
Plasmid p3JM-BpsGlu1 be used for converting B. subtilis cell (degUHy32, Δ nprB, Δ vpr, Δ epr, Δ scoC, Δ wprA, Δ mpr, Δ ispA, Δ bpr), and the cell of conversion is coated on is supplemented with 5ppm chloromycetin On Luria agar plate.Select to be correctly inserted into the bacterium colony of fragment and make it have with tool as confirm by PCR and sequencing (MOPS- base defined medium is supplemented with extra 5mM CaCl to MBD culture medium2) 250-mL shaking flask in fermented, with Expression BpsGlu1 protein (SEQ ID NO：30).
The sequence of bifidobacterium thermophilum alpha-Glucosidase BthGlu1
Identify alpha-Glucosidase gene " BthGlu1 " from bifidobacterium thermophilum RBL67.BthGlu1 gene (SEQ ID NO：31, GENBANK accession number NC_020546.1,150690 to 152495) nucleotide sequence and by SEQ ID NO：31 volumes Putative protein (the SEQ ID NO of code：32) aminoacid sequence is present in GENBANK accession number YP_007592840.1.
The expression of bifidobacterium thermophilum alpha-Glucosidase BthGlu1
To coding whole BthGlu1 protein (SEQ ID NO：32) DNA sequence is optimized with bacillus subtilis Express in bacterium, then synthesized by Generay Biotech Co. and (obtain SEQ ID NO：33) and be inserted in p3JM plasmid, lead Cause p3JM-BthGlu1.P3JM-BthGlu1 plasmid comprises aprE promoter to drive optimized BthGlu1 sequence (SEQ ID NO：33) express.
Plasmid p3JM-BthGlu1 be used for converting B. subtilis cell (degUHy32, Δ nprB, Δ vpr, Δ epr, Δ scoC, Δ wprA, Δ mpr, Δ ispA, Δ bpr), and the cell of conversion is coated on is supplemented with 5ppm chloromycetin On Luria agar plate.Select to be correctly inserted into the bacterium colony of fragment and make it have with tool as confirm by PCR and sequencing (MOPS- base defined medium is supplemented with extra 5mM CaCl to MBD culture medium2) 250-mL shaking flask in fermented, with Expression BthGlu1 protein (SEQ ID NO：32).
The sequence of bifidobacterium breve alpha-Glucosidase BbrGlu2
Identify alpha-Glucosidase gene BbrGlu2 from bifidobacterium breve.Aminoacid sequence (the SEQ ID of BbrGlu2 NO：34) it is present in ncbi database (the GENBANK number of logging in WP_003827971.1).
The expression of bifidobacterium breve alpha-Glucosidase BbrGlu2
Coding BbrGlu2 protein DNA sequence is optimized to express in bacillus subtilises, Ran Houyou Generay Biotech Co. synthesis (obtains SEQ ID NO：35) and be inserted in p3JM plasmid, lead to p3JM-BbrGlu2. SEQ ID NO：35 coding SEQ ID NO：36 aminoacid sequence.P3JM-BbrGlu2 plasmid comprises aprE promoter to drive Optimized BbrGlu2 sequence (SEQ ID NO：35) express.
Plasmid p3JM-BbrGlu2 be used for converting B. subtilis cell (degUHy32, Δ nprB, Δ vpr, Δ epr, Δ scoC, Δ wprA, Δ mpr, Δ ispA, Δ bpr), and the cell of conversion is coated on is supplemented with 5ppm chloromycetin On Luria agar plate.Select to be correctly inserted into the bacterium colony of fragment and make it have with tool as confirm by PCR and sequencing (MOPS- base defined medium is supplemented with extra 5mM CaCl to MBD culture medium2) 250-mL shaking flask in fermented, with Expression SEQID NO：36.
The sequence of bifidobacterium breve alpha-Glucosidase BbrGlu5
Identify alpha-Glucosidase gene " BbrGlu5 " from bifidobacterium breve ACS-071-V-Sch8b.BbrGlu5 gene (SEQ ID NO：37, GENBANK accession number NC_017218.1, the complementary seriess of 2241075 to 2242895) nucleic acid sequence Arrange and by SEQ ID NO：Putative protein (the SEQ ID NO of 37 codings：38) aminoacid sequence is present in GENBANK accession number In YP_005583701.1.
The expression of bacillus bifiduss alpha-Glucosidase BbrGlu5
To coding whole BbrGlu5 protein (SEQ ID NO：38) DNA sequence is optimized with bacillus subtilis Express in bacterium, then synthesized by Generay Biotech Co. and (obtain SEQ ID NO：39) and be inserted in p3JM plasmid, lead Cause p3JM-BbrGlu5.P3JM-BbrGlu5 plasmid comprises aprE promoter to drive optimized BbrGlu5 sequence (SEQ ID NO：39) express.
Plasmid p3JM-BbrGlu5 be used for converting B. subtilis cell (degUHy32, Δ nprB, Δ vpr, Δ epr, Δ scoC, Δ wprA, Δ mpr, Δ ispA, Δ bpr), and the cell of conversion is coated on is supplemented with 5ppm chloromycetin On Luria agar plate.Select to be correctly inserted into the bacterium colony of fragment and make it have with tool as confirm by PCR and sequencing (MOPS- base defined medium is supplemented with extra 5mM CaCl to MBD culture medium2) 250-mL shaking flask in fermented, with Expression BbrGlu5 protein (SEQ ID NO：38).
From expression culture purification alpha-Glucosidase
AclGlu1 and NcrGlu1
Carry out purification AclGlu1 (SEQ ID NO using two chromatographic steps：6) and NcrGlu1 (SEQ ID NO：12) α-Portugal Both glycosidase.For each purification, concentrate the thick hair zymotic fluid of shaking flask, hereafter adding ammonium sulfate to ultimate density is 2M.Will Solution stowage is to the 50-mL phenyl HP post through 20mM Tris (pH 8.0), 2M ammonium sulfate pre-equilibration.With 1M ammonium sulfate, 20mM Tris (pH 8.0) elutes target protein (SEQ ID NO from post：6 or SEQ ID NO：12).Merge corresponding fraction, use VIVAFLOW 200 ultrafiltration apparatuss (Sartorius Stedim) so that it is concentrated and buffered liquid exchanges to 20mM Tris (pH 8.0) in (buffer A).Resulting solution is put on the 40-mL Q HP post of buffered liquid A pre-equilibration.With containing 0.3M NaCl's Buffer A elutes target protein from post.Then the fraction comprising target protein is merged and use 10K AMICON ULTRA-15 equipment concentrates, and is stored in 40% glycerol at -20 DEG C until using.
NfiGlu1
Carry out purification NfiGlu1 alpha-Glucosidase (SEQ ID NO using two hydrophobic interaction chromatography steps：9).Concentrate shaking flask Thick hair zymotic fluid, hereafter add ammonium sulfate to ultimate density be 1M.By solution stowage to through 20mM Tris (pH 8.0), 1M sulfur The 50-mL phenyl HP post of sour ammonium pre-equilibration.Make target protein (SEQ ID NO：9) post is passed through in flowing.Merge and flow out fraction, this Adding ammonium sulfate to ultimate density afterwards is 2M.By solution stowage to the phase through 20mM Tris (pH 8.0), 2M ammonium sulfate pre-equilibration With on phenyl HP post.Elute target protein with 1M ammonium sulfate, 20mM Tris (pH 8.0) from post.Then target will be comprised The fraction of protein merges and uses 10K AMICON ULTRA-15 equipment to concentrate, and be stored in 40% glycerol at -20 DEG C until Use.
TauSec098 and TauSec099
Carry out purification TauSec098 (SEQ ID NO via hydrophobic interaction chromatography：15) and TauSec099 (SEQ ID NO： 18) both alpha-Glucosidases.For each purification, the about 180mL that ammonium sulfate is added to 7-L fermentation tank concentrates thick hair zymotic fluid In to ultimate density be 1M.Then by this solution stowage to pre- flat through 20mM sodium acetate (pH5.0), 1M ammonium sulfate (buffer A) 50-mL HIPREP phenyl-FF agarose column (GE Healthcare) of weighing apparatus.In the same buffer with three column volumes (CV) After washing, using 75%, 50% and 0% buffer A respectively with three CV progressively eluting posts, afterwards with the MILLIQ H of two CV2O Eluting.All fraction are analyzed by SDS-PAGE.Target protein (SEQ ID NO：15 or SEQ ID NO：18) it is primarily present In flowing out in fraction, it adopts 10KDa AMICON ULTRA-15 equipment to be concentrated and buffered fluid exchange to remove excess of sulfur Sour ammonium.At -80 DEG C, the final product that purity is more than 90% is stored in 40% glycerol until using.
BloGlu1, BloGlu2 and BloGlu3
Make BloGlu1 (SEQ ID NO：20)、BloGlu2(SEQ ID NO：24)、BloGlu3(SEQ ID NO：26)α- Glucosidase is all with three step purification.For each purification, concentrate the thick hair zymotic fluid of 1-L DASGIP fermentation tank, hereafter add Plus ammonium sulfate is to 60% saturation.Solution is stirred at 4 DEG C 1 hour, be then centrifuged 30 minutes at 8,000 xg.Make gained Precipitate is resuspended in 20mM Tris (pH 8.0, buffer A).Ammonium sulfate is added in final solution and to ultimate density is 1M；Then said preparation is loaded into the 40-mL through 20mM Tris (pH 8.0), 1M ammonium sulfate (buffer B) pre-equilibration HiPrepTMPhenyl-FF post.After washing, with 75%, 50% and 0% buffer B and H2O respectively carried out with three column volumes by Step eluting.All fraction are analyzed using SDS-PAGE and determination of activity.Merge and comprise target protein (SEQ ID NO： 20、SEQ ID NO：24 or SEQ ID NO：26) fraction, concentrate and followed by through 20mM sodium phosphate (pH7.0), The HiLoad of 0.15M NaCl pre-equilibrationTM26/60 SuperdexTM75 posts.Then the outflow fraction of target protein will be comprised Merge and use 10K AMICON ULTRA-15 equipment to concentrate, and be stored in 40% glycerol at -20 DEG C until using.
BpsGlu1 and BthGlu1
With two step purification BpsGlu1 (SEQ ID NO：30) and BthGlu1 (SEQ ID NO：32) alpha-Glucosidase Both.For each purification, concentrate the thick hair zymotic fluid of 1-L DASGIP fermentation tank, hereafter add ammonium sulfate to 60% saturation Degree.Solution is stirred at 4 DEG C 1 hour, be then centrifuged 30 minutes at 8,000 xg.Gained sediment is made to be resuspended in 20mM In Tris (pH 8.0, buffer A).By ammonium sulfate be added in final solution to ultimate density be 1M；Then said preparation is filled It is downloaded to the 40-mL HiPrep through 20mM Tris (pH 8.0), 1M ammonium sulfate (buffer B) pre-equilibrationTMPhenyl-FF post.Washing After washing, with 75%, 50% and 0% buffer B and H2O respectively carries out progressively eluting with three column volumes.All fraction adopt SDS-PAGE and determination of activity are analyzed.Target protein (SEQ ID NO：30 or SEQ ID NO：32) it is present in 0% to delay Rush in the eluate of liquid B elution step；Merge this eluate and concentrated with 10K AMICON ULTRA-15 equipment.At -20 DEG C Under, the final product that purity is more than 95% is stored in 40% glycerol until using.
BbrGlu2 and BbrGlu5
With four step purification BbrGlu2 (SEQ ID NO：36) and BbrGlu5 (SEQ ID NO：38) alpha-Glucosidase Both.For each purification, concentrate the thick hair zymotic fluid of 1-L DASGIP fermentation tank, hereafter add ammonium sulfate to 60% saturation Degree.Solution is stirred at 4 DEG C 1 hour, be then centrifuged 30 minutes at 8,000 xg.Gained sediment is made to be resuspended in 20mM In HEPES (pH 7.0, buffer A).By ammonium sulfate be added in final solution to ultimate density be 1M；Then said preparation is filled It is downloaded to the HiPrep through 20mM HEPES (pH 7.0), 1M ammonium sulfate pre-equilibrationTMOn phenyl-FF post.With 0.5M ammonium sulfate from post Elute target protein (SEQ ID NO：36 or SEQ ID NO：38).Merge corresponding fraction, 200 surpassed with VIVAFLOW Filter equipment (Sartorius Stedim) so that it is concentrated and buffered liquid exchanges in buffer A.By resulting solution put on through The HiPrep of buffer A pre-equilibrationTMQ FF16/10 post.With the buffer A of the 0-0.5 M NaCl containing linear gradient from post Elute target protein.The fraction comprising target protein is merged, concentrates and followed by through 20mM HEPES (pH7.0), the HiLoad of 0.15M NaCl pre-equilibrationTM26/60SuperdexTMOn 75 posts.Then target protein will be comprised Fraction merges and uses 10K AMICON ULTRA-15 equipment to concentrate, and is stored in 40% glycerol at -20 DEG C until using.
Therefore, expression the various other alpha-Glucosidase of purification.Test these alpha-Glucosidases to α -1,5 glucose Base-Fructose key and α -1,3 and/or α -1, the hydrolysing activity of 6 glucityls-glucose key (examples provided below 11,12, In 15 and 16).
Embodiment 11
The test hydrolysing activity to various glycosidic bonds for the alpha-Glucosidase
This embodiment disclose test alpha-Glucosidase and whether have that to surmount previous this fermentoid (EC3.2.1.20) related Hydrolysing activity.The alpha-Glucosidase of embodiment 10 illustrates to α -1,5 glucityls-Fructose key and α -1,3 and α -1,6 glucityls - Glucose key has hydrolysing activity.
The substrate specificity of alpha-Glucosidase
(dextrinose, maltose, panose, bright from specific substrates based on the glucose when substrate is incubated with alpha-Glucosidase Beading bacterium disaccharide or 3-O-alpha-D-Glucopyranosyl-D-glucose) release, the substrate specificity of analysis embodiment 10 each alpha-Glucosidase disclosed.Glucose Rate of release is using coupling glucose oxidase/peroxidase (GOX/HRP) method (1980, Anal.Biochem.105： 389-397) it is measured.Glucose discharges quantification of coupling GOX/HRP enzyme and peroxide pair produced by glucose response 2, the speed that 2 '-azine group-two 3- ethyl benzo thiazole phenanthroline -6- sulfonic acid (ABTS) is aoxidized.
By making the substrate solution (1%, w/v in water) of 9mL and 0.5M (pH 5.0) sodium acetate buffer of 1mL and 40 μ L 0.5M calcium chloride be mixed with each substrate solution in 15-mL conical pipe.In 50mM sodium acetate buffer (pH 5.0) Preparation has conjugate enzyme (GOX/HRP) solution of ABTS, and ultimate density is 2.74mg/mL ABTS, 0.1U/mL HRP and 1U/mL GOX.A series of diluents of each alpha-Glucosidase sample and Glucose standards are made in MILLIQ water.For aspergillus niger Sugar, to test alpha-Glucosidase sample only with a kind of dosage 10ppm, and reason is that the stock solution of substrate solution is limited.By each α- Glucosidase sample (10 μ L) transfer to comprise 90 μ L under 50 DEG C and 600rpm the precincubation substrate solution of 5 minutes newly micro- In amount titer plate (Corning 3641).Under shake (600rpm), make reaction in THERMOMIXER at 50 DEG C (Eppendorf) carry out in 10 minutes (for dextrinose, maltose, panose and 3-O-alpha-D-Glucopyranosyl-D-glucose substrates) or 60 minutes (for Lucrose substrate).Then respectively by the standard glucose being serially diluted of each reactant mixture of 10 μ L and 10 μ L It is quickly transferred in new microtitration plate (Corning 3641), then correspondingly add the ABTS/GOX/HRP of 90 μ L thereto Solution.It was spaced in quick mensure at 405nm using SOFTMAX PRO plate reading machine (Molecular Devices) with 11 seconds to comprise The microtitration plate of reactant mixture 5 minutes.For each enzyme concentration, it is output as reaction rate Vo.Surveyed using linear regression Determine curve VoSlope with enzyme dosage.Calculate the specific activity of each alpha-Glucosidase using formula 1 based on glucose standard curve：
Specific activity (unit/mg)=slope (enzyme)/slope (standard) × 1000 (1),
Wherein 1 unit=1 μm ol glucose/minute.
For 3-O-alpha-D-Glucopyranosyl-D-glucose, under the enzyme dosage of 10ppm, reaction rate value is directly used in indicative of enzyme activity.
Using preceding method, measure the specificity to each substrate for each alpha-Glucosidase.Also measured were widow -1,6- glucose Glycosides enzyme (purchased from Megazyme, referring to table 4) and transglucosidase (TG L-2000, referring to the table 4) activity to each substrate.Should The result of analysis is provided in table 17.
Table 17
The activity to different substrates for the various alpha-Glucosidases
A, for 3-O-alpha-D-Glucopyranosyl-D-glucose, uses each enzyme with a kind of dosage (10ppm).
Interestingly it was found that alpha-Glucosidase is except showing to α-Isosorbide-5-Nitrae glucityl-glucose key (maltose) Hydrolysing activity outside, also show to α -1,6 glucityls-glucose key (dextrinose), α -1,3 glucityls-glucose key (3-O-alpha-D-Glucopyranosyl-D-glucose) and α -1, the hydrolysing activity (table 17) of 5 glucityls-Fructose key (lucrose).
Therefore, alpha-Glucosidase has the hydrolysing activity surmounting previous EC 3.2.1.20 enzyme correlation.Specifically, α-glucose Glycosides enzyme to α -1,5 glucityls-Fructose key and α -1,3 and α -1,6 glucityls-glucose key has hydrolysing activity.
Embodiment 12
With lucrose and oligosaccharide in alpha-Glucosidase hydroglucan reaction filtrate
This embodiment describes and be present in available from bright in the filtrate of glucosan synthetic reaction using alpha-Glucosidase hydrolysis Beading bacterium disaccharide and other oligosaccharide.Specifically, have studied alpha-Glucosidase disclosed in embodiment 10 (poly- to insoluble glucan α -1,3- glucosan) synthetic reaction filtrate in lucrose and oligosaccharide DP2, DP3 and HS (high sugar, DP4+) hydrolysis Impact.
Point analysis of variance of the oligosaccharide for being tested to alpha-glucosidase activity
Prepare the concentration filtrate of glucosan synthetic reaction first according to embodiment 1.
In brief, sugared from concentrating filtrate separating oligomeric by chromatographic isolation, and analyze glycosidic bond key feature.Using strong acid Property cation exchange resin chromatographic isolation be used for separate concentrate filtrate oligosaccharide fraction.Physics ginseng for this detached post Number is as follows：FINEX CS11GC, #227 resin；Na+Ionic speciess；5% divinylbenzene (crosslinked)；0.34mm granularity；1.64m Bed length；0.093m column diameter.
More specifically, making the sugar juice (concentrate filtrate) concentrating described in table 3 filter and to be diluted to 25g with tap water solid Body/100g solution.Before adding this sugar juice to post resin, with sodium chloride solution (10 weights of six bed volumes (BV) Three BV, three BV of 5 weight % sodium chloride afterwards of amount % sodium chloride) wash this resin to convert the resin to na form. Then sugar juice (0.6L) is fed to post, the water elution post being 50L/ hour with flow velocity thereafter.The service condition of chromatographic isolation It is summarized as follows：0.6L feed volume (size), 25g dry solid/100g solution, 65 DEG C of column temperatures, 50mL/min flow velocity.11 to 21 Oligosaccharide solution is eluted between minute.A small amount of salt instruction conductivity increase is eluted out simultaneously.Divided by HPLC Analyse the oligosaccharide fraction thus prepared to determine its products distribution.In a word, what fraction comprised ＞ 89% contains three or more The oligosaccharide of hexose and the discernible monosaccharide less than 1.5% and disaccharide.Using membrane evaporator (LCI Corporation, Charlotte, NC) this fraction is concentrated into the gross dry weight of 317g/L, use ROTAVAPOR (R-151 afterwards； Buchi, New Castle, DE) carry out rotary evaporation.The products distribution concentrating fraction as HPLC is surveyed is shown in table 18.
Table 18
Concentrate the products distribution of oligosaccharide fraction
The alpha-Glucosidase that preliminary screening hydrolyzes to glucan oligomer
It is respectively directed to a kind of activity of difference alpha-Glucosidase of purification oligosaccharide fraction (table 18) assessment ten made above (embodiment 10) and two kinds of benchmark enzymes are widow -1,6- glucosidase (purchased from Megazyme) and transglucosidase (TG L- 2000) activity.At pH 5.0 and 60 DEG C, in comprising oligosaccharide substrate (2.9% dry solid) and the solution of 2mM calcium chloride Incubate each alpha-Glucosidase (dosage is 1mg/mL).Incubating 24 hours afterwards, will be each by adding the 0.5M NaOH of 50 μ L Individual reaction is quenched.
Measure through the oligosaccharide/monosaccharide content of reaction is quenched as follows.With water by the diluted sample 5 of each reaction again for HPLC analyzes.Using the serial HPLC of Agilent 1200 with AMINEX HPX-42A post (300mm × 7.8mm) at 85 DEG C System carries out HPLC and separates.Sample (10mL) is put on HPLC column and adopts constant gradient MILLI-Q under 0.6mL/min flow velocity Water carries out separating as mobile phase.Detect oligosaccharide product using RI-detector.Be provided in the following table 19 numeral reflection Each DPnAverage peak area percentage ratio (obtained by each sample, duplicate) as the sum of DPI to DP7 a part.
Table 19
Analysis with alpha-Glucosidase process after glucosan filtrate oligosaccharide
As used represented by shade in table 19, compared to the control reaction (" blank ") that there is not enzyme, reaction oligomeric Sugared content is generally partial to the sugar of smaller particle size.These results are pointed out, alpha-Glucosidase can be used for hydrolyzing anti-in glucosan synthesis Should interior contained oligosaccharide and its fraction.In addition, the key feature (embodiment 3 and 4) based on oligosaccharide and alpha-Glucosidase pair The activity of the various glycosidic bonds in addition to α-Isosorbide-5-Nitrae key (embodiment 11) is it is obvious that alpha-Glucosidase can be used for decomposing tool There is α -1,5 glucityls-Fructose key and/or α -1,3 and α -1, the oligosaccharide of 6 glucityls-glucose key.The result that table 19 provides is also Show, compared to antibacterial alpha-Glucosidase, funguses alpha-Glucosidase has more preferable hydrolysing activity to soluble oligosaccharide.
The confirmation of the hydrolysing activity of the oligosaccharide product to glucosan synthetic reaction for the alpha-Glucosidase
Carry out such a reaction, it includes one or two alpha-Glucosidases and available from poly- α -1, and 3- glucosan synthesizes The concentration filtrate (table 3) of reaction.The dosage of alpha-Glucosidase reaction is the enzyme of 4ppm, or for blend, each enzyme is with 1：1 Ratio use, final dose be 4ppm.It is loaded into concentrating filtrate in each reaction with 10% dry solid.Each reaction is also wrapped Calcium chloride containing 2mM (pH 5.0), and carry out at 60 DEG C or 65 DEG C.Incubating 23 hours afterwards, by adding the 0.5M of 50 μ L NaOH is quenched reaction.
Measure through the oligosaccharide/monosaccharide content of reaction is quenched as follows.The diluted sample 25 each being reacted with water again with For HPLC analysis.Using Agilent 1200 series with AMINEX HPX-42A post (300mm × 7.8mm) at 85 DEG C HPLC system carries out HPLC and separates.Sample (10mL) is put on HPLC column and adopts constant gradient under 0.6mL/min flow velocity MILLI-Q water carries out separating as mobile phase.Detect oligosaccharide product using RI-detector.Be provided in the following table 20 number Word reflects each DPnPeak area average percent (from duplicate each sample) as sum a part.Table 20 is carried For result generally confirm the activity (with regard to being provided in the result of table 19) of specific alpha-Glucosidase as discussed above.
Therefore, alpha-Glucosidase can be used for hydrolysis and is present in available from for example poly- α -1 of glucosan synthetic reaction, and 3- glucosan closes Become the lucrose in the fraction (such as filtrate) of reaction and other oligosaccharide.
Embodiment 13
From gtf-S/MUT3325 knock back thing, separation of oligomeric/lucrose pole is divided
It is 9.5L that sucrose (4.50kg) is dissolved in distilled deionized water to final total volume, and under agitation, Resulting solution is heated 5 minutes and is subsequently cooled to 47 DEG C at 80 DEG C.Under agitation, add the gtf-S enzyme comprising 0.6g/L (GTF0459, SEQ ID NO：42) 500 grams of crude extract and comprise 10g/L mutant enzyme (MUT3325, SEQ ID NO：47) 15.0mL crude extract (conventional method referring to preparing for enzyme).Under agitation, by being slowly added 37 weight %HCl 1: 10 (v/v) diluent adjusts the pH of the mixture of gained immediately 6.0 to pH 5.5 to pH.Make reaction temperature and pH respectively It is maintained at 47 DEG C and pH 5.5-6.0, until Sucrose conversion ＞ 95% is analyzed according to HPLC, hereafter by reactant mixture immediately Adjust and to 7.5 and be heated to 90 DEG C 20 minutes to pH 7.0, be subsequently cooled to 25 DEG C to remove granule and precipitation to filter immediately. Using following resin and condition, before IEX/SEC column chromatography, gained filtrate is maintained at 5 DEG C：FINEX CS 11GC SAC (Ca2+Form), post i.d=9.3cm, post bed height 1.58m, T=70 DEG C, flow velocity=51mL/min, linear flow rate=0.44m/ H, feed volume=0.6L=171g, feed RI-DS=25.1g/100g, sampling interval=3min.It is incorporated in 30min extremely The post fraction collected between 67min, the solid being 66% dissolving by evaporation and concentration and passing through as described in General Method HPLC is analyzed.Table 21 illustrates the oligosaccharide of separation fraction thus prepared and monosaccharide component.
Table 21
Derive from the analysis of the oligomer/lucrose fraction of gtf-S/MUT3325 reaction
In this embodiment, glucosan synthetic reaction is used for producing at least one solubility alpha-glucanses product.This is solvable Property product is by glucosyltransferase (GTF0459, the SEQ ID NO being present in glucosylation enzyme reaction：42) gather with α-Portugal Glycosylhydrolase (MUT3325, SEQ ID NO：47) both synergism produce.This embodiment also illustrates that anti-by glucosan synthesis The chromatograph fraction that should prepare.This fraction thus be accordingly used in example 1 below 5 and 16, to test the activity of alpha-Glucosidase.
Embodiment 14
Separation of oligomeric/lucrose fraction from Gtf-C reactant
It is 9.5L that sucrose (4.50kg) is dissolved in distilled deionized water to final total volume, and under agitation, Resulting solution is heated 5 minutes and is subsequently cooled to 47 DEG C at 80 DEG C.Under agitation, add the gtf-C enzyme comprising 0.41g/L (GTF0088BsT1, SEQ ID NO：45) 500 grams of crude extract (conventional method referring to preparing for enzyme).Under agitation, By be slowly added 1: 10 (v/v) diluents of 37 weight %HCl by the pH of the mixture of gained adjust immediately to pH 5.5 to pH 6.0.Reaction temperature and pH is made to be maintained at 47 DEG C and pH 5.5-6.0 respectively, until analyzing Sucrose conversion ＞ according to HPLC 95%, hereafter reactant mixture is adjusted immediately and to 7.5 and is heated to 90 DEG C 20 minutes to pH 7.0, be subsequently cooled to 25 DEG C with Filter immediately to remove granule and precipitation.Using following resin and condition, before IEX/SEC column chromatography, gained filtrate is kept At 5 DEG C：FINEX CS 11GC SAC(Ca2+Form), post i.d=9.3cm, post bed height 1.58m, T=70 DEG C, flow velocity= 50mL/min, linear flow rate=0.44m/h, feed volume=0.6L=171g, feed RI-DS=25.8g/100g, between sampling Every=3min.Be incorporated between 34min to 72min collect post fraction, the solid being 67% dissolving by evaporation and concentration and It is analyzed by HPLC as described in General Method.Table 22 illustrates the oligosaccharide of separation fraction thus prepared and monosaccharide group Point.
Table 22
Derive from the analysis of the oligomer/lucrose fraction of Gtf-C reaction
In this embodiment, glucosan synthetic reaction is used for producing at least one solubility alpha-glucanses product.This enforcement Example also illustrates that by the chromatograph fraction of the glucosan synthetic reaction preparation producing solubility alpha-glucanses product.This fraction thus be accordingly used in Example 1 below 5 and 16, to test the activity of alpha-Glucosidase.
Embodiment 15
With deriving from the oligomer/lucrose fraction preliminary screening α-glucose of Gtf-S/MUT3325 and Gtf-C reaction Glycosides enzyme
This embodiment describes and be present in available from the Portugal producing solubility alpha-glucanses product using alpha-Glucosidase hydrolysis Lucrose in the chromatograph fraction of polysaccharide synthetic reaction and other oligosaccharide.Specifically, to α disclosed in embodiment 10- Glucosidase is carried out for the impact of the hydrolysis of lucrose and oligosaccharide in the fraction prepared by embodiment 13 and 14 Research.
With deriving from oligomer/leukonid two that gtf-S/MUT3325 (embodiment 13) and gtf-C (embodiment 14) reacts Sugared fraction to screen 12 kinds of alpha-Glucosidases and two kinds of benchmark enzymes (widow -1,6- glucosidase and TG altogether as substrate materials L-2000 transglucosidase).All enzyme (alpha-Glucosidase and benchmark enzyme) consumptions are identical protein concentration.In pH 5.5 At 47 DEG C, in comprising oligomer/lucrose substrate (10% dry solid) and the solution of 2mM calcium chloride, incubate each Alpha-Glucosidase (dosage is 100ppm).Incubating 21 hours afterwards, by adding the 0.5M NaOH of 50 μ L, each reaction is being quenched Go out.
Measure through the oligosaccharide/monosaccharide content of reaction is quenched as follows.Make each react sample centrifugation and with water by its In supernatant dilute 25 times for HPLC analyze (conventional method).The percentage ratio being recorded in table 23 reflects each DPnFlat All peak area percent (being obtained by analysis each sample, duplicate) is as a part for sum.Result shows, when with bacterial alpha- When glucosidase compares, funguses alpha-Glucosidase has more preferable hydrolysing activity to glucan oligomer.
As used represented by shade in table 23, compared to the control reaction (" blank ") that there is not enzyme, reaction oligomeric Sugared content is generally partial to the sugar of smaller particle size.These results are pointed out, alpha-Glucosidase can be used for hydrolysis and is included in glucosan conjunction Become the oligosaccharide in reaction and its fraction, particularly produce the chromatographic grade of the glucosan synthetic reaction of solubility alpha-glucanses product Point.In addition, key feature (embodiment 13 and 14) based on oligosaccharide and alpha-Glucosidase to except α-Isosorbide-5-Nitrae key (embodiment 11) it The activity of outer various glycosidic bonds has α -1 it is obvious that alpha-Glucosidase can be used for decomposition, 5 glucityls-Fructose key It is likely to α -1,3 and α -1, the oligosaccharide of 6 glucityls-glucose key.The result that table 23 provides is it is also shown that compared to antibacterial Alpha-Glucosidase, funguses alpha-Glucosidase has more preferable hydrolysing activity to soluble oligosaccharide.
Therefore, alpha-Glucosidase can be used for hydrolysis be present in available from glucosan synthetic reaction such as synthesizing soluble α-Portugal gather Lucrose in the fraction (such as chromatograph fraction) of the reaction of sugared product and other oligosaccharide.
Embodiment 16
With deriving from the oligomer/lucrose level separating sieve alpha-Glucosidase of Gtf-S/MUT3325 and Gtf-C reaction
This embodiment, according to embodiment 15, describes to be present in available from generation solubility α-Portugal using alpha-Glucosidase hydrolysis Lucrose in the chromatograph fraction of the glucosan synthetic reaction of polysaccharide product and other oligosaccharide.
The sugar composite assessment of reaction leading to the enzyme comprising same protein concentration consumption by analysis is for deriving fromOligomer/lucrose the fraction of gtf-S/MUT3325 and gtf-C reactionThe α of (embodiment 15) most hydrolysing activity-Portugal Glycosidase.Respectively at 60 DEG C and 65 DEG C, in the presence of 2mM calcium chloride, under pH 5.5, to alpha-Glucosidase, (consumption is 4ppm；For blend, the ratio of two kinds of enzymes is 1: 1 and total consumption is 4ppm) and oligomer/lucrose substrate (10%ds) incubated.Incubating 23 hours afterwards, reaction is being quenched by adding the 0.5mM NaOH of 50ul.
Measure through the oligosaccharide/monosaccharide content of reaction is quenched as follows.The sample deriving from each reaction is carried out being centrifuged simultaneously With water, supernatant therein is diluted 25 times and analyze (conventional method) for HPLC.The percentage ratio being recorded in table 24 (hereafter) is anti- Reflect each DPnAverage peak area percentage ratio (being obtained by analysis each sample, duplicate) as a total part.Result table Bright when being incubated at 65 DEG C, TauSec098 is effective for hydrolysis DP2 to DP7 oligomer, and TauSec099 Hydrolysis for lucrose is better than TG L-2000.The blend of TauSec098 and TauSec099 (or TG L-2000) Hydrolyzed oligomers and lucrose are producing DP1 effectively.
Therefore, alpha-Glucosidase can be used for hydrolysis be present in available from glucosan synthetic reaction such as synthesizing soluble α-Portugal gather Lucrose in the fraction (such as chromatograph fraction) of the reaction of sugared product and other oligosaccharide.

Claims (15)

1. one kind makes to comprise at least one α -1, α -1 in the sugar of 5 glucityls-Fructose key, the side of 5 glucityls-Fructose key hydrolysis Method, wherein said sugar is disaccharide or oligosaccharide, and wherein said method includes：
Described sugar is made to contact under suitable conditions with alpha-Glucosidase, wherein said alpha-Glucosidase hydrolyzes described sugar at least One α -1,5 glucityls-Fructose key,
And the amount of wherein said sugar reduces compared to the amount of the sugar existing before described contact.
2. method according to claim 1, wherein said alpha-Glucosidase is fixing.
3. method according to claim 1, wherein said sugar is lucrose.
4. method according to claim 3, the concentration of the wherein lucrose after described contact procedure is less than The 50% of the concentration of the lucrose existing before described contact.
5. method according to claim 1, wherein said suitable condition includes：
(i) glucosan synthetic reaction, or
(ii) fraction obtaining from described glucosan synthetic reaction；
Wherein said sugar is the by-product of described glucosan synthetic reaction.
6. method according to claim 5, wherein said glucosan synthetic reaction produces at least one insoluble α-Portugal and gathers Sugared product.
7. method according to claim 6, wherein said level is divided into the filtrate of described glucosan synthetic reaction.
8. method according to claim 5, wherein said glucosan synthetic reaction produces at least one solubility α-Portugal and gathers Sugared product, it is：
The product of (i) glucosyltransferase, or
(ii) glucosyltransferase and the synergistic product of alpha-glucanses hydrolytic enzyme, described alpha-glucanses hydrolytic enzyme can Hydrolysis has one or more α -1,3- glycosidic bond or one or more α -1, the dextran polymer of 6- glycosidic bond.
9. method according to claim 8, wherein said level is divided into the chromatograph fraction of described glucosan synthetic reaction.
10. method according to claim 1, wherein said alpha-Glucosidase is transglucosidase or glucoamylase.
11. a kind of compositionss by making sugar contact with alpha-Glucosidase and produce,
Wherein said sugar for disaccharide or oligosaccharide and comprises at least one α -1,5 glucityls-Fructose key,
At least one α -1 of sugar described in wherein said enzyme hydrolysiss, 5 glucityls-Fructose key,
And the sugar amount that wherein said compositionss comprise reduces compared to the sugar amount existing before described contact.
12. compositionss according to claim 11, wherein said sugar is lucrose.
13. compositionss according to claim 11, wherein said sugar is in (i) glucosan synthetic reaction, or (ii) is from described In the fraction that glucosan synthetic reaction obtains；
Wherein said sugar is the by-product of described glucosan synthetic reaction.
14. a kind of method that enrichment is present in Fructose in the fraction of glucosan synthetic reaction, methods described includes：
A () makes the fraction obtaining from glucosan synthetic reaction contact under suitable conditions with alpha-Glucosidase, and wherein said α- Glucosidase hydrolyzes at least one α -1 of contained disaccharide or oligosaccharide in described fraction, 5 glucityls-Fructose key；And
B () separating levulose from step (a) is through hydrolysis fraction is dense with the Fructose that obtains the fraction that fructose concentration is than step (a) The compositionss of Du Genggao.
A kind of 15. fermentation process, methods described includes：
A () makes the fraction obtaining from glucosan synthetic reaction contact under suitable conditions with alpha-Glucosidase, and wherein said α- Glucosidase hydrolyzes at least one α -1 of contained disaccharide or oligosaccharide in described fraction, 5 glucityls-Fructose key；
B (), with the fraction of microbial fermentation step (a) to obtain product, wherein said fermentation is after step (a) or and step A () is carried out simultaneously；And
C () optionally, separates the product of (b)；
Wherein receive compared to the product that the fraction of the glucosan synthetic reaction not contacted with described alpha-Glucosidase is fermented Rate, the product yield of (b) increases.
CN201580010439.5A 2014-02-27 2015-02-26 Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes Pending CN106460023A (en)

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