CN106068327A - α glucosidase is used to come enzyme hydrolysis disaccharides and compound sugar - Google Patents

α glucosidase is used to come enzyme hydrolysis disaccharides and compound sugar Download PDF

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Publication number
CN106068327A
CN106068327A CN201580010441.2A CN201580010441A CN106068327A CN 106068327 A CN106068327 A CN 106068327A CN 201580010441 A CN201580010441 A CN 201580010441A CN 106068327 A CN106068327 A CN 106068327A
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sugar
alpha
glucosidase
glucan
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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    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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Abstract

The invention discloses a kind of method of α 1,3 or α 1,6 glucobiose key for hydrolysis sugar (disaccharides or compound sugar).The method includes making sugar contact under suitable conditions with α glucosidase such as transglucosidase, during this contact procedure, and at least one α 1,3 or α 1,6 glucobiose key of enzyme hydrolysis sugar.The method can be used for for example reducing the amount of the compound sugar in the filtrate being isolatable from glucan synthetic reaction.

Description

Alpha-Glucosidase is used to come enzyme hydrolysis disaccharides and compound sugar
This application claims U.S. Provisional Application 61/945,233 (submission on February 27th, 2014), 61/945,241 (2014 February 27 submit to), 62/004,290 (submission on May 29th, 2014), 62/004,308 (submission on May 29th, 2014), 62/ 004,312 (submission on May 29th, 2014), 62/004,300 (submission on May 29th, 2014), 62/004,314 (in May, 2014 Within 29th, submit to) and 62/004, the rights and interests of 305 (submissions on May 29th, 2014), 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 hydrolysis field of less glycopolymers.In particular it relates to use alpha-Glucosidase water Solution comprises one or more α-1,3 or α-1, the disaccharides of 6 glucityls-glucose key and compound sugar.
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 CL6220USNP_SequenceListing_ST25.txt of part, date created is on February 11st, 2015, file size It is 266 kilobytes, and this document is submitted to this specification simultaneously.The sequence table comprising in the file of this ASCII fromat For the part of described specification and be incorporated by reference in its entirety herein.
Background technology
Transglucosidase (EC.2.4.1.24, Isosorbide-5-Nitrae-alpha-glucans 6-alpha-glucosyl transferase) 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 to carry out turning the most preferred substrate that glucosylation reacts with this enzyme.Transfer Occur most frequently in HO-6, thus D-Glucose is produced isomaltose, or maltose is produced panose (6-O-alpha-glucosyl Fructus Hordei Germinatus 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 nigerose.This enzyme can make D-glucosyl units be transferred back to HO-4 further, thus improves maltose.
Use transglucosidase owing to turning glucosylation reaction, so Fructus Hordei Germinatus-oligosaccharide residue is converted into comprises higher ratio Different Fructus Hordei Germinatus-the compound sugar (IMO) of the glucosyl residue being connected by the α-D-1 of non-reducing end, 6 glycosidic bonds of example.IMO sugar is in Asia For numerous food and drink formula.Brier et al. (U.S. Patent Application Publication 2003/0167929) discloses employing and turns Portugal Glycosidase is produced IMO by barley malt juice.
Poulose et al. (U.S. Patent Application Publication 2008/0229514) discloses with transglucosidase degradation of polysaccharide all Such as xanthans and guar gum.Xanthans includes cellulosic backbone, wherein selects glucose 1 else, and 3-is connected to comprise mannose and Portugal On the side chain of uronic 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 marine alga ketone by transglucosidase Sugar (trehalulose).These sugar include via 1,2-(sucrose), 1, and 3-(melezitose) or 1,1-(trehalulose) are bonded and are connected to The glucose of fructose.
Although the various hydrolysing activities of transglucosidase having been disclosed, but this fermentoid being generally viewed as alpha-Glucosidase, Give the ability of α-key between its two glucosyl residue of hydrolysis.For example, transglucosidase (hydrolyzes with having maltase activity α-Isosorbide-5-Nitrae glycosidic bond between two glucosyl residue of maltose) it is associated, which 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 hydrolyzable glucityl-glucose, 3 and α-1,6 glycosidic bonds.Alpha-Glucosidase is disclosed herein for degrading Comprise glucityl-α-1,3-glucose and glucityl-α-1, the disaccharides of 6-glucose and compound sugar.
Content of the invention
In one embodiment, the present invention relates to one makes to comprise at least one α-1,3 or α-1,6 glucityls-grape α-1 of the sugar of sugar key, 3 or α-1, the method for 6 glucityls-glucose key hydrolysis, wherein said sugar is disaccharides or compound sugar, and Wherein the method includes: making sugar contact under suitable conditions with alpha-Glucosidase, wherein alpha-Glucosidase hydrolysis sugar is at least One α-1,3 or α-1,6 glucityls-glucose key, and wherein sugared amount are compared to the amount of the sugar existing before contact procedure Reduce.
In another embodiment, the alpha-Glucosidase of method for hydrolysis is fixing.
In another embodiment, the sugar of method for hydrolysis has the degree of polymerization of 3 to 7 before hydrolysis.Real at another Executing in scheme, the concentration of sugar is less than the 50% of the concentration of the sugar existing before contact procedure after the contacting step.
In another embodiment, the suitable condition of method for hydrolysis includes: (i) glucan synthetic reaction, or (ii) The fraction being obtained by glucan synthetic reaction；The accessory substance that wherein sugar is glucan synthetic reaction.In another embodiment, Glucan synthetic reaction produces at least one insoluble alpha-glucans product.In another embodiment, level is divided into glucan The filtrate of synthetic reaction.In another embodiment, glucan synthetic reaction produces at least one solubility alpha-glucans product Thing, which is: the product of (i) glucosyltransferase, or (ii) glucosyltransferase and alpha-glucans hydrolase synergy Product, this alpha-glucans hydrolase 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 another embodiment, level is divided into the chromatographic isolation fraction of glucan synthetic reaction, its Middle glucan synthetic reaction produces at least one solubility alpha-glucans product.
In another embodiment, alpha-Glucosidase is transglucosidase.In another embodiment, glucoside is turned Enzyme comprises the amino acid sequence with SEQ ID NO:1 with at least 90% homogeneity.
In another embodiment, the present invention relates to a kind of combination being produced by making sugar contact with alpha-Glucosidase Thing, wherein sugar is disaccharides or compound sugar and comprises at least one α-1,3 or α-1,6 glucityls-glucose key, wherein α-glucose At least one α-1 of glycosides enzyme hydrolysis sugar, 3 or α-1,6 glucityls-glucose key, and the sugar amount that wherein composition comprises are compared The sugar amount existing before contact procedure reduces.
In another embodiment, the sugar of composition has the degree of polymerization of 3 to 7 before hydrolysis.For example, contact procedure The concentration of sugar is less than the 50% of the concentration of the sugar existing before contact procedure afterwards.
In another embodiment, the sugar of composition is in (i) glucan synthetic reaction, or (ii) is synthesized by glucan In the fraction that reaction obtains；The accessory substance that wherein sugar is glucan synthetic reaction.In another embodiment, level is divided into Portugal to gather The filtrate of sugar synthetic reaction or the chromatographic isolation fraction of glucan synthetic reaction.
In another embodiment, the present invention relates to a kind of enrichment and be present in fructose in the fraction of glucan synthetic reaction Method, the method includes: (a) makes to connect under suitable conditions with alpha-Glucosidase available from the fraction of glucan synthetic reaction Touch, wherein at least one α-1 of contained disaccharides or compound sugar in alpha-Glucosidase hydrolysis fraction, 3 or α-1,6 glucityls-Portugal Grape sugar key；And (b) from step (a) through hydrolysis fraction separating levulose, to obtain the fraction than step (a) for the fructose concentration The high composition of fructose concentration.
In another embodiment, the present invention relates to a kind of fermentation process, described method includes: (a) makes to gather available from Portugal The fraction of sugar synthetic reaction contacts under suitable conditions with alpha-Glucosidase, wherein contained in alpha-Glucosidase hydrolysis fraction At least one α-1 of disaccharides or compound sugar, 3 or α-1,6 glucityls-glucose key；B () uses the level of microbial fermentation step (a) Point to produce product, wherein ferment can be after step (a) or carry out with step (a) simultaneously；And (c) is optionally, separate (b) Product；Wherein, compared to not compared with the product yield that the fraction of glucan synthetic reaction of enzyme contact is fermented, B the product yield of () increases.
Accompanying drawing and BRIEF DESCRIPTION OF THE SEQUENCES
Fig. 1: (raw material) and the filter of (treated material) glucan reaction afterwards before processing with NOVO 188 enzyme hydrolysis Liquid material1H NMR spectra (sees embodiment 2-3).
Fig. 2: before by TG L-2000 transglucosidase hydrolysis process (raw material) and afterwards (treated material) Portugal gather Sugar reaction filtrate material1H NMR spectra (sees embodiment 2-3).
Table 1 nucleic acid and the general introduction of protein sequence identification number
Detailed description of the invention
The disclosure of the patent of all references and non-patent literature is incorporated by reference in its entirety herein.
As used herein, term " invent " or " disclosed in this invention " be not intended to limit 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 Disaccharides or compound sugar." disaccharides " refers to the carbohydrate with two monose being connected by glycosidic bond herein." oligomeric herein Sugar " refers to by the carbohydrate for example consisting of 2 to 9 monose that glycosidic bond connects.Herein, compound sugar also can claim For " oligomer ".Monose contained in disaccharides or compound sugar can be described as such as " monosaccharide unit " or " monomeric unit ".Excellent herein The monose 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.
Term " α-1,3 glucityls-glucose key ", " α-1,3 glucose-glucose keys " and " glucose-α-1,3-herein Glucose " refers to α-1 between two alpha-D-glucose molecules, 3-glycosidic bond.Term " α-1,6 glucityls-glucose herein 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 herein, 3 glucityls-glucose key and/or α-1,6 Portugals Glycosyl-glucose key is included in disaccharides or compound sugar.
Term " α-1,5 glucityls-fructose key ", " α-1,5 Glucose-Fructose keys " and " glucose-α-1,5-fruit herein Sugar " refers to α-1 between alpha-D-glucose molecule and fructose molecule, 5-glycosidic bond.In certain embodiments, α-1 herein, 5 Glucityl-fructose key is included in disaccharides or compound sugar.
" alpha-D-glucose " of this paper is alternatively referred to as " glucose ".
Comprising α-1, the disaccharides of 5 glucityls-fructose key is referred to herein as lucrose.Term " lucrose " " D-glycopyranosyl-α (1-5)-D-fructopyranose " exchanges herein and uses.Lucrose has a structure that
Term " alpha-Glucosidase ", " α-Isosorbide-5-Nitrae-glucosidase " and " α-D-glucoside glucohydralase " exchange herein 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 sugar (such as disaccharides) and the end of polysaccharide substrate non-reducing (Isosorbide-5-Nitrae)-connection.Now public herein The alpha-Glucosidase opened is also to α-1, and 5 glucityls-fructose key has hydrolysing activity, and to α-1,3 and α-1,6 glucityls-really Sugar key has hydrolysing activity.Transglucosidase and glucoamylase are the example of the alpha-Glucosidase with this type of activity.
Term " transglucosidase " (TG), " transglucosidase " and " Isosorbide-5-Nitrae-alpha-glucans 6-alpha-glucosyl transferase " exist Interchangeably used herein.Transglucosidase (EC 2.4.1.24) had previously been accredited as and some α-D-glucose-compound sugar one Act the D-glucosyltransferase of catalyzing hydrolysis and transfer reaction when incubating.Herein presently disclosed transglucosidase also to α- 1,5 glucityl-fructose key has hydrolysing activity, and to α-1,3 and α-1,6 glucityls-fructose key has hydrolysing activity.
Term " glucoamylase " (GA), " glucoamylase " and " α-Isosorbide-5-Nitrae-glucan glucohydralase " are herein It is used interchangeably.Glucoamylase (EC 3.2.1.3) had previously been accredited as the disaccharides containing glucose for the catalyzing hydrolysis, compound sugar With α-Isosorbide-5-Nitrae and α-1 of the non-reducing end of polysaccharide, the outer effect enzyme of both 6 glycosidic bonds.Presently disclosed glucoamylase is also herein To α-1,5 glucityls-fructose key has hydrolysing activity.
Enzyme hydrolysis is that wherein in the case of adding key element water, enzymatic enters the process of the bond fission in molecule." water herein Solve ", " hydrolysis " α-1,3 or α-1,6 glucityls-glucose key or " to α-1,3 or α-1,6 glucityls-glucose key has water Solve activity " refer to come α-1 between two glucose molecules of enzyme hydrolysis by alpha-Glucosidase such as transglucosidase, 3 or α- 1,6 glycosidic bond.This type of hydrolysis is comprising α-1,3 and/or α-1, and the disaccharides of 6 glucityls-glucose key or compound sugar are with this paper's Occur when alpha-Glucosidase contacts under suitable conditions.Therefore, " hydrolysis " at least includes herein: (i) comprise one or Multiple α-1,3 and/or α-1, the disaccharides of 6 glucityls-glucose key or compound sugar, and (ii) alpha-Glucosidase.
Term " saccharification " refers to resolve into sugar (disaccharides or compound sugar) process of its monosaccharide component herein.Can be in this Wenshui Solve and reaction makes sugar saccharification occurs.
For making to comprise at least one α-1,3 and/or α-1, the sugar (disaccharides or compound sugar) of 6 glucityls-glucose key with " the suitable condition " of the alpha-Glucosidase contact of this paper refers to one or more α-1 of alpha-Glucosidase hydrolysis sugar, and 3 And/or α-1, those conditions (such as temperature, pH, time) of 6 glucityls-glucose key.Suitable condition can include " aqueous bar Part ", for example, include at least 20 weight % water.Aqueous conditions can characterize solution or mixture.Wherein make to comprise at least one α-1,3 And/or α-1, it is anti-that the solution that the sugar of 6 glucityls-glucose key contacts with alpha-Glucosidase or mixture can be described as alpha-Glucosidase Answer (for example, transglucosidase or glucose starch enzyme reaction).
" fix " enzyme herein and refer to be connected to the enzyme of the soluble material of inertia.Such as United States Patent (USP) 5541097 discloses For the method preparing immobilised enzymes, the disclosure of which is herein incorporated by reference.
Term " glucan " and " dextran polymer " are used interchangeably herein, and refer to the Portugal connecting through glycosidic bond The polysaccharide of grape sugar monomer." alpha-glucans " refers to dextran polymer herein, and the 81%th, the 82%th, at least about the 80%th, it comprise 83%th, the 84%th, the 85%th, the 86%th, the 87%th, the 88%th, the 89%th, the 90%th, the 91%th, the 92%th, the 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98%th, the α-glycosidic bond of 99% or 100%.
" insoluble glucan " refers to the dextran polymer insoluble in aqueous conditions herein.Insoluble glucan herein One example is poly-α-1 that DP is at least 8 or 9,3-glucan.In certain embodiments, such as presently disclosed glucityl Transferase reaction produces at least one insoluble glucan product.
Term " soluble glucan ", " solubility alpha-glucans ", " Soluble Fiber ", " soluble glucan fiber ", " soluble dietary fiber " etc. used interchangeably herein, to refer to dissolve in the dextran polymer of aqueous conditions.Solvable herein Property glucan example be some compound sugar, poly-α-1 less than 8 for the such as DP, 3-glucan, and in examples provided below Disclosed in some compound sugar.In certain embodiments, as presently disclosed glucosylation enzyme reaction produces at least one Plant soluble glucan product.In certain embodiments, another stack features of solubility alpha-glucans compound herein is characterized Be: it is (i) Water-Soluble Glucose oligomer, has the degree of polymerization of 3 or bigger, (ii) digestion resistance (i.e. show pole Slowly digestible or there is no digestibility), few at people's intestinal absorption or do not absorb, and (iii) at least portion in lower gastrointestinal tract Divide fermentable.The digestibility of soluble glucan fiber composition can for example be measured by AOAC method 2009.01.
Term " poly-α-1,3-glucan " and " α-1,3-dextran polymer " are used interchangeably herein.Poly-α-1,3- Glucan is the polymer comprising the glucose monomer unit linking together through glycosidic bond, the glycosidic bond of wherein at least about 50% For α-1,3-glycosidic bond.As used herein, term " α-1,3-glycosidic bond " refers to by carbon 1 He on adjacent alpha-D-glucose ring The 3 class covalent bonds that alpha-D-glucose molecule is engaged with each other.
" molecular weight " of glucan is represented by number-average molecular weight (M hereinn) or weight average molecular weight (Mw).Or, molecular weight Be represented by dalton, gram/mol, DPw(weight average degree of polymerization) or DPn(number-average degree of polymerization).For calculating these molecular weight determinations Various methods be well known in the art, such as use high pressure lipuid chromatography (HPLC) (HPLC), SEC Or gel permeation chromatography (GPC) (SEC).
Term " glucosyltransferase ", " gtf enzyme ", " gtf enzyme catalyst ", " gtf ", " dextransucrase " etc. are herein It is used interchangeably.Gtf enzymatic activity catalysing sucrose substrate reactions is with prepared product glucan and fructose herein.Gtf reaction other Product (accessory substance) can include glucose (obtaining when from glucityl-gtf enzyme intermediate hydrolyzation of glucose), various solvable Property compound sugar (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-extreme direction is to C-extreme direction) signal peptide, varistructure Territory, catalyst structure domain and glucan-binding domain.According to CAZy (carbohydrate-active enzymes) database, glucosyltransferase herein Classify as glycoside hydrolase Families 70 (GH70) (Cantarel et al., Nucleic Acids Res.37:D233-238, 2009)。
Term " sucrose " refers to through α-1, the alpha-D-glucose molecule that 2-glycosidic bond connects and β-D-Fructose group of molecules herein The non-reducible disaccharide becoming.Sucrose is commonly referred to sugar.
Term " glucan synthetic reaction ", " glucan reaction ", " gtf reaction " etc. are used interchangeably herein, and are Refer to the reaction being carried out by glucosyltransferase.As used herein, glucan synthetic reaction is usually directed to such a solution: The solution comprising sucrose and water and other optional components comprises at least one activity glucosyltransferase.Can be at this Other components in literary composition glucan synthetic reaction include such as fructose, glucose, lucrose, soluble oligosaccharide (example Such as DP2-DP7) and one or more soluble glucan products.In addition, in some respects, glucan synthetic reaction can be wrapped Include one or more alpha-glucans hydrolases.It should be appreciated that some beta-glucan products such as degree of polymerization (DP) is at least 8 or 9 Poly-α-1,3-glucan is water-insoluble and therefore insoluble in glucan synthetic reaction, but can separate out solution.
Term " alpha-glucans hydrolase " and " glucan hydrolase " are used interchangeably herein, and refer to hydrolysis The enzyme of alpha-glucans oligomer.Alpha-glucans hydrolase can be determined by its endo hydrolysis activity to some α-D-glycosidic bond Justice.The example of alpha-glucans hydrolase includes dextranase (EC 3.2.1.11 herein；Can endo hydrolysis α-1,6-connect Glycosidic bond), mutant enzyme (EC3.2.1.59；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 the degree of branching in some alpha-glucans, branched type and opposed branch length, alpha-glucans water can be negatively affected Solve the ability of enzyme endo hydrolysis some glucoside key.
" the dry solid percentage " of glucan synthetic reaction refers to weight % of all sugar in glucan synthetic reaction.Can example As calculated the dry solid percentage of gtf reaction based on the sucrose amount for preparing product.
" fraction " of the glucan synthetic reaction of this paper refers to the liquid solution part of glucan synthetic reaction.Fraction can be Derive from part or all of liquid solution of glucan synthetic reaction, and be isolatable from the solubility or insoluble of synthesis in reaction Property beta-glucan products.In some embodiments, fraction is optional is referred to as " mother liquor ", and wherein product is insoluble (solid) Portugal Glycan product.One example of fraction is the filtrate of glucan synthetic reaction.Because fraction can comprise dissolve sugar such as sucrose, Fructose, glucose, lucrose, soluble oligosaccharide (such as DP2-DP7), so fraction can also referred to as come from glucan " sugar juice of mixing " of synthetic reaction.Refer to that the alpha-Glucosidase through this paper is processed with water herein " through the fraction of hydrolysis " Lucrose that solution is present in fraction and/or the fraction of compound sugar.
Term " filtrate ", " glucan react to filtrate ", " glucan filtrate " etc. used interchangeably herein, and refer to from The fraction filtering out in the solid beta-glucan products of synthesis in glucan synthetic reaction.Refer to " through the filtrate of hydrolysis " herein Process through alpha-Glucosidase herein to hydrolyze the filtrate of the lucrose being present in filtrate and/or compound sugar.
Term " percentage by volume ", " percent by volume ", " volume % ", " v/v% " etc. make interchangeable herein With.In solution, the percent by volume of solute can be measured by following formula: [(solute volume)/(liquor capacity)] × 00%.
Term " weight % ", " percentage by weight (weight %) ", " weight-weight percentages (w/w %) " etc. exist Used interchangeably herein.Weight % refers to material hundred when it is comprised in composition, mixture or solution in mass Proportion by subtraction.Except as otherwise noted, all percentages of this paper are all weight percentage.
As used herein, " polydispersity index ", " PDI ", " heterogeneity index ", " dispersed " etc. refer to the given of measurement Polymer (such as glucose oligomer, such as solubility alpha-glucans) sample middle-molecular-weihydroxyethyl is distributed, and can be by by weight average Molecular weight calculates (PDI=M divided by number-average molecular weightw/Mn)。
Term " increase ", " enhanced " and " improvement " is used interchangeably herein.These terms refer to bigger amount Or activity, it is such as slightly larger than the amount of primary quantity or activity or activity or compared to primary quantity or active large excess of amount or work Property, and include intervenient all amounts or activity.Or, these terms can refer to for example this amount or activity with and it compared with Amount or activity compare, height at least 1%, the 2%th, the 3%th, the 4%th, the 5%th, the 6%th, the 7%th, the 8%th, the 9%th, the 10%th, the 11%th, the 12%th, the 13%th, 14%th, the 15%th, the 16%th, the 17%th, the 18%th, 19% or 20%.
As used herein, for polynucleotides 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 is for obtaining maximum corresponding and comparison.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 comparing with reference sequences (it does not comprise to add or disappearance), the many nucleosides 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: determine the number of the position occurring identical nucleic acid base or amino acid residue in the two sequences 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.
Can use online in National Center for Biotechnology Information (NCBI) website Basic Local Alignment Search Tool (BLAST) algorithm can for example be used for measuring 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 use Clustal algorithm (such as ClustalW or ClustalV) to calculate.For Using the multiple ratio pair of Clustal comparison method, default value may correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10.With Clustal method carry out in contrast to and protein sequence percentage identities calculate default parameters 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.Or, sequence it Between homogeneity percentage EMBOSS algorithm (such as pin (needle)) can be used to carry out, parameter be such as GAP OPEN=10, GAP EXTEND=0.5, END GAP PENALTY=false, END GAP OPEN=10, END GAP EXTEND=0.5, Use BLOSUM matrix (such as BLOSUM62).
The feature as some embodiment for the multiple polypeptides amino acid sequence is disclosed herein.Can use with disclosed herein Sequence has the variant of at least about these sequences of 70%-85%, 85%-90% or 90%-95% homogeneity.Or, variant Amino acid sequence and sequence disclosed herein can have at least 70%, the 71%th, the 72%th, the 73%th, the 74%th, the 75%th, the 76%th, the 77%th, 78%th, the 79%th, the 80%th, the 81%th, the 82%th, the 83%th, the 84%th, the 85%th, the 86%th, the 87%th, the 88%th, the 89%th, the 90%th, the 91%th, the 92%th, 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98% or 99% homogeneity.Variant amino acid sequences and open sequence have herein Identical function/activity, or there is open sequence at least about the 85%th, the 86%th, the 87%th, the 88%th, the 89%th, the 90%th, the 91%th, 92%th, the 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98% or 99% function/activity.
As term " separation " used in certain embodiments refer to be kept completely separate with its natural origin any carefully Born of the same parents' component (polynucleotides for example separating or peptide molecule).In some cases, polynucleotides or the peptide molecule of separation is A part for bigger composition, buffer system or reagent mixture.For example, polynucleotides or the peptide molecule of separation can be with allos Mode is included in cell or biological interior.Another example (such as glucoamylase, turns glucoside for the alpha-Glucosidase separating Enzyme) or glucosyltransferase.Enzyme reaction disclosed herein (such as alpha-Glucosidase reaction, glucosylation enzyme reaction) is Synthesis, the process of non-naturally-occurring.
Disclosed embodiments of the present invention relate to one makes to comprise at least one α-1,3 or α-1,6 glucityls-glucose α-1 of the sugar of key, 3 or α-1, the method for 6 glucityls-glucose key hydrolysis.Sugar is disaccharides or compound sugar.The method includes making sugar Contact under suitable conditions with alpha-Glucosidase.In contact procedure, at least one α-1 of alpha-Glucosidase hydrolysis sugar, 3 or α-1,6 glucityls-glucose 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 the method being alternatively referred to as reducing sugar amount in composition.
Significantly, it is believed that alpha-Glucosidase hydrolyzable α-1,3 and α-1,6 glucityls-glucose key is previously the unknown.Root Can thus be accordingly used in from glucan synthetic reaction and/or from the fraction being obtained by it according to the alpha-Glucosidase reaction of this method for hydrolysis Remove the compound sugar accessory substance comprising these glucose-glucose keys.This type of is removed and illustrates relative to may result in beta-glucan products The accessory substance of degraded is removed chemical process (such as acid hydrolysis) and is improved to some extent.Finally, gather according to the Portugal that above method for hydrolysis is processed Sugar reaction fraction is more suitable for such as downstream application and such as ferments, because the content of glucose monosaccharide increases in fraction.Under for Swimming across journey, monose is generally more disposable compared to compound sugar accessory substance.
Alpha-Glucosidase (EC 3.2.1.20) hydrolysis sugar using in embodiments herein (comprises in these keys At least one) α-1,3 and/or α-1,6 glucityls-glucose key.Alpha-Glucosidase is previously identified for catalyzing hydrolysis Release compound sugar (such as disaccharides) and the alpha-D-glucose residue of the end of polysaccharide substrate non-reducing (Isosorbide-5-Nitrae)-connection.Existing herein At these enzymes disclosed also to such as α-1,3 and α-1,6 glucityls-glucose key has hydrolysing activity.
Alpha-Glucosidase is available from for example any source (such as plant, animal, microorganism, such as bacterium or fungi/ferment Female), transglucosidase such as disclosed below and/or glucoamylase may originate from its those sources.For example, alpha-Glucosidase The alpha-Glucosidase of fungi can be.In United States Patent (USP) the 6355467th, other examples of alpha-Glucosidase suitable herein include 5922580th, the 5795766th, those disclosed in 5763252 and 8633006, above-mentioned document is all herein incorporated by reference.
In certain embodiments, alpha-Glucosidase can comprise SEQ ID NO:5, the 6th, the 8th, the 9th, the 11st, the 12nd, the 14th, the 15th, the 17th, 18th, the 20th, the 22nd, the 24th, the 26th, the 28th, the 30th, the 32nd, the 34th, the 36th, 38 amino acid sequence, or DIAZYME RDF ULTRA (DuPont Industrial Biosciences) amino acid sequence.Or, alpha-Glucosidase can comprise and SEQ ID NO:5, the 6th, the 8th, the 9th, 11st, the amino acid sequence of the 12nd, the 14th, the 15th, the 17th, the 18th, the 20th, the 22nd, the 24th, the 26th, the 28th, the 30th, the 32nd, the 34th, the 36th, 38 or DIAZYME RDF ULTRA At least about the 90%th, row have the amino acid of the 91%th, the 92%th, the 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98% or 99% homogeneity Sequence, and to sugared α-1,3 and/or α-1,6 glucityls-glucose key has hydrolysing activity.For example several foregoing sequences are Lack the ripe alpha-Glucosidase of N-terminal signal peptide.For this type of sequence, it will be appreciated that if carrying out expressing to it not Use signal peptide (such as using the wherein expression system at cell inner expression and available from cell lysate for the enzyme), be then usually added into N- End initial methionine (if necessary) (directly or via insert heterologous amino acid sequence such as epi-position).
Transglucosidase (EC 2.4.1.24；Isosorbide-5-Nitrae-alpha-glucans 6-alpha-glucosyl transferase) can be real in some of this paper Execute and scheme is used as alpha-Glucosidase, with α-1 of hydrolysis sugar (comprising at least one in these keys), 3 and/or α-1,6 glucose Base-glucose key.This fermentoid had previously been accredited as when incubating together with some α-D-glucose-compound sugar catalyzing hydrolysis and had turned Move and react both D-glucosyltransferases.Transglucosidase as presently disclosed in this paper is also to such as α-1,3 and α-1,6 glucose Base-glucose key has hydrolysing activity.
The transglucosidase of this paper can derive from any microbial source, such as bacterium or fungi.The transglucosidase of fungi Example include but is not limited to trichoderma (Trichoderma) species (such as trichoderma reesei (T.reesei)), aspergillus (Aspergillus) species and Xin Satuo Pseudomonas (Neosartorya) species (such as Fei Shi Xin Satuo bacterium (N.fischeri)) Those.The example of the Aspergillus sp that transglucosidase may originate from it includes but is not limited to aspergillus niger (A.higger), bubble is contained Aspergillus (A.awamori), aspergillus oryzae (A.oryzae), Aspergillus terreus (A.terreus), rod aspergillus (A.clavatus), aspergillus fumigatus And aspergillus nidulans (A.nidulans) (A.fumigatus).Can be used for other examples of transglucosidase of this paper at Barker Et al. (1953, J.Chem.Soc.3588-3593)；Pazur et al. (1986, Carbohydr.Res.149:137-147), In Nakamura et al. (1997, J.Biotechnol.53:75-84) and U.S. Patent Application Publication 2008/0229514 Describing, these patents are all herein incorporated by reference.Other examples of the transglucosidase that can be used for this paper are heat-staple Those；United States Patent (USP) 4689296 discloses the method for preparing heat endurance transglucosidase, and the disclosure of which is with the side of quoting Formula is expressly incorporated herein.More examples of the transglucosidase that can be used for this paper can be in those of in GENBANK database (NCBI) Any one, the such as number of logging in: D45356 (GID:2645159, aspergillus niger), BAD06006.1 (GID:4031328, bubble contain Aspergillus), 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, rod aspergillus), XP_ 751811.1 (GID:70993928, aspergillus fumigatus), XP_659621.1 (GID:67523121, aspergillus nidulans), XP_ (GID:119473371, Fei Shi are new for 001266999.1 (GID:119500484, Fei Shi Xin Satuo bacterium) and XP_001258585.1 Sa torr bacterium), it is all herein incorporated by reference.Or, the transglucosidase of this paper can have and any aforementioned disclosed turn The amino acid sequence of glucosidase sequence has an amino acid sequence of at least 90% or 95% homogeneity, and to sugared α-1,3 And/or α-1,6 glucityls-glucose key has hydrolysing activity.When all aforementioned transglucosidases are for the hydrolysis of this paper When, preferably lack the mature form of N-terminal signal peptide.
In some embodiment of this paper, transglucosidase can comprise the amino acid of SEQ ID NO:1 (TG L-2000) Sequence, which is aspergillus niger transglucosidase (U.S. Patent Application Publication 2008/0229514).Or, transglucosidase can comprise Have at least about the 90%th, the 91%th, the 92%th, the 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98% or 99% same with SEQ ID NO:1 The amino acid sequence of property, and to sugared α-1,3 and/or α-1,6 glucityls-glucose 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 prepare, this application with Way of reference is expressly incorporated herein.SEQ ID NO:1 is the ripe transglucosidase lacking N-terminal signal peptide.Because SEQ is ID NO: 1 does not starts with methionine residues, it will be appreciated that if carrying out expressing to it and not using signal peptide (such as to use wherein The expression system at cell inner expression and available from cell lysate for the enzyme), then usual by N-end initial methionine addition SEQ ID NO:1 (directly or via insert heterologous amino acid sequence such as epi-position).
Glucoamylase (EC 3.2.1.3；α-Isosorbide-5-Nitrae-glucan glucohydralase) can be used as in certain embodiments α- Glucosidase.For example, glucoamylase can be with transglucosidase included together in each hydrolysis setting/bar disclosed herein In part.In this linguistic context, glucoamylase can be used for hydrolyzing (i) α-1 being present in the sugar comprising these type bonds any, and 5 Glucityl-fructose key, and/or (ii) α-1,3 and/or α-1,6 glucityls-glucose key.This fermentoid is previously accredited as urging Change α-Isosorbide-5-Nitrae and α-1, the outer effect of both 6 glycosidic bonds of the non-reducing end of the disaccharides containing glucose for the hydrolysis, compound sugar and polysaccharide Enzyme.As herein, presently disclosed glucoamylase is also to α-1, and 5 glucityls-fructose key has hydrolysing activity.Some embodiment party In case, alpha-Glucosidase is not glucoamylase.
The glucoamylase of this paper can derive from any microbial source, such as bacterium or fungi.The glucoamylase of bacterium Example include but is not limited to bacillus (Bacillus) species (such as Alkaliphilic bacillus (B.alkalophilus), Bacillus amyloliquefaciens (B.amyloliquefaciens), bacillus lentus (B.lentus) lichens spore bacillus (B.licheniformis), bacillus stearothermophilus (B.stearothermophilus), bacillus subtilis (B.subtilis), bacillus thuringiensis (B.thuringiensis)) and streptomyces (Streptomyces) species (example As (muta lead mycillin (S.lividans)).The example of the glucoamylase of fungi includes but is not limited to trichoderma thing Plant (such as trichoderma reesei, long shoot wood mould (T.longibrachiatum), T.strictipilis, trichoderma asperellum (T.asperellum), Kang Changmu mould (T.konilangbra), Trichoderma harzianum (T.hazianum)), Aspergillus sp (for example Aspergillus niger, aspergillus oryzae, Aspergillus terreus, rod aspergillus, aspergillus nidulans, aspergillus albicans, aspergillus awamori), Rhizopus (Rhizopus) species (such as Rhizopus oryzae (R.oryzae), snow head mold (R.niveus)), Talaromyces (Talaromyces) species (such as Ai Mosen Blue shape bacterium (T.emersonii), thermophilic actinomycete (T.thermophilus), T.duponti), Mucor (Mucor) thing Kind, Hypocrea (Hypocrea) species (such as H.gelatinosa, east meat seat bacterium (H.orientalis), wine red meat seat Bacterium (H.vinosa), H.citrina), Fusarium (Fusarium) species (such as Fusarium oxysporum (F.oxysporum), rose Look fusarium (F.roseum), F.venenatum), Neurospora (Neurospora) species (such as Neuraspora crassa (N.crassa)), Humicola (Humicola) species (such as ash humicola lanuginosa (H.grlsea), H.insolens, pubescence detritus Mould (H.lanuginose)), Penicillium (Penicillium) species (such as penicillium notatum (P.notatum), penicillium chrysogenum ) and saccharomyces (Saccharomycopsis) species (such as saccharomycopsis fibuligera (P.chrysogenum) (S.fibuligera)) those.It is disclosed in United States Patent (USP) for these bacteriums of this paper and the example of Fungal Glucoamylases Study Application discloses in 2013/0102035, and the document is hereby incorporated herein by.Can be used for its of glucoamylase of this paper Its example is at Svensson et al. (1983, Carlsberg Res.Commun.48:529-544), Boel et al. (1984, EMBO J.3:1097-1102), Hayashida et al. (1989, Agric.Biol.Chem.53:923-929)；United States Patent (USP) 5024941st, United States Patent (USP) the 4794175th, United States Patent (USP) the 4247637th, United States Patent (USP) the 6255084th, United States Patent (USP) No.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) is the 4618579th, Houghton-Larsen et al. (2003, Appl.Microbiol.Biotechnol.62:210-217) and United States Patent (USP) Being described in 7413887, these patents are all herein incorporated by reference.Or, the glucoamylase of this paper can have with The amino acid sequence of any aforementioned disclosed glucose starch enzyme sequence has the amino acid sequence of at least 90% or 95% homogeneity, And to (i) α-1,5 glucityls-fructose key and/or (ii) α-1,3 and/or α-1,6 glucityls-glucose key has hydrolysis and lives Property.When all aforementioned glucoamylases are for the hydrolysis of this paper, 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 the 321st, G ZYME G9904X, OPTIMAX7525, DEXTROZYME, DISTILLASE and GLUCZYME.
In some embodiment of this paper, glucoamylase can comprise the amino acid sequence of SEQ ID NO:2 (GC321) Row, which is trichoderma reesei glucoamylase.Or, glucoamylase can comprise to have at least about the 90%th, with SEQ ID NO:2 91%th, the amino acid sequence of the 92%th, the 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98% or 99% homogeneity, and to (i) α-1, 5 glucityls-fructose key and/or (ii) α-1,3 and/or α-1,6 glucityls-glucose key has hydrolysing activity.Any SEQ ID NO:2 or its variant can for example according to United States Patent (USP) 7413887 or U.S. Patent Application Publication 2013/0102035 disclosure Preparing, these applications are herein incorporated by reference.SEQ ID NO:2 is the ripe glucose starch lacking N-terminal signal peptide Enzyme.Because SEQ ID NO:2 does not starts with methionine residues, it will be appreciated that if carrying out expressing to it and not using letter Number peptide (such as using the wherein expression system at cell inner expression and available from cell lysate for the enzyme), then usual initiate N-end Methionine adds SEQ ID NO:2 (directly or via insert 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).Or, this fermentoid can be prepared by any method known in the art, such as in United States Patent (USP) Shen Please disclose the 2008/0229514th, described in United States Patent (USP) 7413887 or U.S. Patent Application Publication 2013/0102035, this A little applications are hereby incorporated herein by.For example, alpha-Glucosidase can be recombinated generation in heterologous expression system, such as micro-life Thing or fungi heterologous expression system.The example of heterologous expression system includes bacterium (such as Escherichia coli (E.coli), bacillus Belong to (Bacillus sp.)) and eukaryotic system.Eukaryotic system can use such as yeast (for example, pichia (Bacillus Sp.), saccharomyces (Bacillus sp.)) or fungi (for example, trichoderma (Trichoderma sp.), such as trichoderma reesei；Bent Mould species, such as aspergillus niger) expression system.The transglucosidase of SEQ ID NO:1 and the glucoamylase of SEQ ID NO:2 And their variant can for example in trichoderma reesei host express.
When alpha-Glucosidase is for the hydrolysis of this paper, preferably lack the mature form of N-terminal signal peptide.With The polynucleotides of codase, the polynucleotides of this codase can be used in the expression system of ripe alpha-Glucosidase producing this paper Also comprise the sequence of coding N-terminal signal peptide to instruct exocytosis.In this type of embodiment, believe during secretion process Number peptide cuts down from enzyme.Signal peptide can be natural or allos for transglucosidase or glucoamylase.Or Person, the alpha-Glucosidase of mature form can be by for example with the expression at cell inner expression and available from cell pyrolysis liquid for the wherein enzyme System carries out expressing (not using signal peptide) to it to be provided.In either case (secretion or cell inner expression), allos ammonia Base acid sequence such as epi-position may be optionally contained in the N-end of alpha-Glucosidase.
In certain embodiments, can be by directly using the cell expressing one or more enzymes in this paper hydrolysis Alpha-Glucosidase is provided.In other words, the alpha-Glucosidase contacting with carbohydrate can be because it be by the suitable bar being placed in for hydrolysis Cell in part is expressed and is existed.Therefore this type of cell can be used for the alpha-Glucosidase system replacing adding the separation to hydrolysis Agent.Cell for the purpose can be such as bacterium, yeast or fungal cell.The example of yeast includes deriving from following those: Saccharomyces (such as saccharomyces cerevisiae (S.cerevisiae)), Kluyveromyces (Kluyveromyces), candida (Candida), pichia (Pichia), fission yeast (Schizosaccharomyces), Hansenula (Hansenula), Kloeckera (Kloeckera) and perhaps prosperous saccharomyces (Schwanniomyces).Can be used for this paper's Other expression systems be disclosed in U.S. Patent Application Publication 2013/0323822, this application is herein incorporated by reference this Literary composition.
The sugar of this paper comprises at least one α-1,3 or α-1,6 glucityls-glucose key.Therefore, according to sugared length, its For example the 1st, the 2nd, the 3rd, the 4th, the 5th, the 6th, 7 or 8 α-1,5 glucityls-glucose key can be comprised.Sugar preferably comprises the 1st, 2 or 3 this generic keys.? In other preferred embodiments, sugar only has α-1,3 and/or α-1,6 glucityls-glucose key.In other embodiments, Sugar can have one or more α-1,5 glucityls-fructose key.
Because the sugar of this paper comprises at least one α-1,3 or α-1,6 glucityls-glucose key, so sugar comprises at least two Individual glucose unit.In certain embodiments, the sugar of this paper only comprises a glucose unit, or glucose and fructose units Both.Such composition can characterize disaccharides and the compound sugar accessory substance of glucan synthetic reaction.Or, except glucose and fructose Outside, the sugar of this paper also can comprise other monose, such as galactolipin, ribose and wood sugar.
In certain embodiments, the sugar of hydrolysis disclosed by the invention can be compound sugar.The compound sugar of this paper can have example Such as the 2nd, the 3rd, the 4th, the 5th, the 6th, the 7th, 8 or 9 monosaccharide units.As understood in the art, the compound sugar of this paper can refer to its degree of polymerization (DP) number this specify the number of monomeric unit in compound sugar.For example, DP3 compound sugar has 3 monomeric units.Cause This, compound sugar can for example, DP3, DP4, DP5, DP6, DP7, DP8 or DP9 compound sugar.In certain embodiments, the DP of sugar is 3 to 7 (that is, DP 3-7).
Except at least one α-1,3 or α-1, outside 6 glucityls-glucose key, there are 3 or more monosaccharide units Compound sugar also can comprise such as other keys herein.For example, compound sugar can exist one or more α-1,5 glucityls-fructose Key, it is also easy to be hydrolyzed by alpha-Glucosidase as illustrated herein.
In certain embodiments, sugar only comprises through α-1,3 and/or α-1, the glucose monomer that 6 glycosidic bonds connect.Cause This, this type of compound sugar only comprises α-1,3 glucityls-glucose key and/or α-1,6 glucityls-glucose key.This type of compound sugar Example only comprises α-1,3 keys or α-1,6 keys.In certain embodiments, compound sugar can comprise at least 85%, the 86%th, the 87%th, 88%th, the 89%th, the 90%th, the 91%th, the 92%th, the 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98% or 99% glucityl-glucose key. In other embodiments, can there is α-1 of about 75%-85%, 3 glucityls-glucose key peace treaty in the compound sugar of this paper α-1 of 15%-25%, 6 glucityls-glucose key.Or, the compound sugar of this paper can comprise any percentage (between 1% He Any integer value between 99%) α-1,3 glucityls-glucose key and any percentage (appointing between 1% and 99% Meaning integer value) α-1,6 glucityls-glucose key, as long as these percentages are not more than 100%.These compound sugar any Can be at deriving among the fraction of glucan synthetic reaction, this glucan synthetic reaction produces such as (i) insoluble alpha-glucans (for example poly-α-1,3-glucan), or (ii) solubility alpha-glucan product.This linkage content can characterize: (i) is single various oligomeric Sugar, or (ii) one group of compound sugar (i.e. average linkage content).Only comprise by α-1,3 and/or α-1, the glucose list that 6 glycosidic bonds connect The compound sugar of body can for example, DP2-DP7 or DP3-DP7.It should be appreciated that concrete distribution in compound sugar for the key can be according to generation The condition (such as gtf enzyme) of the glucan synthetic reaction of compound sugar accessory substance and change.It is also understood that the concrete distribution of key is right Not vital in presently disclosed method.
The embodiments herein illustrates alpha-Glucosidase (such as transglucosidase and glucoamylase) hydrolyzable (i) and comprises α-1, the lucrose of 5 glucityls-fructose key, and (ii) only comprise α-1,3 glucityls-glucose and/or α-1,6 glucose Both compound sugar 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, use alpha-Glucosidase in the reaction of 6 glucityls-glucose key.
At least one α-1 of the sugar of this paper, 3 or α-1,6 glucityls-glucose key can be by the alpha-Glucosidase water of this paper Solve.Or, it is believed that sugar the 2nd, the 3rd, the 4th, 5 or more this generic keys can for example be hydrolyzed by alpha-Glucosidase.In certain embodiments, At least one α-1,3 or α-1, the hydrolysis of 6 glucityls-glucose key can occur at sugared non-reducing end.
In disclosed method for hydrolysis, the amount of sugar reduces compared to the amount of the sugar existing before contact procedure.This minimizing due to At least one α-1 of sugar, 3 or α-1, the hydrolytic cleavage of 6 glucityls-glucose key.In this paper method for hydrolysis, contact procedure it After sugar amount (such as concentration) be smaller than contact procedure before (contact it making alpha-Glucosidase under suitable conditions with sugar Before) existing for sugar amount about the 1%th, the 2%th, the 3%th, the 4%th, the 5%th, the 10%th, the 20%th, the 30%th, the 40%th, the 50%th, the 60%th, the 70%th, 80% Or 90% (or any integer value between 1% and 90%).
In disclosed method for hydrolysis, the amount of sugar reduces compared to the amount of the sugar existing before contact procedure.Should be understood this ratio Relatively can carry out in any manner.For example, before and after the method for being hydrolyzed, both sugar concentration can be measured.Or, except Outside not adding such as presently disclosed alpha-Glucosidase to control reaction, can enter relative to the control reaction with the same terms Row compares.
In certain embodiments, alpha-Glucosidase can be fixing.Can use any method known in the art and/ Or mode fixes enzyme, such as those disclosed in United States Patent (USP) 5541097 and 4713333, the two patent is all to quote Mode is expressly incorporated herein.For example, can by make one or more enzymes contact with the solution of amine reactive explosive (such as glutaraldehyde) with Formed adduct (such as enzyme-glutaraldehyde adduct), thereafter this adduct is attached to through polyamine (such as ethylene imine, all Such as EPOMIN P-1050) fix one or more enzymes on the solid carrier that processes.
In certain embodiments, the solid carrier (solid support) that alpha-Glucosidase can be made to be fixed to the upper can be nothing Machine or organic material.This type of material include for example gama-alumina, titanium dioxide, granular active carbon, granular silicon diatomaceous earth, bead, Cellular glass, foam, silica gel, metal oxide and aluminum oxide.
Polyamine can be used for processing solid carrier so that solid carrier is subsequently exposed to comprise adding of enzyme and amine reactive explosive Compound, causes enzyme to be bound to solid carrier.The example of the polyamine that can be used for this paper includes: polyethylenediamine, ethylene imine (example Such as poly-diethylenetriamines, poly-trien, poly-penten, polyhexamethylene diamines), polymethylene two hexamethylene Two or more in base amine, polymethylene diphenylamines, poly-tetren, polyphenylene diamines and these polyamine Blend.Preferred polyamine is water miscible and/or has about 500 dalton to 100,000 daltonian molecular weight.Can be at certain A little embodiments use ethylene imine such as EPOMIN P-1050.
The amine reactive explosive that can be used for preparing the adduct of the enzyme comprising this paper can for example, aldehyde, organohalogen compounds, acid Acid anhydride, azo-compound, isothiocyanates and/or isocyanates.The example of these amine reactive explosives includes: glutaraldehyde, amber Aldehyde, terephthalaldehyde, two-diazo benzidine-2,2 '-disulfonic acid, 4,4 '-two fluoro-3,3 '-diphenylsulfone dinitro, diphenyl-4, 4 '-two thiocyanates-2,2 '-disulfonic acid, 3-methoxyl group diphenyl methane-4,4 '-diisocyanate, Toluene-2,4-diisocyanate-isocyanates- 4-isothiocyanates, Toluene-2,4-diisocyanate ,-4-diisocyanate resin, diazo benzidine, diazo benzidine-3,3 '-dianisidine, N, N '- Hexa-methylene two iodoacetamide, hexamethylene diisocyanate, Cyanuric Chloride and/or 1,5-bis-fluoro-2,4-dinitro benzene.Excellent Selection of land, amine reactive explosive is aldehyde, such as glutaraldehyde.
The alpha-Glucosidase with amine reactive compound adduction can be made to contact with the solid carrier processing through polyamine, so that Enzyme is fixed on solid carrier.Immobilised enzymes can be used in various reactor assembly herein, such as post (such as packed column) or stir Mix groove reactor, to carry out hydrolysis as disclosed herein.
Suitable condition for making the sugar of this paper and alpha-Glucosidase (such as transglucosidase) contact is to support sugar One or more α-1,3 or α-1, those conditions that 6 glucityls-glucose key is hydrolyzed by alpha-Glucosidase.Suitable condition Example is disclosed in following example.For making alpha-Glucosidase and the sugar condition (such as temperature, pH, time) that contacts of substrate also It is disclosed in U.S. Patent Application Publication the 2008/0229514th, United States Patent (USP) 7413887 and U.S. Patent Application Publication 2013/ It in 0102035 (these patents are all herein incorporated by reference), and is equally applicable to disclosed method for hydrolysis.
In disclosed method for hydrolysis, disaccharides and compound sugar are usually dissolved in water or aqueous solution.Therefore, make this paper's Carry out under the sugared suitable aqueous conditions contacting dissolved sugar preferably wherein with alpha-Glucosidase.Aqueous conditions can characterize comprise to The solution of few about 20 weight % water or mixture.Or, the aqueous conditions of this paper for example, at least about 20 weight %, 30 weights Amount %, 40 weight %, 50 weight %, 60 weight %, 70 weight %, 80 weight %, 85 weight %, 90 weight % or 95 weight % Water (or any integer value between 20 weight % and 95 weight %).Aqueous conditions may also include the slow of such as suitable concn Rush liquid, such as acid, neutral or alkaline buffer, and select based on the pH scope being provided by buffer solution.Buffer solution/buffering The example of agent includes citrate, acetate (such as sodium acetate), KH2PO4, MOPS, CHES, borate, sodium carbonate and bicarbonate Sodium.
The pH of the hydrolysis of this paper for example can be about 3.0 to 9.0.The 3.5th, the 4.0th, the 3.0th, hydrolysis pH for example can be about 4.5th, the 5.0th, the 5.5th, the 6.0th, the 6.5th, the 7.0th, the 7.5th, the 8.0th, 8.5 or 9.0.Or, pH can be about 4-5.For setting the technology bag of pH Include and use such as buffer solution, alkali and/or acid, and known to being in the 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 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C or 80 DEG C (or any integer value between 20 DEG C and 80 DEG C).Real at some Executing in scheme, the hydrolysis temperature of about 60 DEG C, 65 DEG C or 60 DEG C-65 DEG C 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 0.5 hour can be at least about, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 little When, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours or 90 Hour (or any integer value between 0.5 hour and 72 hours).In certain embodiments, hydrolysis can for example enter Row is less than 4 hours (such as 0.5 hour to 4 hours).Realize the time period needed for desired hydrolysis level will according to used specifically Condition changes, and it will be appreciated by those skilled in the art that.For example, make interpolation to react or be fixed to for reaction solid Enzyme amount on carrier increases will reduce time of contact.
In certain embodiments, one or more alpha-Glucosidases of this paper can be used for hydrolysis.For example, glucose is turned Glycosides enzyme and glucoamylase can be used for reacting.In the hydrolysis of this paper, the amount of alpha-Glucosidase can such as ratio any Amount add drop 10% to 20% (or 5% to 10%) for following example (such as embodiment 2).Or, about 0.1-0.5 body The alpha-Glucosidase of long-pending % or 0.1-1.0 volume % can be used for hydrolysis.Or, the alpha-Glucosidase of this paper with about or At least about 1ppm, 2ppm, 3ppm, 4ppm, 5ppm, 6ppm, 7ppm, 8ppm, 9ppm, 10ppm, 11ppm, 12ppm, 13ppm, 14ppm or 15ppm is used for hydrolysis.Transglucosidase unit (TGU) can for example be defined as under conditions of following mensuration every Minute produces the amount of the transglucosidase of micromole's panose.Transglucosidase activity can for example measure as follows: will turn glucoside Enzyme introduces and comprises 4mM p-nitrobenzophenone-α-glucoside and the 100mM sodium acetate buffer of 1mg/ml bovine serum albumin(BSA) (BSA) In (pH 4.5).After incubating 30 minutes at 30 DEG C, terminate reaction, and record by adding isopyknic 1M sodium carbonate OD405.Glucoamylase unit (XU) can for example be defined as the amount of the glucoamylase by producing 1g reduced sugar, is calculated as at pH Per hour from the glucose of soluble starch substrate (4%DS [substitution value]) at 4.2 and 60 DEG C.
In some embodiment disclosed by the invention, in hydrolysis, the initial concentration of sugar can for example be about 1 weight % To 50 weight %.For example, the concentration of lucrose can be about 5 weight %, 10 weight %, 15 weight %, 20 weight %, 25 Weight %, 30 weight %, 35 weight % or 40 weight % (or any integer value between 5 weight % and 40 weight %). And for example, in the hydrolysis of this paper, one or more compound sugar (such as DP2, DP3, DP4, DP2-DP7, DP3-DP7) dense Degree can be about 1 weight %, 2 weight %, 3 weight %, 4 weight %, 5 weight %, 6 weight %, 7 weight %, 8 weight %, 9 weights Amount %, 10 weight %, 11 weight %, 12 weight %, 13 weight %, 14 weight % or 15 weight %.Those skilled in the art Recognizing, the activity of alpha-Glucosidase can be had an impact by the concentration of total reducing sugar (including disaccharides and compound sugar)；In some respects, at water Solving in reaction makes the maximized preferred total sugar concentration of enzymatic activity be smaller than 50 weight % dry solids (DS), and most preferred concentration is 20 weight %-35 weight %DS.
In certain embodiments, the suitable condition for making sugar contact with the alpha-Glucosidase of this paper mays include: (i) Glucan synthetic reaction, or the fraction that (ii) is obtained by glucan synthetic reaction；The by-product that wherein sugar is glucan synthetic reaction Thing.In other words, the hydrolysis of this paper can be in the scope of glucan synthetic reaction or the part of glucan synthetic reaction In carry out, although it is generally carried out among the latter.It is insoluble that the glucan synthetic reaction of this paper can for example produce one or more Property and/or solubility alpha-glucans product.Therefore, in some embodiments of this paper, glucan synthetic reaction may be characterized as " alpha-glucans synthetic reaction ".
Glucan synthetic reaction is usually directed to such a solution: it comprises at least one sucrose, water and one activity Portugal Glycosyl transferase and other optional components.Other components that can be in glucan synthetic reaction include fructose, grape Sugar, lucrose, soluble oligosaccharide (such as DP2-DP7) and one or more soluble glucan products.Separately Outward, in some respects, glucan synthetic reaction can include one or more alpha-glucans hydrolases.It should be appreciated that some Portugal gathers Sugar product such as DP is poly-α-1 of at least 8 or 9, and 3-glucan can be water-insoluble and therefore in glucan synthetic reaction In insoluble, but solution can be separated out.Therefore, the glucan being produced by the glucan synthetic reaction of this paper can be insoluble 's.The alpha-Glucosidase of this paper can be added to wherein in any stage of glucan synthetic reaction, such as in the initial system of reaction Standby period, wherein latter two time point was preferred or when reaction is nearly completed (for example, completing 80% to 90%) or completes.
The glucan synthetic reaction of this paper, in addition to producing beta-glucan products, also can produce accessory substance such as leukonid Disaccharides and/or soluble oligosaccharide.In some respects, glucan is poly-alpha-glucans.Therefore, the glucan synthetic reaction of this paper Can for example be used for producing poly-α-1,3-glucan or become glycan, its generally in glucan synthetic reaction with at least one bright beading Bacterium disaccharides and/or compound sugar accessory substance produce jointly.
In certain embodiments, glucan synthetic reaction includes producing such as poly-α-1 of poly-alpha-glucans, 3-glucan Glucosyltransferase.The example of this type of glucosyltransferase that can be used for this paper is disclosed in United States Patent (USP) 7000000 and the U.S. is special Profit application disclose 2013/0244288th, 2013/0244287 and 2014/0087431, and these patents are all incorporated by reference Literary composition.
The glucosyltransferase of this paper can derive from any microbial source, such as bacterium or fungi.Bacterium glucosylation The example of enzyme is for deriving from streptococcus (Streptococcus) species, Leuconostoc (Leuconostoc species) thing Those of kind or lactobacillus (Lactobacillus) species.The example of Streptococcus species includes Lactobacillus salivarius (S.salivarius), Streptococcus sobrinus (S.sobrinus), S.dentirousetti, sobrinus (S.downei), change Different streptococcus (S.mutans), Streptococcus oralis (S.oralis), solution gallic acid streptococcus (S.gallolyticus) and blood Streptococcus (S.sanguinis).The example of Leuconostoc (Leuconostoc) species includes Leuconostoc mesenteroides (L.mesenteroides), L.amelibiosum, Argentina leukonid (L.argentmum), meat leukonid (L.carnosum), addicted to citric acid leukonid (L.citreum), streptococcus cremoris (L.cremoris), the bright beading of glucan Bacterium (L.dextranicum) and L.fructosum.The example of lactobacillus (Lactobacillus) species includes addicted to yogurt bar Bacterium (L.acidophilus), Lactobacillus delbrueckii (L.delbrueckii), Lactobacillus helveticus (L.helveticus), saliva breast bar Bacterium (L.salivarius), Lactobacillus casei (L.casei), lactobacillus curvatus (L.curvatus), Lactobacillus plantarum (L.plantarum), Lactobacillus saki (L.sakei), Lactobacillus brevis (L.brevis), Bu Shi lactobacillus (L.buchneri), Lactobacillus fermenti (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 glucan synthesis.During dextran polymer synthesizes, the glucosyltransferase relying on primer needs Reaction solution exists the starting molecule serving as enzyme primer.As used herein, term " primer " refers to any potentially act as glucose The molecule of the initiator of based transferase.The primer that can be used for some embodiment includes such as dextran and other carbon hydrate Thing base primer, the glucan such as hydrolyzing.U.S. Patent Application Publication 2013/0244287 (it is herein incorporated by reference) Disclosing poly-α-1,3-glucan is used as the glucan that hydrolysis prepared by raw material.Dextran as primer can be for example right Rotation sugar acid anhydride T10 (i.e. there is the dextran of 10kD molecular weight).
Glucosyltransferase for the glucan synthetic reaction of this paper can be produced by any method being known in the art Raw.For example, glucosyltransferase can be at heterologous expression system, and restructuring in such as microorganism heterologous expression system produces.Allos table The example reaching system includes bacterium (such as Escherichia coli (E.coli), such as TOP10 or MG1655；Bacillus (Bacillus sp.)) and eucaryon (such as yeast, such as pichia (Pichia sp.) and saccharomyces (Saccharomyces sp.)) expression system.
Glucosyltransferase described herein can be with state (for example pure or impure) use of any purifying.For example, glucityl Transferase can be to purify and/or separate at it before use.The example of impure glucosyltransferase includes cell pyrolysis liquid Those of form.Cell pyrolysis liquid or extract can be prepared by the bacterium (such as Escherichia coli) for heterogenous expression enzyme.For example, French crushing apparatus can be used to destroy bacterium.In the embodiment of alternative, available homogenizer (such as APV, Rannie, Gaulin) make bacterium homogenize.Glucosyltransferase is usually dissolved in the preparation of these types.The bacterium of this paper 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-glucans, such as poly-α-1,3-glucan.
If so desired, the temperature of the glucan 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 embodiment, temperature is about 20 DEG C to about 40 DEG C.
In the glucan synthetic reaction of this paper, the initial concentration of sucrose can be e.g., from about 20g/L to about 400g/L.Or Person, the initial concentration of sucrose can be about 75g/L to about 175g/L or about 50g/L to about 150g/L.Or, sucrose initial Concentration can be e.g., from about 40g/L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, 100g/L, 110g/L, 120g/L, 130g/ L, 140g/L, 150g/L or 160g/L (or any integer value between 40g/L and 160g/L)." initial concentration of sucrose " Refer to only at the sucrose concentration adding after all reaction solution components (at least water, sucrose, gtf enzyme) in gtf reaction solution.
Can be highly purified (>=99.5%) or for any other pure for the sucrose of the glucan synthetic reaction of this paper Degree or grade.For example, sucrose can have the purity of at least 99.0% or can be SILVER REAGENT sucrose.And for example, can use incomplete Refined sucrose.The sucrose not refined completely of this paper refers to the undressed sucrose for refining white sucrose.Therefore, completely not refined Sucrose can be refined for completely unpurified or part.The example of unpurified sucrose is " melada " (" raw sugar ") and molten Liquid.The example of the refined sucrose of part does not experiences one, two, three or more crystallisation step.This paper's is completely not refined ICUMSA (the International Commission for Uniform Methods of Sugar of sucrose Analysis) 150 can be greater than.Sucrose can derive from any renewable sugar source herein, such as sugarcane, sugar beet, cassava, Sugar grass or corn.Can be used for sucrose for example, crystal form or non-crystalline forms (the such as syrup, sugarcane of the suitable form of this paper Juice, beet juice).The other suitable form of the sucrose not refined completely is 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 Sugar Analysis(ICUMSA)(H.C.S.de Whalley compiles Volume, Elsevier Pub.Co., 1964), the document is herein incorporated by reference.ICUMSA can for example as by R.J.McCowage, R.M.Urquhart and 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 glucan synthetic reaction can be about 4.0 to about 8.0.Or, the 4.0th, pH can be about 4.5th, the 5.0th, the 5.5th, the 6.0th, the 6.5th, the 7.0th, 7.5 or 8.0.Can be regulated by adding or mixing suitable buffer solution or control PH, this buffer solution includes but is not limited to: phosphate, tris, citrate or combinations thereof.Delaying of glucan synthetic reaction Rushing liquid concentration can for example, 0mM to about 100mM or about 10mM, 20mM or 50mM.
Poly-α-1 producing in the glucan synthetic reaction of this paper, the 60%th, at least about the 50%th, 3-glucan can have 70%th, the 80%th, the 90%th, the 95%th, the 96%th, the 97%th, the 98%th, 99% or 100% (or arbitrarily whole between 50% and 100% Numerical value) α-1,3 glycosidic bonds.Therefore, in this type of embodiment, poly-α-1, the 40%th, less than about the 50%th, 3-glucan have 30%th, the 20%th, the 10%th, the 5%th, the 4%th, the 3%th, the 2%th, 1% or 0% (or any integer value between 0% and 50%) is non- α-1,3 glycosidic bonds.
Poly-α-1 of this paper, 3-glucan preferably have straight chain/main chain of non-branched.In certain embodiments, poly-α- 1,3-glucan does not have branching-point or has less than about the 10%th, the 9%th, the 8%th, the 7%th, the 6%th, the 5%th, the 4%th, the 3%th, 2% or 1% point Scolus (as the percentage of glycosidic bond in polymer).The example of branching-point includes α-1,6 branching-points.
Poly-α-1 producing in the glucan synthetic reaction of this paper, the molecular weight of 3-glucan can be determined as number-average molecular weight (Mn) or weight average molecular weight (Mw).Or, molecular weight can by dalton or gram/mol based on mensuration.It also can be used to refer to poly-α-1,3- The DP of dextran polymerw(weight average degree of polymerization) or DPn(number-average degree of polymerization).
Poly-α-1 of this paper, the M of 3-glucannOr Mw1000 can be at least about.Or, MnOr MwCan for example, at least about 1000 to about 600000 (or any integer value between 1000 and 600000).Or, poly-α-1,3-glucan can have There is such molecular weight: at least about 100 or at least about 100 to 1000 (or any integer value between 100 and 1000) DPnOr DPw。
The fraction of glucan synthetic reaction is suitable configurable for make sugared and such as presently disclosed alpha-Glucosidase contact Condition.Fraction can be part or all of liquid solution deriving from glucan synthetic reaction.Generally, in making fraction and reacting One or more solubilities of synthesis or insoluble glucan product separate.For example, fraction can be made to close at it with one or more Separate from the water-fast beta-glucan products (for example poly-α-1,3 glucans) that solution separates out during one-tenth.In some of the disclosure Fraction in preferred embodiment obtains autohemagglutination α-1,3-glucan synthetic reaction.
In certain embodiments, the volume of fraction can be at (before optionally dilution or concentration stage are divided, see below) Therefrom obtain the glucan synthetic reaction of this fraction volume at least about the 10%th, the 20%th, the 30%th, the 40%th, the 50%th, the 60%th, 70%th, 80% or 90% (or any integer value between 10% and 90%).Generally, insoluble glucan (example is being produced Such as poly-α-1,3 glucans) glucan synthetic reaction in, fraction is by (not complete for the part of liquid solution component for reaction Portion).Fraction can be obtained in any stage of glucan 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 glucan synthetic reaction includes filtrate and supernatant.Therefore, at it Middle synthesis insoluble glucan product those embodiments in, can use funnel, filter (such as pressure filter), centrifuge, Or known in the art allow from solid, remove any other method of some or all liquid or equipment closes from glucan Reaction is become to obtain the fraction of (separation) this paper.Filtration can for example be carried out by gravity, vacuum or press filtration.Filter and preferably remove Wholly or largely insoluble glucan；Average cell size (e.g., from about 40-50 micron) can be used to be enough to from liquid remove admittedly Any filtering material (such as filter paper) of body.Fraction generally retains wholly or largely its component dissolved, and such as glucan closes Become the accessory substance of reaction.
If so desired, the fraction of this paper optionally can be diluted or concentrates.The concentration of fraction can use 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 such as be used by evaporation As set the temperature being about 40-50 DEG C) concentrate fraction.At some aspects of this paper, fraction can be made to be concentrated into such body Long-pending: about the 75%th, the 80%th, the 85%th, the 90% or 95% of initial level partial volume.Concentrated fraction (for example concentrated filtrate) can It is optionally referred to as syrup.
Fraction can comprise water in some respects, and this water substitutes water present in the composition therefrom obtaining fraction.For example, may be used In some chromatography separating method that initial solvent is substituted by another kind of solvent wherein from glucan synthetic reaction separate a kind of or Multiple sugar accessory substances (the sugared accessory substance [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 making sugar and α-glucoside Any appropraite condition (for example, temperature, pH and time) of enzyme contact.For example, added before fraction at alpha-Glucosidase, 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 DEG C-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-glucan synthetic reaction in some preferred embodiment of this paper；Such as fraction is excellent Selection of land is filtrate.Poly-α-1 of this paper, the fraction of 3-glucan synthetic reaction is including at least water, fructose and one or more classes The sugar (lucrose and/or compound sugar, such as DP2-DP7) of type.Other components that can be in such fraction are for example wrapped Include sucrose (the remaining sucrose i.e. not consumed in gtf reaction), one or more gtf enzymes, glucose, buffer solution, salt,Borate, NaOH, hydrochloric acid, cell pyrolysis liquid component, protein and/or nucleic acid.Minimally, Autohemagglutination α-1, the component of the fraction of 3-glucan synthetic reaction includes such as water, fructose, glucose, one or more types Sugar (lucrose and/or compound sugar, such as DP2-DP7) and optional sucrose.It should be appreciated that the composition portion of fraction Divide the condition depending on therefrom obtaining the glucan synthetic reaction of fraction.In those fractions comprising one or more gtf enzymes, Preferably, before using fraction in the hydrolysis of this paper, this type of one or more gtf enzyme inactivates (such as heat inactivation).
It should be appreciated that being specifically distributed of the sugared accessory substance producing via polymerising sucrose in glucan 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 in glucan synthetic reaction Fraction in particular make-up not vital for disclosed method for hydrolysis.In general, as the amount of sucrose increases, The selectivity to lucrose and compound sugar for the reaction will increase.On the contrary, as temperature increases, reaction is to bright beading The selectivity of bacterium disaccharides tends to reducing, and the selectivity for compound sugar is generally not affected by impact.It should be appreciated that by by sugar The ratio of the sugar that calculates divided by total solution weight of quality and water i.e. weight % dry solid (DS) can be by evaporating the water (preferably Under vacuo with less than at a temperature of 50 DEG C) or add water and regulate, and substantially without impact sugar in glucan synthetic reaction Fraction in Relative distribution.Can also be increased by terminating gtf reaction before realizing conversion (being converted into glucan) completely The percentage 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 glucan synthetic reaction of this paper can produce one or more solubility alpha-glucanses product Thing.Solubility alpha-glucans product (or " Soluble Fiber ") can be: the direct product of (i) glucosyltransferase, or (ii) Glucosyltransferase and the synergistic product of alpha-glucans hydrolase, described alpha-glucans hydrolase 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-glucans of this paper can include, for example:
A) α-1 of at least 75%, 3-glycosidic bond；
B) α-1 less than 25%, 6-glycosidic bond；
C) α-1 less than 10%, 3,6-glycosidic bonds；
D) it is less than 5000 daltonian Mw；
E) at 20 DEG C, in water under 12 weight %, less than the viscosity of 0.25 pascal second (Pa s)；
F) scope is the dextrose equivalent (DE) of 4 to 40；
G) digestibility less than 10%, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) measured by method 2009.01；
H) at 25 DEG C, the solubility of at least 20% (w/w) in pH 7 water；With
I) polydispersity index (PDI) less than 5.
This type of solubility alpha-glucans can be prepared as disclosed in U. S. application 62/004,290.
For example, solubility alpha-glucans fiber composition can comprise at least 75%, preferably at least the 80%th, more preferably at least 85%th, the α of even more desirably at least 90% and most preferably at least 95%-(1,3) glycosidic bond.
And for example, outside above-mentioned α-(1,3) glycosidic bond embodiment, solubility alpha-glucans fiber composition also can wrap Containing less than the 25%th, be preferably less than the 10%th, more preferably 5% or less, α-(1,6) glycosidic bond of even more preferably less than 1%.
And for example, outside above-mentioned α-(1,3) and α-(1,6) glycosidic bond content embodiment, solubility alpha-glucans is fine Dimension composition also can comprise less than α-(1,3, the 6) glycosidic bond the 10%th, being preferably less than the 5%th, most preferably in less than 2.5%.
And for example, solubility alpha-glucans fiber composition can comprise α-(1, the 3) glycosidic bond of 93% to 97% and be less than 3% α-(1,6) glycosidic bond, and have corresponding to 3 to 7 mixing DP weight average molecular weight.In another embodiment, solvable Property alpha-glucans fiber composition can include about α-(1, the 3) glycosidic bond of 95% and α-(1,6) glycosidic bond of about 1%, and has There is the weight average molecular weight corresponding to 3 to 7 mixing DP.In the another aspect of embodiments above, solubility alpha-glucans fiber group Compound can include about α-(1,3, the 6) key of 1% to 3% or α-(1,3,6) key of preferably from about 2%.
And for example, in addition to above-mentioned glycosidic bond content embodiment, solubility alpha-glucans fiber composition is also Can comprise less than α-(Isosorbide-5-Nitrae) glycosidic bond the 5%th, being preferably less than 1% and most preferably in less than 0.5%.
And for example, in addition to above-mentioned glycosidic bond content embodiment, solubility alpha-glucans fiber composition is also Can have less than 5000 dalton, preferably smaller than 2500 dalton, be more preferably between 500 dalton and 2500 dalton, And most preferably from about 500 dalton to about 2000 daltonian weight average molecular weight (Mw)。
And for example, in addition to any features above, at 20 DEG C and in water under 12 weight %, solubility alpha-glucans fiber Composition also can have 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, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) measured by method 2009.01, solubility alpha-glucans fiber composition can have less than the 10%th, excellent Choosing is less than the digestibility of the 9%th, the 8%th, the 7%th, the 6%th, the 5%th, the 4%th, the 3%th, 2% or 1% digestibility.On the other hand, digestibility Relative level or may be used without AOAC2011.25 (comprehensive total dietary fiber detection is fixed, Integrated Total Dietary Fiber Assay) (McCleary et al., 2012, J.AOAC Int., 95 (3), 824-844) mensuration.
In addition to any embodiments above, solubility alpha-glucans fiber composition can have in pH 7 water at 25 DEG C Have at least 20% (w/w), preferably at least the 30%th, the 40%th, the 50%th, 60% or 70% solubility.
In one embodiment, the content of the reduced sugar that solubility alpha-glucans fiber composition can comprise is less than 10 weights Amount %, preferably smaller than 5 weight % and most preferably 1 weight % or less.
In one embodiment, solubility alpha-glucans fiber composition can have less than 4 kilocalories/g, be preferably less than 3 The heat content of kilocalorie/g, more preferably less than 2.5 kilocalories/g and most preferably from about 2 kilocalories/g or less.
And for example, the solubility alpha-glucans of this paper mays include:
A) α-1 of 10% to 30%, 3-glycosidic bond；
B) α-1 of 65% to 87%, 6-glycosidic bond；
C) α-1 less than 5%, 3,6-glycosidic bonds；
D) it is less than 5000 daltonian weight average molecular weight (Mw)；
E) at 20 DEG C, in water under 12 weight %, less than the viscosity of 0.25 pascal second (Pa s)；
F) scope is the dextrose equivalent (DE) of 4 to 40, preferably 10 to 40；
G) digestibility less than 10%, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) measured by method 2009.01；
H) at 25 DEG C, the solubility of at least 20% (w/w) in pH 7 water；With
I) polydispersity index (PDI) less than 5.
This type of solubility alpha-glucans can be prepared such as U. S. application 62/004 disclosed in 308.
And for example, the solubility alpha-glucans of this paper mays include:
A) α-1 of 25-35,3-glycosidic bond；
B) α-1 of 55%-75%, 6-glycosidic bond；
C) α-1 of 5%-15%, 3,6-glycosidic bonds；
D) it is less than 5000 daltonian weight average molecular weight；
E) at 20 DEG C, in water under 12 weight %, less than the viscosity of 0.25 pascal second (Pa.s)；
F) scope is the dextrose equivalent (DE) of 4 to 40；
G) digestibility less than 10%, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) measured by method 2009.01；
H) at 25 DEG C, the solubility of at least 20% (w/w) in water；With
I) polydispersity index less than 5.
This type of solubility alpha-glucans can be prepared as disclosed in U. S. application 62/004,312.
And for example, the solubility alpha-glucans of this paper mays include:
A) α-1 of at least 95%, 6-glycosidic bond；
B) α-1 of 1% or less, 3-glycosidic bond；
C) α-1 less than 2%, 3,6-glycosidic bonds；
D) α-Isosorbide-5-Nitrae-glycosidic bond less than 1.5%；
E) it is less than 20000 daltonian weight average molecular weight；
F) at 20 DEG C, in water under 12 weight %, less than the viscosity of 0.25 pascal second (Pa s)；
G) scope is the dextrose equivalent (DE) of 1 to 30；
H) digestibility less than 10%, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) measured by method 2009.01；
I) at 25 DEG C, the solubility of at least 20% (w/w) in pH7 water；With
J) polydispersity index less than 5.
This type of solubility alpha-glucans can be prepared as disclosed in U. S. application 62/004,314.
And for example, the solubility alpha-glucans of this paper mays include:
A) scope is:
I) α-1 of 1% to 50%, 3-glycosidic bond；Or
Ii) it is more than 10% but be less than the α-Isosorbide-5-Nitrae-glycosidic bond of 40%；Or
Iii) i) and ii) any combination；
B) α-1 of 1% to 50%, 2-glycosidic bond；
C) α-1 of 0% to 25%, 3,6-glycosidic bonds；
D) α-1 less than 98%, 6-glycosidic bond；
E) weight average molecular weight less than 300kDa；
F) at 20 DEG C, in water under 12 weight %, less than the viscosity of 0.25 pascal second (Pa s)；
G) digestibility less than 20%, such as analytical chemistry Shi Xiehui (Association of Analytical Communities, AOAC) measured by method 2009.01；
H) at 25 DEG C, the solubility of at least 20% (w/w) in pH 7 water；With
I) it is less than 26, the polydispersity index of preferably smaller than 5.
This type of solubility alpha-glucans can be prepared as disclosed in U. S. application 62/004,305.
In certain embodiments, solubility alpha-glucans is the direct product of glucosyltransferase.Poly-in suitable Portugal In sugar synthetic reaction, this type of glucosyltransferase and can be as disclosed herein for its condition, or as in United States Patent (USP) Shen Please for example the 62/004,290th, the 62/004,308th, the 62/004,312nd, public in any one in 62/004,314 and/or 62/004,305 Open.
Solubility alpha-glucans or alternatively such as glucosyltransferase and alpha-glucans hydrolase synergy Product, described alpha-glucans hydrolase can hydrolyze has one or more α-1,3-glycosidic bond or one or more α-1, The dextran polymer of 6-glycosidic bond.In some respects, for producing the glucan synthetic reaction of solubility alpha-glucans product At least one glucosyltransferase and at least one alpha-glucans hydrolase can be comprised.In other side, glucan synthesizes Reaction can initially comprise one or more glucosyltransferases as unique enzyme component.This type of reaction generation not yet passes α-Portugal The first alpha-glucans that endohydrolase is modified.Then, at least one alpha-glucans hydrolase is added to reaction properly Time period, to allow to be modified to the first product solubility alpha-glucans product.Accordingly, there exist and turned via glucityl by it Move enzyme and the different modes of alpha-glucans hydrolase synergy synthesizing soluble alpha-glucans product.In glucan synthesis It during reaction and/or after glucan synthesis, is used for carrying out glucan synthetic reaction and (wherein comprises one or more alpha-glucanses Hydrolase) condition can as disclosed herein, or as at U.S. Patent application such as 62/004, the 290th, 62/004, the 308th, 62/ 004,312nd, in 62/004,314 and/or 62/004,305 disclosed in any one.
The alpha-glucans hydrolase of this paper can for such as Dextranase (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-glucan of (1-3)-and (1-4)-key；EC 3.2.1.61), glucan 1,6-alpha-Glucosidase (EC3.2.1.70) and alternan enzyme (EC 3.2.1.-；Can inscribe water Solve cracking alternan；E.C.3.2.1.-；See United States Patent (USP) 5786196).
The mutant enzyme including SEQ ID NO:47 can be used in certain aspects.Or, mutant enzyme can for example comprise and SEQ ID NO:47 has at least 90%, the ammonia of the 91%th, the 92%th, the 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98% or 99% homogeneity Base acid sequence, and there is mutant enzyme activity.
As presently disclosed can for the glucan synthetic reaction producing one or more solubility alpha-glucans products Being directly used as wherein carrying out the suitable condition of the hydrolysis of this paper, wherein alpha-Glucosidase is used for hydrolyzing alpha-1, and 5 glucityls- Fructose key.Can be according to regard to for example poly-α-1 of generation, any above public affairs of the hydrolysis process of the glucan synthetic reaction of 3-glucan The condition opened carries out this type of hydrolysis.Or, the glucan synthesis for producing one or more solubility alpha-glucans products is anti- The fraction (such as chromatographic isolation fraction) answered can be used as wherein to α-1, and 5 glucityls-fructose key carries out alpha-Glucosidase mediation The suitable condition of hydrolysis.
In some embodiment of this paper, fraction can be the chromatographic isolation fraction of glucan synthetic reaction.For example, fraction Can be for producing the chromatographic isolation level of the glucan synthetic reaction of one or more solubility alpha-glucans products as disclosed herein Point.This type of reaction can glucan synthesis during and/or glucan synthesis complete after optionally comprise one or more α-Portugals Endohydrolase.In the embodiment of these types any, generally obtain fraction, in order to make wholly or largely (for example, extremely Few about the 60%th, the 70%th, the 80%th, the 90%th, 95%) solubility alpha-glucans product separates with producing its response composite.Once Separate with wholly or largely solubility alpha-glucans product, so that it may use one or more alpha-glucanases to make fraction stand this Any α-1 disclosed in Wen, 5 glucityls-fructose water solution preocess.
The chromatographic isolation fraction of this paper can generally use the liquid chromatography of proper types to obtain.Liquid chromatogram can for example make With SEC (SEC), column chromatography, high performance liquid chromatography (HPLC), ion-exchange chromatography, affinity chromatography, Ultrafiltration, micro-filtration or dialysis are carried out.
The invention still further relates to a kind of combination being produced by making sugar contact with alpha-Glucosidase (such as transglucosidase) Thing, wherein (i) sugar is for comprising at least one α-1,3 or α-1, the disaccharides of 6 glucityls-glucose key or compound sugar, and (ii) At least one α-1 of alpha-Glucosidase hydrolysis sugar, 3 or α-1,6 glucityls-glucose key.The composition bag producing in like fashion The sugar amount containing reduces compared to the sugar amount existing before contact.The example of composition includes any those disclosed herein, such as Derive from the filtrate through hydrolysis of glucan synthetic reaction or for producing the warp of the glucan synthetic reaction of solubility alpha-glucans The fraction of hydrolysis.In disclosed above and embodiment, any feature with regard to method for hydrolysis and product thereof can characterize composition. The following characteristics of composition is example.
In some embodiment of composition, alpha-Glucosidase can comprise and SEQ ID NO:5, the 6th, the 8th, the 9th, the 11st, the 12nd, 14th, the 15th, the 17th, the 18th, the 20th, the 22nd, the 24th, the 26th, the 28th, the 30th, the 32nd, the 34th, the 36th, 38, or DIAZYME RDF ULTRA (DuPont Industrial Biosciences) have at least 90%, the 91%th, the 92%th, the 93%th, the 94%th, the 95%th, the 96%th, the 97%th, 98% or The amino acid sequence of 99% homogeneity.In some embodiment of composition, transglucosidase can comprise and SEQ ID NO:1 There is the amino acid sequence of at least 90% homogeneity.Or, it is disclosed that any alpha-Glucosidase disclosed herein can be used for generation Composition.
In some embodiment of composition, sugar has the degree of polymerization of 3 to 7 before hydrolysis.
Before the sugared concentration of the composition being produced by the method for hydrolysis of this paper can for example contact with alpha-Glucosidase less than sugar The 50% of the sugared concentration existing.
In some embodiment of this paper, the composition that produced by method for hydrolysis can be glucan synthetic reaction thing or Its fraction, wherein the accessory substance of glucan synthetic reaction contacts with alpha-Glucosidase.In this embodiment, fraction can be for example, The filtrate of glucan synthetic reaction or for producing the fraction of the glucan synthetic reaction of solubility alpha-glucans.In this enforcement In scheme, sugar can have the degree of polymerization of such as 3 to 7 before hydrolysis.
It will be appreciated by the skilled person that presently disclosed embodiments part is otherwise likely difficult to for saccharification decompose Disaccharides and compound sugar.For example, it is possible to utilize this feature to implement enhanced method: the enrichment of (i) fructose and (ii) fermentation.
Example 6 below shows, compared to using unhydrolysed filtrate, when use is by alpha-Glucosidase (transglucosidase) Fructose enrichment is enhanced by chromatography during the glucan filtrate hydrolyzing.
Therefore, invention disclosed further relates to the side that a kind of enrichment is present in the fructose in the fraction of glucan synthetic reaction Method.The method includes: (a) makes fraction and the alpha-Glucosidase (such as transglucosidase) available from glucan synthetic reaction properly Under conditions of contact, at least one α-1 of disaccharides contained in wherein enzyme hydrolysis fraction or compound sugar, 3 or α-1,6 glucityls- Glucose key；And (b) from step (a) through hydrolysis fraction separating levulose, to obtain the fraction than step (a) for the fructose concentration The high composition of fructose concentration.
Disclosed in for example with regard to the fructose of alpha-Glucosidase (such as transglucosidase) and the fraction of glucan synthetic reaction The feature of enrichment method can be according to any disclosure relating to every kind of these features 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 In lower embodiment disclosed, or use chromatography according to European Patent Publication EP2292803B1, this patent is with way of reference simultaneously Enter herein.
Derive from the composition (such as fructose soln or fructose syrup) with higher fructose concentration of disclosed enrichment method Can have at least about 90 weight %, 91 weight %, 92 weight %, 93 weight %, 94 weight %, 95 weight %, 96 weight %, 97 The fructose of weight %, 98 weight % or 99 weight %.
The fructose enrichment method of this paper is than the method for the filtrate utilizing without the hydrolysis of such as presently disclosed alpha-Glucosidase More preferable expressively.This type of performance increasing 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 fraction and the α available from glucan synthetic reaction- Glucosidase (for example, transglucosidase or glucoamylase) contacts, wherein alpha-Glucosidase hydrolysed grade under suitable conditions Disaccharides contained in point or at least one α-1 of compound sugar, 5 glucityls-fructose key；B () uses the level of microbial fermentation step (a) Divide to produce product；And (c) is optionally separated the product of (b).The fermentation step of step (b) can after step (a) or with Step (a) is carried out simultaneously.Significantly, the method can be used for example by the filtrate through hydrolysis of fermentation glucan synthetic reaction Prepare ethanol.Derive from the ethanol yield higher than acquisition during fermentation still unhydrolysed glucan filtrate for the ethanol yield of this process.
Synthesize anti-with regard to alpha-Glucosidase (for example, transglucosidase or glucoamylase), disaccharides and compound sugar, glucan The fraction answered and the feature of the disclosed fermentation process of suitable contact conditions for example can relate to every kind according to provided herein Any disclosure of these features.
Microorganism for the fermentation process of this paper can be such as bacterium, yeast or fungi.Can be used for the bacterium of this paper Example includes Lactobacillus species, Streptococcus species, Bifidobacteria, leukonid species, Escherichia (Escherichia) species (such as Escherichia coli) and Bacillus spec.The example of the yeast that can be used for this paper includes ferment Female species, such as saccharomyces cerevisiae (S.cerevislae) and saccharomyces bayanus (S.bayanus).
The fermentation process of this paper can produce product such as ethanol or acid (such as lactic acid).However, it is believed that if so desired, can produce Other products raw.It will be appreciated by those skilled in the art that use such as disclosed fermentation process produces specific product and will depend on respectively The condition of kind, such as one or more microorganisms for fermentation.For example, the condition for fermentation of this paper can be such as following example Disclosed, or 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, both is herein incorporated by reference.
In some embodiment of the fermentation process of this paper, product yield is higher than the alpha-Glucosidase fermenting without this paper The product yield obtaining during the glucan filtrate hydrolyzing.This compares the non-hydrolysed grade that can refer to for example use glucan synthetic reaction The control fermentation divided.The product yield fermenting herein can for example increase at least about the 10%th, the 20%th, the 40%th, the 60%th, 80% or 100% (or any integer value between 10% and 100%).Can increase additionally, the fermentation of this paper is formed the speed of product Greatly.
Example 7 below demonstrates, lucrose can be provided that the charging comprising unhydrolysed glucan filtrate Yeast fermentation be ethanol.Therefore, there is further disclosed herein a kind of fermentation lucrose with microorganism is product (example Such as ethanol) method.The method can include fermenting (i) or (ii) Portugal of hydrolyzing without alpha-Glucosidase as disclosed herein Glycan filtrate.No matter whether lucrose is provided in glucan filtrate or provides (such as half purifying in another form Or enriched form), the method for the lucrose that ferments can include making microorganism (such as yeast, such as saccharomyces cerevisiae) fit Lucrose should be utilized.This type of adapts to can include making microorganism raw in the presence of lucrose and other optional sugar Long at least 2 or 3 growth cycles, microorganism utilizes more lucrose to carry out tunning thereafter.In some embodiment In, microorganism (i) can grow (1 complete cycle) in the first charging comprise lucrose, and (ii) is from the first charging Middle removal, (two complete cycles, (iv) appoints from the second charging in (iii) growth in the second charging comprise lucrose Selection of land is removed, and (v) optionally grows (three complete cycles) in the 3rd charging.In certain embodiments, with the party The microorganism that formula adapts to can increase the ability of fermentation lucrose.
Example 9 below demonstrates, when glucan filtrate is fermented by yeast while being hydrolyzed by transglucosidase, deposits Nearly all (such as the ＞ 98% or ＞ 99%) lucrose being in glucan filtrate can be used for being fermented by yeast.Cause This, enhanced lucrose fermentation process can include with alpha-Glucosidase (for example, transglucosidase or glucose starch herein Enzyme) hydrolysis lucrose, use fermentable lucrose simultaneously.
The non-limiting example of the compositions disclosed herein and method includes:
1. one kind makes to comprise at least one α-1,3 or α-1, α-1 of the sugar of 6 glucityls-glucose key, 3 or α-1,6 glucose The method of base-glucose key hydrolysis, wherein said sugar is disaccharides or compound sugar, and the method comprise the steps that
Sugar is made to contact under suitable conditions with alpha-Glucosidase, at least one of wherein said alpha-Glucosidase hydrolysis sugar α-1,3 or α-1,6 glucityls-glucose key,
And wherein sugar amount reduces compared to the sugar amount existing before contact.
2. the method according to embodiment 1, wherein said alpha-Glucosidase is fixing.
3. the method according to embodiment 1 or 2, the degree of polymerization of wherein said sugar is 3 to 7 before hydrolysis.
4. the method according to embodiment the 1st, 2 or 3, wherein the concentration of sugar after the contacting step is less than in contact The 50% of the concentration of the sugar before existing.
5. the method according to embodiment the 1st, the 2nd, 3 or 4, wherein suitable condition includes:
(i) glucan synthetic reaction, or the fraction that (ii) is obtained by glucan synthetic reaction；
Wherein said sugar is the accessory substance of glucan synthetic reaction.
6. the method according to embodiment 5, at least one insoluble α of wherein said glucan synthetic reaction generation- Beta-glucan products.
7. the method according to embodiment 6, wherein level is divided into the filtrate of described glucan synthetic reaction.
8. the method according to embodiment 5, wherein said glucan synthetic reaction produces at least
A kind of solubility alpha-glucans product, which is:
The product of (i) glucosyltransferase, or
(ii) glucosyltransferase and the synergistic product of alpha-glucans hydrolase, described alpha-glucans hydrolase Can hydrolyze and there is 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 said level is divided into the chromatographic isolation of described glucan synthetic reaction Fraction.
10. the method according to according to any one of embodiment 1-9, wherein said alpha-Glucosidase is transglucosidase.
11. 1 kinds of compositions being produced by making sugar contact with alpha-Glucosidase,
Wherein said sugar is disaccharides or compound sugar and comprises at least one α-1,3 or α-1,6 glucityls-glucose key,
Wherein at least one α-1 of enzyme hydrolysis sugar, 3 or α-1,6 glucityls-glucose key,
And the sugar amount that wherein composition comprises reduces compared to the sugar amount existing before contact.
12. compositions according to embodiment 11, wherein the degree of polymerization of sugar is 3 to 7 before hydrolysis.
13. compositions according to embodiment 11 or 12, wherein sugar is in (i) glucan synthetic reaction, or (ii) In the fraction being obtained by glucan synthetic reaction；
The accessory substance that wherein sugar is described glucan synthetic reaction.
14. 1 kinds of enrichments are present in the method for the fructose in the fraction of glucan synthetic reaction, and described method includes:
A () makes to contact under suitable conditions with alpha-Glucosidase available from the fraction of glucan synthetic reaction, wherein α-Portugal At least one α-1 of contained disaccharides or compound sugar, 3 or α-1,6 glucityls-glucose key in glycosidase hydrolysis fraction；And
(b) from step (a) through hydrolysis fraction separating levulose, to obtain the fruit of the fraction than step (a) for the fructose concentration The high composition of sugar concentration.
15. 1 kinds of fermentation process, described fermentation process includes:
A () makes to contact under suitable conditions with alpha-Glucosidase available from the fraction of glucan synthetic reaction, wherein α-Portugal At least one α-1 of contained disaccharides or compound sugar, 3 or α-1,6 glucityls-glucose key in glycosidase hydrolysis fraction；
B () is wherein fermented after step (a) or and step by the fraction of microbial fermentation step (a) to produce 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 glucan synthetic reaction not contacted with alpha-Glucosidase is fermented Rate is compared, and the product yield of (b) increases.
Embodiment
Present invention disclosed will be further elaborated in the following embodiments.Although it should be understood that these embodiments are said Understand some preferred aspect of the present invention, but be only given in an exemplary manner.By above-mentioned discussion and these embodiments, this The technical staff in field can determine that the essential feature of the present invention, and without departing from the premise in the spirit and scope of the invention Under, variations and modifications can be carried out to adapt to multiple use and condition to the present invention.
Abbreviation
Some abbreviations used herein implication as follows: " g " refers to gram, and " h " refers to hour, and " mL " refers to milliliter, " psi " Referring to pound per square inch, " wt% " refers to percentage by weight, and " μm " refers to micron, and " % " refers to percentage, and " DEG C " refers to take the photograph Family name's degree, " mg " refers to milligram, and " mm " refers to millimeter, and " mL/min " refers to that milliliter is per minute, and " m " refers to rice, and " μ L " refers to microlitre, " mmol " refers to mM, and " min " refers to minute, and " mol% " refers to molar percentage, and " M " refers to mole, and " mg/g " refers to milli Gram 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 CE of glucosyltransferase (gtf)
Use the expression system of isopropyl ss-D-1-thiogalactoside (IPTG)-induction at coli strain DH10B Middle expression streptococcus salivarius gtfJ enzyme (SEQ ID NO:3).Compared to streptococcus salivarius gtfJ amino acid sequence, (GENBANK knows Alias 47527), SEQ ID NO:3 has 42 residue deletions of N-end, but includes initial methionine.In brief, greatly Enterobacteria DH10B cell is converted to be expressed SEQ ID NO:3 by codon optimized DNA sequence dna, thus in Escherichia coli Express gtfJ enzyme.This DNA sequence dna includes at expression vectorIn (DNA 2.0, Menlo Park CA).Will The cell of conversion is inoculated in initial optical density, and (OD, 600nmPlace) be 0.025 LB culture medium (10g/L tryptone；5g/L ferment Female extract, 10g/L NaCl) in, and make it grow 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.Be placed in the culture of induction on shaking machine and Gather in the crops after inducing 3 hours.
Existed by making the cell of cultivationIn centrifuge, centrifugal (25 DEG C, 16000rpm) results obtain enzyme GtfJ (SEQ ID NO:3), makes cell be resuspended in 5.0mM phosphate buffer (pH7.0) and in cooled on ice to 4 DEG C.Adopt Bead beater is used to make clasmatosis by 0.1-mm silica bead, then centrifugal so that not under 4 DEG C and 16000rpm Smudge cells and pellet cell debris.Make crude extract (comprising soluble g tfJ enzyme, SEQ ID NO:3) and precipitate and separate, and lead to Cross Bradford protein determination analysis to measure protein concentration (mg/mL).
Following preparation streptococcus (Streptococcus sp.) C150gtf-S enzyme (SEQ ID NO:40).SG1184 is Bacillus subtilis (Bacillus subtilis) expresses bacterial strain, and it expresses the streptococcus truncating pattern (Streptococcus sp.)C150(GI:321278321) glycosyl transferase Gtf-S (“GTF0459”).Will be from the coding of colibacillus expression plasmid pMP79 (SEQ ID NO:41) under aprE promoter effect The gene of the protein G TF0459 (SEQ ID NO:42) that N-end truncates is cloned into the integrated expression plasmid of bacillus subtilis In NheI and the HindIII site of p4JH and merge with bacillus subtilis AprE signal peptide on carrier.First by construct It is transformed in Escherichia coli DH10B and select on the LB flat board containing ampicillin (100 μ g/mL).Then by channel syndrome Construct pDCQ984 of real expression GTF0459 is transformed in the bacillus subtilis BG6006 comprising nine protease deficiencies (amyE::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 select on the LB flat board of chloride mycin (5 μ g/mL). Make the bacterium colony growing on the LB flat board containing 5 μ g/mL chloramphenicol streak inoculation on the LB flat board containing 25 μ g/mL chloramphenicol several Secondary.The bacillus subtilis making gained is expressed bacterial strain SG1184 and first grows simultaneously on the LB culture medium containing 25 μ g/mL chloramphenicol Then squamous subculture in the GrantsII culture medium containing 25 μ g/mL chloramphenicol, grows 2-3 days at 30 DEG C.At 15,000g and Make culture centrifuge at 4 DEG C 30 minutes and make supernatant liquid filtering pass through 0.22-μm of filter.Supernatant after filtering is divided into Part and-80 DEG C of freezings.
Made the bacillus subtilis SG1184 of expression GTF0459 (SEQ ID NO:42) by conventional fed batch fermentation Bacterial strain grows under aerobic submerged conditions.Use containing 0-0.25% corn steep solids (Roquette), 5-25g/L sodium phosphate and The solution of potassium phosphate, 0.3-0.6M ferrous sulfate, manganese chloride and calcium chloride, 0.5-4g/L magnesium sulfate and 0.01-3.7g/L sulfuric acid The nutrient medium of the solution of zinc, cuprous sulfate, boric acid and citric acid.Add defoamer FOAMBLAST 882 with 2-4mL/L Control foaming.When the initial glucose sugar that can't detect in batch, add 10-L fermentation with the charging of 50% (w/w) glucose.Portugal Grape sugar feed rate rose in several hours.Make ferment control in 30 DEG C and 20%DO, and initially stirring is 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%.When end of run, results obtain culture fermentation broth and are centrifuged it to obtain supernatant. Then make to comprise the supernatant of GTF0459 (SEQ ID NO:42) stored frozen at-80 DEG C.
Prepare Streptococcus mutans MT-4239gtf-C enzyme (SEQ ID NO:43) as follows.Use optimized with at withered grass gemma In bacillus express codon composite coding truncate pattern glucosyltransferase (gtf) (It is accredited as GI:3130088, SEQ ID NO:43；Derive from the gtf-C of Streptococcus mutans MT-4239) gene and it is by GenScript Synthesis.GENSCRIPT plasmid amplification coding is had N-end and truncates GTF0088BsT1 (the SEQ ID truncating with C-end T1 NO:45) gene (SEQ ID NO:44), and under aprE promoter effect, it is cloned into the integrated expression of bacillus subtilis In NheI and the HindIII site of plasmid p4JH and merge with bacillus subtilis AprE signal peptide on carrier.First by structure Build body to be transformed in Escherichia coli DH10B and select on the LB flat board containing ampicillin (100 μ g/mL).Then will Construct pDCQ1021 of attested expression GTF0088BsT1 is transformed into the bacillus subtilis comprising nine protease deficiencies (amyE::xylRPxylAcomK-ermC, degUHy32, oppA, Δ spoIIE3501, Δ aprE, Δ nprE, Δ in BG6006 Epr, Δ ispA, Δ bpr, Δ vpr, Δ wprA, Δ mpr-ybfJ, Δ nprB) and at the LB flat board of chloride mycin (5 μ g/mL) Upper selection.The bacterium colony making growth on the LB flat board containing 5 μ g/mL chloramphenicol is drawn on the LB flat board containing 25 μ g/mL chloramphenicol Line is inoculated several times.The bacillus subtilis making gained expresses bacterial strain SG1221 first at the LB culture medium containing 25 μ g/mL chloramphenicol Upper growth then squamous subculture in the GrantsII culture medium containing 25 μ g/mL chloramphenicol, grow 2-3 days at 30 DEG C.? Make culture centrifuge 30 minutes at 15,000g and 4 DEG C and make supernatant liquid filtering pass through 0.22-μm of wave filter.Will filter after upper Clear liquid is divided into equal portions and-80 DEG C of freezings.
Made the bacillus subtilis of expression GTF0088BsT1 (SEQ ID NO:45) by conventional fed batch fermentation SG1221 bacterial strain grows under aerobic submerged conditions.Use and contain 0-0.25% corn steep solids (Roquette), 5-25g/L phosphorus The solution of acid sodium and potassium phosphate, 0.3-0.6M ferrous sulfate, manganese chloride and calcium chloride, 0.5-4g/L magnesium sulfate and 0.01- The nutrient medium of the solution of 3.7g/L zinc sulfate, cuprous sulfate, boric acid and citric acid.Add defoamer with 2-4mL/L FOAMBLAST882 controls foaming.When the initial glucose sugar that can't detect in batch, mended by the charging of 50% (w/w) glucose Add 2-L fermentation.Glucose feed rate rose in several hours.Make ferment control in 30 DEG C and 20%DO, and initially stir For 400rpm.With 50% (v/v) ammonium hydroxide, pH is controlled in 7.2.In the fermentation running through 2-days runs, monitor fermentation parameter Such as pH, temperature, air mass flow and DO%.When end of run, results obtain culture fermentation broth and are centrifuged it to obtain Obtain supernatant.Then make to comprise the supernatant of GTF088BsT1 (SEQ ID NO:45) stored frozen at-80 DEG C.
GlucosyltransferaseGTF0459Mensuration with GTF0088BsT1 activity
Glucosyl transferase activity measure carried out by procedure below: presence or absence of 25g/L dextran (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 or the 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 at 90 DEG C of heating 5min so that GTF inactivates when 1h, 2h and 3h.By 13, Centrifuge under 000xg and within 5 minutes, remove insoluble material, make it be filtered through 0.2 μm of RC (regenerated cellulose) film afterwards.85 Use two series connection AMINEX HPX-87C posts to analyze gained filtrate (Bio-Rad, Hercules, CA) by HPLC at DEG C to determine Amount sucrose concentration.Draw the sucrose concentration of each time point relative to the reaction time and determined by the slope of linearity curve initial 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 CE of α-(1,3)-glucan hydrolase (mutant enzyme)
Coding Ma Erneifei mould (Penicillium marneffei)18224TMMutant enzyme (Be accredited as GI:212533325) gene by GenScript (Piscataway, NJ) synthesize.Open at 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 and the AscI restriction site of plasmid pTrex3, this plasmid is i.e. designed for expressing Richter scale The carrier of the target gene in wood mould (Trichoderma reesei), uses aspergillus niger acetamidase to be used for selecting.Pass through base Because rifle injects in the Plastid transformation of gained to trichoderma reesei.The concrete grammar of via Particle Bombardment Transformation is described in international PCT patent Shen Please disclose WO2009/126773 A1, the disclosure of which is herein incorporated by reference.Use the spore having from stable clone The 1-cm of son2Agar rod TRM05-3 inoculates and prepares culture medium (the following stated).Under 28 DEG C and 220rpm, culture is made to exist Shaking flask grows 4-5 days.In order to gather in the crops the protein obtaining secretion, first pass through centrifugal 10 minutes under 4000g and remove cell Group, and make supernatant liquid filtering pass through 0.2-μm of sterilizing filter.The expression of mutant enzyme MUT3325 (SEQ ID NO:47) is passed through SDS-PAGE is confirmed.
It is listed below preparing nutrient media components.
NREL-Trich Lactose Defined
Trichoderma reesei trace element
By expressing the freezing spore suspension of bacterial strain TRM05-3 in the flask with baffle plate for the 2-L with the MUT3325 of 1.0mL The minimal medium of liquid inoculation 0.5L prepares fermentation seed culture, and (minimal medium is by 5g/L ammonium sulfate, 4.5g/L phosphoric acid Potassium dihydrogen, 1.0g/L epsom salt, 14.4g/L anhydrous citric acid, 1g/L calcium chloride dihydrate, 25g/L glucose, and trace Element includes 0.4375g/L citric acid, 0.5g/L ferrous sulfate heptahydrate, 0.04g/L white vitriol, 0.008g/L five water sulfuric acid Copper, 0.0035g/L Manganous sulfate monohydrate and 0.002g/L boric acid composition.PH is 5.5).8L in transferring to 14-L fermentation tank Before preparing culture medium, make culture growth 48 hours under 32 DEG C and 170rpm.Prepare culture medium by 75g/L glucose, 4.5g/L potassium dihydrogen phosphate, 0.6g/L CALCIUM CHLORIDE DIHYDRATE, 1.0g/L epsom salt, 7.0g/L ammonium sulfate, 0.5g/L are anhydrous Citric acid, 0.5g/L ferrous sulfate heptahydrate, 0.04g/L white vitriol, 0.00175g/L cupric sulfate pentahydrate, 0.0035g/L mono- Hydrated manganese sulfate, 0.002g/L boric acid and 0.3mL/L FOAMBLAST 882 form.
For under 34 DEG C and 500rpm in the batch of grown on glucose 24h, first run fermentation.At the end of 24h, Temperature is made to be reduced to 28 DEG C and make mixing speed increase to 1000rpm.Then biological at 0.030g glucose-sophorose solid/g Under the specific feed rate of matter/hour, with mixture (62%w/w) the charging fermentation tank of glucose and sophorose.At end of run When, by centrifugal removal living beings, and use 10-kD molecular weight to retain ultrafiltration core (UFP-10-E-35；GE Healthcare, Little Chalfont, Buckinghamshire, UK) will be containing MUT3325 mutant enzyme (SEQ by filtration ID NO:47) supernatant concentration about 10 times.Make condensing protein in-80 DEG C of storages.
αThe mensuration of-glucan hydrolase (mutant enzyme) activity
Use by streptococcus sobrinus (Streptococcus sobrinus)33478TMThe enzyme of the secretion producing Carry out the insoluble change chitosan polymer needed for formation determination mutant enzyme activity.Specifically, (Brain Heart on BHI agar plate Infusion agar, Teknova, Hollister, CA) streak inoculation one ring streptococcus sobrinus33478TMGlycerine Original seed, and make flat board incubate 2 days at 37 DEG C.Choose several bacterium colony with ring with in initial medium bottle (deriving from Teknova) Inoculation 2X100mL BHI fluid nutrient medium, and at 37 DEG C, incubate culture, static holding 24h.Thin by centrifugal removal gained Born of the same parents simultaneously make gained supernatant liquid filtering pass through 0.2-μm of sterilizing filter；Collect the filtrate of 2X101mL.Add 2X in filtrate The 200g/L sucrose (final sucrose 20g/L) of 11.2mL.Under non-stirring state, make to react on 37 DEG C and incubate 67h.By Under 5000xg, centrifugal 10min collects gained polysaccharide polymer.Drain supernatant carefully.With the sterilized water of 40mL by insoluble Polymer washs 4 times.The change chitosan polymer making gained is lyophilized 48h.Change chitosan polymer (390mg) is made to be suspended in the nothing of 39mL To prepare 10mg/mL suspension in bacterium water.By ultrasonically treated (40% amplitude until compared with large crumb disappear, total～10min) Homogenizing becomes glycan suspension.Homogenized suspension is divided into equal portions and 4 DEG C of storages.
By at pH 5.5 and 37 DEG C with containing 0.5mg/mL become chitosan polymer (prepared as described above) 25mM KOAc buffer solution incubates proper amount of enzyme, starts mutant enzyme and measures.At each time point, take out the reactant mixture of aliquot And with isopyknic 100mM glycine buffer (pH 10) cancellation.Gone unless each through sudden by 5min centrifugal under 14,000xg Go out the insoluble material in sample.By P-hydroxybenzoic acid hydrazides solution (PAHBAH) (Lever M., Anal.Biochem., (1972) 47:273-279) measure the compound sugar producing each time point and the reducing end of polysaccharide polymer is carried out quantitatively, and And initial rate is determined by the slope of the junior three in time course or the linear diagram of four time points.By by 10 μ L Response sample supernatant add to the PAHBAH working solution of 100 μ L carrying out PAHBAH mensuration and heats 5min at 95 DEG C. By a reagent A (concentrated hydrochloric acids of 0.05g/mL P-hydroxybenzoic acid hydrazides and 5 volume %) of mixing and four parts of reagent B (0.05g/mL NaOH, 0.2g/mL sodium potassium tartrate tetrahydrate) carrys out preparation work solution.Absorbance at record 410nm, and pass through Deduct suitable background absorption and the calibration curve using the glucose (as reference material) of various concentration to produce calculates reduction The concentration of end.
Carry out response feature ana lysis by HPLC
Periodic sample using equipped with RI-detector from reaction1260HPLC is analyzed. At the flow velocity of 0.6mL/min and 85 DEG C, use and there is deionized waterHP-87C post (BioRad, Hercules, CA) quantitative determine sucrose, glucose, the content of lucrose and fructose in gtf reaction.At 0.6mL/ At the flow velocity of min and 85 DEG C, use and there is deionized waterHP-42A post (BioRad) quantitative determines Soluble oligosaccharide accessory substance (DP2-DP7) in gtf reaction.
Equipped with RI-detectorUltiMateTM3000 HPLC (ThermoScientific) are used for Including the sample of immobilised enzymes (embodiment 4).At the flow velocity of 0.3mL/min and 85 DEG C, use and there is deionized waterRezexTMSugar analyzed by calcium monose post.
Analyze compound sugar by NMR
Use 5-mm low temperature three to ally the communists to shake pulse field gradient (PFG) pops one's head in 500MHz (for1H) work under NMR data is gathered on Agilent DD2 spectrometer.It is real by observation transmitter frequency is carefully placed in " Pu Shi (presat) " At the resonance of the residual water signal tested, and then use 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, 65536 data points, the 90-degree pulse of 4s presaturation and 5.85 μ s.Sample temperature is made to be held in 25 DEG C. By the D by 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 together and prepares sample to 5-mm NMR pipe, 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.Reduce for α RE End (RE) is assigned as 5.2, is 4.65 for β RE.
Embodiment 1
Prepare syrup by polymerising sucrose
This embodiment disclose and make sucrose polymerization produce soluble sugar by using gtf enzyme in glucan synthetic reaction The general fashion of mixture.Specifically, being prepared for the filtrate of glucan synthetic reaction, then being concentrated is syrup.
Add sucrose (3000g) to the PEF bucket of the 5-gallon of cleaning.By water (18.1L) and FermasureTM (10mL) add to bucket, and by adding 5 volume %NaOH and 5 volume %H2SO4Regulate pH to 7.0.Final volume is About 20L, and as by measured by HPLC, the initial concentration of sucrose is 152.5g/L.By adding as in conventional method part Rough gtf enzyme (the SEQ ID NO:3) extract of 0.3 volume % of described preparation causes glucan polymerisation.This extraction Thing comprises the protein of about 2.9mg/mL.The overhead machinery motor equipped with glass axle and PTFE blade is used to carry to reaction solution For stirring.
After 48 hours, HPLC analyzes display, has consumed the sucrose of 96% and has thought that reaction completes.Use 325-mesh Steel wire and 40-urn filter use Bu Shi filter funnel to filter insoluble poly-α-1 removing reaction, 3-beta-glucan products. Then use Rotary Evaporators (bath temperatures of 40 DEG C-50 DEG C) that mother liquor (filtrate) is concentrated into the total sugar concentration of about 320g/L sugar.Dense The composition of contracting filtrate is provided in table 2.
Table 2
The composition of the concentrated filtrate of glucan 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 concentrated filtrate of glucan synthetic reaction comprises sucrose, fructose, glucose, lucrose and low Glycan DP2-DP7.
Embodiment 2
The impact on syrup solution in the filtrate of glucan synthetic reaction for the enzyme
This embodiment determines various glucoamylase (EC 3.2.1.3), transglucosidase (EC2.4.1.24), β-Portugal The activity of glycosidase (EC 3.2.1.21), AMS (EC 3.2.1.1) and glucosidase (EC 3.2.1), it is therefore intended that subtract The concentration of lucrose and/or compound sugar accessory substance in the concentrated filtrate of little glucan synthetic reaction.Some enzyme is such as 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 accessory substances, causes the monose (glucose and fructose) in treated filtrate Corresponding increase.
First brief outline of procedure according to embodiment 1 prepares the filtrate of glucan synthetic reaction and is concentrated as syrup.Should The composition of concentrated filtrate is provided in table 3.NMR analyzes display, and the α (1,3) being present in syrup is 78 with the ratio of (1,6) key ∶22。
Table 3
The composition of the concentrated filtrate of glucan synthetic reaction
Sucrose Lucrose Glucose Fructose DP2 DP3+ Total amount
g/L 161 210 93 302 33 61 860
Wt% 18.7 24.4 10.8 35.1 3.8 7.1 100.0
The syrup of table 3 is for testing various enzyme to the lucrose of glucan synthetic reaction and compound sugar accessory substance Hydrolysing activity.Assuming that lucrose comprises special keys [α (1,5)-glucityl fructose] and compound sugar mainly comprise α (1, 3) and α (1,6) glucityl-glucose key, when these experiments start which kind of enzyme can be used for hydrolysis both accessory substances this not It is apparent from.For should analyzing, select the enzyme (table 4) with different activities.
Table 4
The enzyme assessment hydrolyzing for lucrose and compound sugar
a DuPont Industrial Biosciences
The condition of the syrup processing table 3 with above each enzyme is provided in table 5 (carrying enzyme amount, time, temperature, pH, sugar concentration) 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 hydrolysis Bacterium disaccharides and the percentage of DP3+ (at least DP3-DP7) compound sugar.DP3+ percent hydrolysis is calculated as (1-(DP3 in final syrup Weight % of+compound sugar)/weight % of DP3+ compound sugar (in the initial syrup)).Similarly, lucrose hydrolysis percentage Ratio is calculated as (1-(lucrose weight % in final syrup)/(lucrose weight % in initial syrup)).
Table 5
By lucrose and compound sugar in various enzyme hydrolysis concentrated filtrates
The sugared concentration (total concentration of sucrose, glucose, fructose, lucrose and compound sugar) that a is measured by HPLC； The value of record is rounded up to closest to 10g/L increment.
BDP3+ comprises DP3-DP7, but also can comprise bigger soluble oligosaccharide, and it has when being prepared by some gtf enzyme Have higher α-1,6 keys and α-1, the ratio of 3 keys.
Table 5 shows Isosorbide-5-Nitrae-alpha-Glucosidase and 1, and 6-alpha-Glucosidase demonstrates part (embodiment 2.1) or few (enforcement Example 2.2) hydrolysis lucrose, but from compound sugar, discharge some glucose.AMS (embodiment 2.3 and reality Execute example 2.4) use demonstrate that the activity to target compound is minimum.Similarly, the use of Pullulanase (embodiment 2.5) is shown Go out minimum activity.
Cellulase (embodiment 2.14 and 2.15) is largely invalid when hydrolyzing lucrose, but hydrolysis Some compound sugar.
Although compound sugar does not comprise β key, but astoundingly, β-glucosyl enzym also shows that extremely low (ACCELERASE BG, embodiment 2.9) to the hydrolysis scope of high (NOVO 188, embodiment 2.10 and 2.11).The relative potency of these enzymes It is widely varied.In some cases, the amount of the compound sugar of hydrolysis substantially exceeds (embodiment 2.11) or close to (embodiment 2.12) percentage of the lucrose hydrolyzing.In other cases, lucrose is by β-glucosyl enzym height water Solving, compound sugar is hydrolyzed (embodiment 2.13) by moderate simultaneously.Relatively High Defferential between the result of viewed β-glucosyl enzym Showing, present in tested β-glucosyl enzym preparation, other enzymes such as glucoamylase 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 hydrolysis compound sugar High activity with lucrose.Seemed in some cases by the lucrose that transglucosidase hydrolyzes It is quantitative, and the DP3+ material more than 95% is hydrolyzed to glucose and DP2 (embodiment 2.7) under higher load enzyme amount. The transglucosidase using purified pattern demonstrates similar activity (embodiment 2.8), show observed hydrolysis owing to Transglucosidase rather than background activity.
Glucoamylase (embodiment 2.16-2.18) illustrates a range of work for lucrose and compound sugar Property.Only have a kind of tested glucoamylase (embodiment 2.18) and give lucrose and the compound sugar water less than 30% Solve.
Result in table 5 shows, alpha-Glucosidase such as DIAZYME RDF ULTRA, glucoamylase and turn glucoside Enzyme hydrolyzable is present in the lucrose accessory substance in glucan reaction 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 When planting activity, it is believed that this activity also extends to comprise α-1, the compound sugar of 5 glucityls-fructose key.
The result of table 5 further demonstrates that, alpha-Glucosidase such as glucoamylase and transglucosidase hydrolyzable are present in Compound sugar accessory substance in glucan reaction filtrate.Because these compound sugar are mainly by through α-1,3 and/or α-1,6 is bonded Glucose monomer unit forms (embodiment 3), so the data of table 5 show alpha-Glucosidase hydrolyzable α-1,3 glucityls-grape Sugar and/or α-1,6 glucityls-fructose key.
Because the lucrose of alpha-Glucosidase usually effective hydroglucan synthetic reaction and/or compound sugar are secondary Product, so these enzymes can be used alone or in combination reduce the Portugal from the monose comprising increments and the sugared accessory substance of reduction amount Formose Reaction filtrate produces the process time needed for high-purity syrup.The example of effective enzyme combination can be for for hydrolyzing bright beading The transglucosidase of bacterium disaccharides such as TG L-2000, and effectively hydrolyze glucoamylase (the such as GC of compound sugar accessory substance 321)。
Therefore, alpha-Glucosidase can hydrolyze (i) α-1 in specific sugar, 5 glucityls-fructose key, and (ii) α-1 independently, 3 and α-1,6 glucityls-glucose key.
Embodiment 3
The comparison of the key distribution of glucan reaction filtrate component before and after enzyme hydrolysis
This embodiment measures transglucosidase (EC 2.4.1.24) and β-glucosyl enzym (EC 3.2.1.21) to being present in Lucrose in the concentrated filtrate of glucan synthetic reaction and the hydrolysing activity of compound sugar accessory substance.Discovery turns glucoside Enzyme decreases the amount of these accessory substances, causes the monose in treated filtrate (glucose and fructose) to increase accordingly.
The compound sugar accessory substance being present in the filtrate of above glucan synthetic reaction comprises the glucose-grape of ＞ 90% Sugar key, such as (conventional method) that measured by NMR.In glucose-glucose key, about 78% expression α-1,3 keys and about 22% expression α-1,6 keys.
NMR is for measuring the key feature of the material generating after hydrolysis in above example 2.11.As it is shown in figure 1, it is corresponding In α-1, the peak of 3 keys reduces by 86%, corresponds to α-1, and the peak of 6 keys only reduces by 2.3%, and corresponding to lucrose peak Peak reduces by 21%.Although sucrose is almost quantitatively by this enzyme hydrolysis, but Novo 188 seem can not hydrolyzing alpha-1,6 keys.
NMR is similarly used for the key feature of the material that mensuration TG L-2000 (SEQ ID NO:1) transglucosidase generates (Fig. 2).By the concentrated filtrate (deriving from the material of table 3) of 210 μ L, the D of 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 balance Time zero spectrum (raw material in Fig. 2), and then add the enzyme of 0.5 volume %.At 60 DEG C, sample is made to put down in probe again Weigh and add pad, and measuring within a few minutes analyzed.With TG L-2000 enzyme (material treated in Fig. 2) After processing 10 hours, corresponding to α-1, the peak of 3 keys reduces by 41%, corresponds to α-1, and the peak of 6 keys reduces by 36%, and corresponds to The peak of lucrose reduces ＞ 95% (Fig. 2).Observing that α-reducing end peak and β-reducing end peak both of which increase, this is corresponding Increase (Fig. 2) in fructose and glucose.
These results show that transglucosidase can will comprise α-1,3 and α-1, the compound sugar of 6 keys be converted into glucose and Lucrose can be converted into fructose and glucose.Therefore, (i) α-1 in the specific sugar of transglucosidase hydrolyzable, 5 Portugals Glycosyl-fructose key and (ii) α-1,3 and α-1,6 glucityls-glucose key.
Embodiment 4
Use the lucrose in immobilized enzyme hydrolysis glucan reaction filtrate and compound sugar
This embodiment describes use immobilization 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 glucan synthetic reaction and other compound sugar.Specifically Ground, (SEQ ID NO:1, available from Genencor/DuPont Industrial to have studied immobilization transglucosidase TG L-2000 And immobilization glucoamylase GC-147 is (available from Genencor/DuPont Industrial Biosciences) Biosciences) to lucrose in the filtrate of glucan synthetic reaction and compound sugar DP2, DP3 and HS (high sugar, DP4 +) impact that hydrolyzes.
Glucoamylase and transglucosidase are fixed by the method according to described in United States Patent (USP) 5541097, and it is public Open content to be herein incorporated by reference.
In the typical method for fixing glucoamylase and transglucosidase, make two batches of porous that about 8.0g/ criticizes Granular silicon diatomaceous earth (EP Minerals, Reno, NV) by distilled water aquation and is then transferred to diameter 1.5-cm, highly 30-cm In glass column reactor.Flow up pumping water with about 6-7mL/min, thus from whole three posts, remove particulate.Generally, exist In one hour, water effluent does not contains particulate.Water is drained into the top of granular bed of diatomaceous earth and by the poly-second of 0.1%w/v from post Alkene imines (PEI, the EPOMIN P-1050) aqueous solution is replaced.Flowing then up pumps the PEI solution of 3500mL and makes effluent Cycle through bed 2 hours.Then, at room temperature, graininess bed of diatomaceous earth is washed 2 hours with the distilled water flowing up, to remove Remove the PEI dissociating.So, it is thus achieved that particulate Si diatomaceous earth-PEI carrier.
Meanwhile, add the 3.5mL glucoamylase GC-147 with the activity limiting in table 4 to the 0.02M second of 315ml In phthalate buffer (pH 4.5).Then, by the 50%w/w glutaraldehyde of 1.575g (GA-50) it is added slowly to It in glucose starch enzyme aqueous solution, is gently mixed, and make glutaraldehyde and glucose starch enzyme aqueous solution at 20 DEG C-25 under being gently mixed Reacting 4 hours at a temperature of DEG C, it results in the treated enzyme-glutaraldehyde adduction comprising treated glucoamylase Thing.Use has the transglucosidase TG L-2000 of the activity limiting in table 4 rather than glucoamylase repeats these steps respectively Suddenly, the treated enzyme-glutaraldehyde adduct comprising treated transglucosidase is thus resulted in.
Then, every kind of treated enzyme-glutaraldehyde adduct is made to comprise self of particulate Si diatomaceous earth-PEI carrier at it 4 hours (20-25 DEG C) of circulation in post (produced above).With water, excessive treated adduct is washed out from carrier.Thus Preparation has the post of immobilization glucoamylase or transglucosidase.
The glucan filtrate of the composition having defined in table 3 is diluted to 180g/L, and regulation is to pH4.5, and makes it lead to Cross the post comprising immobilised enzymes.Column temperature is controlled in 60 DEG C.After column equilibration 16 hours, timing sampling under different in flow rate. Measured sugar composition (table 6) of hydrolysis product by HPLC.When each change flow rate set, post is made to put down before sampling again Weigh at least 1-2 bed volume.Calculate the degree of hydrolysis of lucrose and compound sugar by the mode described in embodiment 2.Test three Plant post to configure: 1) immobilization glucoamylase, 2) immobilization transglucosidase, and 3) solid after immobilization transglucosidase Surely glucoamylase is changed.
Table 6
Immobilization glucoamylase and the application of transglucosidase hydrolysis compound sugar and lucrose
Table 6 shows, as average contact time (being defined as nominal column volume divided by mean flow rate) increases, and leukonid two The degree of hydrolysis of sugar and compound sugar generally increases.It is particularly preferred with immobilization transglucosidase hydrolysis lucrose, because of Even if being surveyed the fastest flow velocity by use also do not observe significant difference.Although each post is shown respectively suitable conversion, but Portugal The combination of saccharogenic amylase and transglucosidase gives the highest hydrolysis of compound sugar.
Therefore, the use of immobilization glucoamylase or transglucosidase or this two classes immobilised enzymes shows hydrolysis Comprise α-1,3 and α-1, the compound sugar of 6 glucityls-glucose key and the effective technology of lucrose.These results Meet those of embodiment 2.The fixing of other alpha-Glucosidases should provide similar result.
Embodiment 5
It is enriched with fructose by chromatography from glucan reaction filtrate
This embodiment disclose the fructose that can how be enriched with further in glucan reaction filtrate by chromatography.
Generally, when by chromatography separation glycan molecule, component wash-out and molecular dimension negative correlation, therefore first elute Maximum molecule.Therefore, the filtrate for glucan synthetic reaction, first elutes compound sugar, is disaccharides afterwards, followed by singly Sugar.Use sodium cation resin to separate and cannot be sufficiently separated fructose and glucose, and all of lucrose, sucrose and DP2 is eluted out jointly.Ion exchange resin separating glucose that cation be calcium and fructose are preferably used.
First brief outline of procedure according to embodiment 1 prepares the filtrate of glucan synthetic reaction and is concentrated as syrup.Should The composition of concentrated filtrate is provided in table 7.
Table 7
The composition of the concentrated filtrate of glucan synthetic reaction
The syrup of table 7 is filtered and is diluted to nonionic exchanged water 25g dry solid/100g solution, and feed to comprising In the post of crosslinked strong-acid ion exchange resin (calcium form).The physical parameter of this post shows in table 8.Syrup by dilution (15.8L) feed to the post being held in 65 DEG C, hereafter with the water elution post that flow velocity is 30L/ hour.
Table 8
The physical parameter of post
In this separation, remaining in the lucrose in post longer than sucrose, this is likely due to lucrose It with calcium cation complex, and is in fact jointly eluted out with glucose.Two fractions comprising fructose are made to separate.Between Elute fraction 5.1 between 47 minutes and 120 minutes, and between 120 minutes and 172 minutes, elute fraction 5.2. In charging to the fructose of chromatographic isolation, the fructose of 95.7% separates with ＞ 90% purity.As measured by HPLC, each fraction Product distribution in (5.1 and 5.2) is shown in table 9.
Table 9
Comprise the product distribution of the chromatographic isolation fraction of significant quantity fructose
Fraction Sucrose Lucrose Glucose Fructose DP3+ Other Total amount The fructose % reclaiming
5.1 31.9 34.8 20.8 3.9 5.4 4.8 100 3.9
5.2 0.0 1.0 0.8 97.7 0.0 0.6 100 95.7
Because the feed composition for this separation comprises 36.0% fructose, so total stream of 34.5% is to have altogether The fructose syrup of ＞ 90 weight %DS fructose reclaims.If the sucrose ignored in charging, then the sugar of 40.7% is to have ＞ 90 weight The fructose syrup of amount %DS fructose reclaims.
Therefore, the fructose in glucan reaction filtrate can be enriched with further by chromatography.Example 6 below demonstrates, The glucan filtrate that transglucosidase hydrolyzes can be used to strengthen this process.
Embodiment 6
It is enriched with fructose by chromatography from the glucan reaction filtrate of hydrolysis
This embodiment illustrates, compared with separating levulose from unhydrolysed glucan filtrate, from compound sugar and leukonid In the glucan filtrate that disaccharides has been hydrolyzed, separating levulose causes the yield of high-purity fructose syrup to increase.
By under 60 DEG C and pH 4.5 to being processed by 1 volume % transglucosidase TG L-2000 (SEQ ID NO:1) The glucan filtrate of 24 hours carries out concentrating (vacuum at 50 DEG C) and prepares syrup.During concentration process, observe that some are low Glycan formation, it can be desired, because it is known that transglucosidase produces compound sugar under the monose of high concentration.Syrup has The end product described in Table A is had to be distributed.
Table A
The composition of the concentration glucan filtrate hydrolyzing before it is concentrated
Sucrose Lucrose Glucose Fructose DP2 DP3+ Total amount
g/L 3 ＜ 10 294 409 73 81 ～870
Weight % 0.3 1.1 33.7 47.0 8.4 9.3 100
Syrup described in Table A filtered and is diluted to 25.4g DS/100g solution with nonionic exchanged water, and feeding to bag In post containing crosslinked storng-acid cation exchange resin (calcium form).The physical parameter of post is shown in table B.Then by dilution Syrup (169g) feeds to the post being held in 65 DEG C, hereafter with the water elution post that flow velocity is 50mL/min.
Table B
The physical parameter of post
Resinous type FINEX CS11GC
Ionic species Ca2+
Crosslinking, divinylbenzene % 5.5
Granularity (mm) 0.34
Bed length (m) 1.69
Column diameter (m) 0.093
Two fractions comprising fructose are made to separate.Fraction 6.1 is being eluted between 73 minutes and 103 minutes, and Fraction 6.2 is being eluted between 103 minutes and 120 minutes.In charging to the fructose of chromatographic isolation, charging is to post The fructose of 93.0% separates with ＞ 90% purity in fraction 6.2.Product as HPLC surveys, in each fraction (6.1 and 6.2) Distribution is shown in table C.
Table C
Comprise the product distribution deriving from the chromatographic isolation fraction of the fructose of the glucan filtrate of hydrolysis
The separative 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, prepare by the glucan filtrate hydrolyzing without transglucosidase compared to by chromatography The embodiment 5 of syrup is obtained, and the chromatographic separation and purification of this material causes the yield of high-purity fructose syrup to increase.Because using Comprise 47% fructose (Table A) in the feed composition of this separation, so total stream of 43.7% is to have ＞ 90 weight %DS fruit The fructose syrup of sugar reclaims.43.7% rate of recovery is significantly better than 34.5% rate of recovery of embodiment 5 herein.
Therefore, compared with from unhydrolysed glucan filtrate during separating levulose, from the Portugal having been hydrolyzed by transglucosidase In glycan filtrate, separating levulose causes the yield of fructose to increase.
Embodiment 7
Prepare ethanol by the filtrate of glucan synthetic reaction of fermenting
This embodiment disclosing the fermentation of glucan filtrate yeast is ethanol.
By making yeast extract be suspended in running water (2.4L, optical density is 65 at 600nm) and then using LEGEND Yeast extract is centrifuged 5 minutes under 4500g and washs yeast (saccharomyces cerevisiae) cream by XTR centrifuge (Thermo Scientific) (Tonon mill, Brazil).After decantation supernatant, made yeast cells resuspended by other centrifuging twice and concentrate. After third time is washed, regulated pH to 2 by adding 5 weight % sulfuric acid.Use GENESYS 20 4001 spectrophotometer (Thermo Scientific) measures optical density and is regulated to 100 (at 600nm) by adding running water.Will be through adjusting The yeast extract (1.5L) of joint adds to 7.5-L BIOFLO310 fermentor vessel (New Brunswick).Fermentation tank is set For keeping the temperature of 30 DEG C, and stir at 100 rpm.Although during fermentation determining pH, but its by add acid or Aqueous slkali controls.
Preparation comprises yeast extract (10g/L), peptone (20g/L) and sugared the entering of 200g/L deriving from glucan filtrate Material solution is simultaneously sterilized 15 minutes at 121 DEG C by PHOENIX AV-250PLUS autoclave.Before fermentation starts, make charging molten Liquid is cooled to 25 DEG C (room temperatures).Under 684mL/ hour speed, sterilized feedstock solution (3.5L) is added little to fermentation tank about 5 When, and make fermentation carry out 22 hours.
During fermentation timing sampling by GENESYS 20 4001 spectrophotometric analysis optical density, is reflected by PAL-3 Brix Scale analyzed by meter (Atago), 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, within 5 minutes, separate yeast by using LEGEND XTR centrifuge to centrifuge under 4500g thin Born of the same parents.After decantation supernatant, made yeast resuspended twice by centrifuging again and concentrate.After third time is washed, by adding 5 Weight % sulfuric acid regulates pH to 2.Use GENESYS 20 4001 spectrophotometric determination optical density and by adding running water Regulated to 100 (at 600nm).According to above-mentioned the same terms, the yeast cells reclaiming from previous fermentation is used to carry out again Fermenting twice circulates, and all uses fresh feed every time.With the fermentation results of the yeast acquisition reclaimed for the first time and for the second time respectively It is provided in table 11 and table 12.
Table 11
Use charging and the time-histories fermenting characteristic of 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
Use charging and the time-histories fermenting characteristic of 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 the first fermentation, but yeast cells reclaims via second and starts to adapt to And consume lucrose.Use reclaim yeast three fermentation cycle after, alcohol fermentation titre by 33g/L (table 10, 22 hours) increase to 54g/L (table 12,21 hours), but even if still there is significant quantity in last circulation wild Oryza species Lucrose.
Therefore, glucan filtrate can be used for sweat to produce ethanol.
Embodiment 8
Prepare ethanol by the glucan filtrate of fermentation hydrolysis
This embodiment illustrates makes wherein lucrose and compound sugar side components previous by the glucan of saccharification Filtrate fermentation causes ethanol yield to increase.
The method that fermentation describes according to embodiment 7 is carried out, but used here as previously by transglucosidase (TG L- 2000, SEQ ID NO:1) the glucan filtrate that processes.The glucan filtrate of following preparation hydrolysis.Glucan filtrate is regulated extremely 300g sugar/L, then uses 1.0M NaOH and 5 weight % sulfuric acid to regulate pH to 4.0.The final volume of said preparation is 6.75L.Then use PHOENIX AV-250PLUS autoclave to sterilize 15 minutes filtrate at 121 DEG C, then temperature is adjusted Joint is to 60 DEG C.Make the TG L-2000 enzyme extract as described in table 4 (135mL) mix with sterilized filtrate, and at 60 DEG C and Solution is made to incubate 72 hours in cultivating shaking table (IKA KS4000) under 100rpm.It is thus prepared for the glucan filtrate of hydrolysis.
By make cream be suspended in running water (2.4L, optical density is 65 at 600nm) and then use LEGEND XTR from Yeast extract is centrifuged 5 minutes under 4500g by scheming washs yeast (saccharomyces cerevisiae) cream (Bom Retiro mill, Brazil). After decantation supernatant, made yeast resuspended by centrifuging twice again and concentrate.After third time is washed, by adding 5 weights Amount % sulfuric acid regulates pH to 4.5, and uses GENESYS 20 4001 spectrophotometric determination optical density and by adding certainly Water is regulated to 100 (at 600nm).Yeast extract (1.5L) will be adjusted add to 7.5-L BIOFLO310 fermentation tank In container.Fermentation tank is set as keep the temperature of 30 DEG C, stirs at 100 rpm, and use 4M ammonium hydroxide aqueous solution or 5 Weight % aqueous sulfuric acid makes pH be held in 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 by PHOENIX AV-250Plus autoclave.Before fermentation starts, make charging molten Liquid is cooled to 25 DEG C (room temperatures).Under 684mL/ hour speed, sterilized feedstock solution (3.5L) is added little to fermentation tank about 5 When, and make fermentation carry out 22 hours.
During fermentation timing sampling by GENESYS 20 4001 spectrophotometric analysis optical density, is reflected by PAL-3 Brix Scale analyzed by meter, analyzes sugar and concentration of alcohol with HPLC (conventional method).These results are summarized in table 13.
Table 13
The charging of the first alcohol fermentation of the glucan filtrate through hydrolysis for the employing 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, within 5 minutes, separate yeast by using LEGEND XTR centrifuge to centrifuge under 4500g thin Born of the same parents.After decantation supernatant, made yeast cells resuspended twice by centrifuging again and concentrate.After third time is washed, pass through Add 5 weight % sulfuric acid to regulate pH to 2.Use GENESYS 20 4001 spectrophotometric determination optical density and by adding Running water is regulated to 100 (at 600nm).According to above-mentioned the same terms, use the yeast cells reclaiming from previous fermentation Carry out fermenting twice circulation again, all use fresh feed every time.The yeast cells reclaiming for the first time and for the second time is used to obtain Fermentation results is respectively provided in table 14 and table 15.
Table 14
The charging of use the first recovery yeast cells and the glucan filtrate through hydrolyzing and time-histories fermenting characteristic
List the dense of in charging and each fermentation time point (0-21 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
Table 15
The charging of use the second recovery yeast cells and the glucan filtrate through hydrolyzing and time-histories fermenting characteristic
List the dense of in charging and each fermentation time point (0-21 hour) ethanol (EtOH) and sugar compounds Degree (g/L).
Starting to ferment about six hours in, all fermentations are basically completed, and cause the ethanol titre of 57-60.0g/L.Will These fermentations compare, with those of embodiment 7, those phases demonstrating and using the fermentation of unhydrolysed glucan filtrate to obtain Ratio, glucan filtrate hydrolyzed before it ferments and causes faster and bigger ethanol yield.
Therefore so that it is middle lucrose and compound sugar side components are caused by the glucan filtrate fermentation of saccharification Ethanol yield increases with faster speed.This saccharification can use such as transglucosidase to carry out.
Embodiment 9
The synchronous saccharification of glucan filtrate and fermentation
This embodiment disclose the synchronous saccharification of the charging comprising glucan filtrate and fermentation may result in enhanced fermentation spy Property.
It is suspended in running water (2.4L, optical density is 65 at 600nm) by making yeast extract and then uses LEGEND XTR centrifuge yeast extract is centrifuged 5 minutes under 4500g wash yeast (saccharomyces cerevisiae) cream (Bom Retiro mill, Brazil).After decantation supernatant, made yeast cells resuspended by centrifuging twice again and concentrate.After third time is washed, Regulated pH to 4.5 by adding 5 weight % sulfuric acid, and use GENESYS 20 4001 spectrophotometric determination optical density And regulated to 100 (at 600nm) by adding running water.Add the yeast extract (1.5L) being adjusted to 7.5-L In BIOFLO310 fermentor vessel.Fermentation tank is set as keep the temperature of 30 DEG C, stirs at 100 rpm, and use 4M hydrogen The amine-oxides aqueous solution or 5 weight % aqueous sulfuric acids make pH be held in 4.5.
Preparation comprises yeast extract (10g/L), peptone (20g/L) and sugared the entering of 200g/L deriving from glucan filtrate Material solution is simultaneously sterilized 15 minutes at 121 DEG C by PHOENIX AV-250PLUS 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 extract (1%v/v) adds to sterilized feedstock solution.At present TG L-2000 enzyme will be comprised 684mL/ is little Feedstock solution (3.5L) is added to fermentation tank about 5 hours, and makes fermentation carry out 48 hours.
During fermentation timing sampling by GENESYS 20 4001 spectrophotometric analysis optical density, is reflected by PAL-3 Brix Scale analyzed by meter (Atago), analyzes sugar and concentration of alcohol with HPLC (conventional method).These results are summarized in table 16.
Table 16
The charging of the synchronous saccharification of glucan filtrate and alcohol fermentation and time-histories fermenting characteristic
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, was similar to the fermentation (embodiment that filtrate before fermentation step has been hydrolyzed 8), and be given compared to use do not hydrolyze the slightly more excellent ethanol titre (62g/L) of filtrate (embodiment 7).Additionally, 6 is little When consume almost all of lucrose (table 16 is compared) with table 13-15.Extremely send out if directly carbohydrase is added Ferment thing, then except before facing fermentation by carbohydrase such as TG L-2000 interpolation in addition to the charging comprising glucan filtrate, it should Obtain similar result.
Therefore, comprise the synchronous saccharification of the charging of glucan filtrate and fermentation may result in enhanced fermentation character, such as increase The consumption of (i) glucan 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 compound sugar of 6 glucityls-glucose key (with The embodiment of lower offer is the 11st, the 12nd, in 15 and 16).
The discovery of rod aspergillus alpha-Glucosidase (Aclglul)
The bacterial strain of rod aspergillus is chosen as can be used for the possible source of other enzymes of various commercial Application.Identify in rod aspergillus A kind of gene code alpha-Glucosidase (referred to as " Aclglul "), and the sequence of this gene is provided in SEQ ID NO:4. The corresponding protein of SEQ ID NO:4 coding is provided in SEQ ID NO:5.Aclglul belongs to based on PFAM search The glycosyl hydrolase family 31 of (pfam.sanger.ac.uk web link).At N-end, protein (SEQ ID NO:5) has Have the signal peptide of a length of 19 amino acid, as SignalP edition 4 .0 predict (Nordahl Petersen et al. 2011, Nature Methods, 8:785-786).The existence of signal peptide shows the enzyme that Aclglul is secretion.The mature form of prediction The amino acid sequence of Aclglul is shown as SEQ ID NO:6.
The expression of rod aspergillus alpha-Glucosidase Aclglul
It is cloned into synthesis Aclglul gene in pTrex3gM expression vector and (be described in U.S. Patent Application Publication 2011/ 0136197, it is herein incorporated by reference) and by named for gained plasmid pJG294.The sequence of Aclglul gene is passed through DNA sequencing is confirmed.
Use 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 (being described in WO05/001036).Prediction comprises SEQ ID The Protein secretion of NO:6 is in extracellular medium, and filtered culture medium is used for carrying out SDS-PAGE and α-glucoside Enzyme assay confirms expression of enzymes.
The discovery of Fei Shi Xin Satuo bacterium (Neosartorva fischeri) alpha-Glucosidase Nfiglul
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 A kind of gene code alpha-Glucosidase (referred to as " Nfiglul ") in family name Xin Satuo bacterium, and the sequence of this gene is provided in SEQ In ID NO:7.It is provided in SEQ ID NO:8 by the corresponding protein of SEQ ID NO:7 coding.Nfiglul belong to based on The glycosyl hydrolase family 31 of PFAM search (pfam.sanger.ac.uk web link).At N-end, protein (SEQ ID NO:8) there is the signal peptide of a length of 19 amino acid, as SignalP edition 4 .0 predicts (Nordahl Petersen et al. 2011, Nature Methods, 8:785-786).The existence of signal peptide shows the enzyme that Nfiglul is secretion.The ripe shape of prediction The amino acid sequence of the Nfiglul of formula is shown as SEQ ID NO:9.
The expression of Fei Shi Xin Satuo bacterium alpha-Glucosidase Nfiglul
It is cloned into synthesis Nfiglul gene in pTrex3gM expression vector and (be described in U.S. Patent Application Publication 2011/ 0136197) and by named for gained plasmid pJG295.The sequence of Nfiglul gene is confirmed by DNA sequencing.
Use 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 (being described in WO05/001036).Prediction comprises SEQ ID The Protein secretion of NO:9 is in extracellular medium, and filtered culture medium is used for carrying out SDS-PAGE and α-glucoside Enzyme assay confirms expression of enzymes.
The discovery of Neuraspora crassa (Neurospora crassa) alpha-Glucosidase Ncrglul
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 A kind of gene code alpha-Glucosidase (referred to as " Ncrglul ") in neurospora, and the sequence of this gene is provided in SEQ ID In NO:10.It is provided in SEQ ID NO:11 by the corresponding protein of SEQ ID NO:10 coding.Ncrglul belong to based on The glycosyl hydrolase family 31 of PFAM search (pfam.sanger.ac.uk web link).At N-end, protein (SEQ ID NO:11) there is the signal peptide of a length of 22 amino acid, as SignalP edition 4 .0 predicts (Nordahl Petersen etc. People, 2011, Nature Methods, 8: 785-786).The existence of signal peptide shows the enzyme that Ncrglul is secretion.The one-tenth of prediction The amino acid sequence of the Ncrglul of ripe form is shown as SEQ ID NO:12.
The expression of Neuraspora crassa alpha-Glucosidase Ncrglul
It is cloned into synthesis Ncrglul gene in pTrex3gM expression vector and (be described in U.S. Patent Application Publication 2011/ 0136197) and by named for gained plasmid pJG296.The sequence of Ncrglul gene is confirmed by DNA sequencing.
Use 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 (being described in WO05/001036).Prediction comprises SEQ ID The Protein secretion of NO:12 is in extracellular medium, and filtered culture medium is used for carrying out SDS-PAGE and α-glucose Glycosides enzyme assay confirms 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 a kind of gene code alpha-Glucosidase (referred to as " TauSec098 ") in Rasamsonia composticola, And the sequence of this gene is provided in SEQ ID NO:13.The corresponding protein of SEQ ID NO:13 coding is provided in SEQ In ID NO:14.TauSec098 belongs to the glycosyl hydrolase based on PFAM search (pfam.sanger.ac.uk web link) Family 31 and comprise N-end CBM 20 domain.At N-end, protein (SEQ ID NO:14) has a length of 22 The signal peptide of amino acid, as SignalP edition 4 .0 predicts (Nordahl Petersen et al. 2011, Nature Methods, 8:785-786).The existence of signal peptide shows the enzyme that TauSec098 is secretion.The mature form of prediction The amino acid sequence of TauSec098 is shown as SEQ ID NO:15.
The expression of Rasamsonia composticola alpha-Glucosidase TauSec098
Synthesis TauSec098 gene is cloned into trichoderma reesei by Generay Biotech Co. (Shanghai, China) In expression vector pGXT (pTTT-plasmid) and by named for gained plasmid pGX256-TauSec098.TauSec098 base The sequence of cause is confirmed by DNA sequencing.
Use protoplast transformation (Te ' o et al., J.Microbiol.Methods 51:393-399,2002) by plasmid PGX256-TauSec098 is transformed in the Li's Trichoderma strains of quadruple disappearance (being described in WO05/001036).At 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；Epsom salt 0.6g/L；Calcium chloride dihydrate 0.6g/L；Ferrous sulfate (II) 5mg/L；Zinc sulfate 1.4mg/L； Cobalt 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 grows, collect the spore of transformant and transfer to new second In acid amides agar plate.After acetamide plates grows 5 days, by 1 × 108Spore inoculating is to the 30ml Portugal in 250-mL shaking flask In grape sugar/sophorose defined medium.Shaking flask is made to shake 5 days at 28 DEG C.Derive from the supernatant of these cultures for demonstrate,proving The expression (SDS PAGE) of real mature T auSec098 enzyme (SEQ ID NO:15) 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 a kind of gene code alpha-Glucosidase (referred to as " TauSec099 ") in Rasamsonia composticola, And the sequence of this gene is provided in SEQ ID NO:16.The corresponding protein of SEQ ID NO:16 coding is provided in SEQ In ID NO:17.TauSec099 belongs to the glycosyl hydrolase based on PFAM search (pfam.sanger.ac.uk web link) Family 31.At N-end, protein (SEQ ID NO:17) has the signal peptide of a length of 17 amino acid, such as SignalP version Originally 4.0 (Nordahl Petersen et al., 2011, Nature Methods, 8:785-786) is predicted.Depositing of burst At the enzyme showing that TauSec099 is secretion.The amino acid sequence of the TauSec099 of the 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 by Generay Biotech Co. (Shanghai, China) In expression vector pGXT (pTTT-plasmid) and by named for gained plasmid pGX256-TauSec099.TauSec0998 The sequence of gene is confirmed by DNA sequencing.
Use protoplast transformation (Te ' o et al., J.Microbiol.Methods 51:393-399,2002) by plasmid PGX256-TauSec099 is transformed in the Li's Trichoderma strains of quadruple disappearance (being described in WO05/001036).At 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；Epsom salt 0.6g/L；Calcium chloride dihydrate 0.6g/L；Ferrous sulfate (II) 5mg/L；Zinc sulfate 1.4mg/L； Cobalt 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 grows, collect the spore of transformant and transfer to new second In acid amides agar plate.After acetamide plates grows 5 days, by 1 × 108Spore inoculating is to the 30ml Portugal in 250-mL shaking flask In grape sugar/sophorose defined medium.Shaking flask is made to shake 5 days at 28 DEG C.Derive from the supernatant of these cultures for demonstrate,proving The expression (SDS PAGE) of real mature T auSec099 enzyme (SEQ ID NO:18) and activity.
The sequence of bifidobacterium longum alpha-Glucosidase BloGlul
Identify alpha-Glucosidase gene " BloGlul " from bifidobacterium longum subspecies-bifidobacterium longum JDM301. BloGlul gene (SEQ ID NO:19, GENBANK accession number NC014169.1, the complementary series of 140600 to 142414) Nucleotide sequence and be present in by the amino acid sequence of putative protein (SEQ ID NO:20) of SEQ ID NO:19 coding In GENBANK accession number YP_003660432.1.
The expression of bifidobacterium longum alpha-Glucosidase BloGlul
To coding, the DNA sequence dna of whole BloGlul protein (SEQ ID NO:20) is optimized with at bacillus subtilis Bacterium is expressed, then Generay Biotech Co. (Shanghai, China) is synthesized (producing SEQ ID NO:21) and insert Enter in p3JM plasmid, cause p3JM-BloGlul.P3JM-BloGlul plasmid comprises aprE promoter with Drive Optimization The expression of BloGlul sequence (SEQ ID NO:21).
Plasmid p3JM-BloGlul 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 chloramphenicol On Luria agar plate.Select as by PCR and order-checking confirmed with the bacterium colony being correctly inserted into fragment and make it have MBD culture medium (based on the defined medium of MOPS, is supplemented with other 5mM CaCl2) 250-mL shaking flask in carry out send out Ferment, to express BloGlul protein (SEQ ID NO:20).
The sequence of bifidobacterium longum alpha-Glucosidase BloGlu2
Identify alpha-Glucosidase BloGlu2 from bifidobacterium longum.The amino acid sequence (SEQ ID NO:22) of BloGlu2 It is present in ncbi database (the GENBANK number of logging in WP_007054665.1).
The expression of bifidobacterium longum alpha-Glucosidase BloGlu2
To coding BloGlu2 protein DNA sequence be optimized with in bacillus subtilis express, then by Generay Biotech Co. synthesis (producing SEQ ID NO:23) is simultaneously inserted in p3JM plasmid, causes p3JM-BloGlu2. The amino acid sequence of SEQ ID NO:23 coding SEQ ID NO:24.P3JM-BloGlu2 plasmid comprises aprE promoter to drive The expression of the BloGlu2 sequence (SEQ ID NO:23) optimizing.
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 chloramphenicol On Luria agar plate.Select as by PCR and order-checking confirmed with the bacterium colony being correctly inserted into fragment and make it have MBD culture medium (based on the defined medium of MOPS, is supplemented with other 5mM CaCl2) 250-mL shaking flask in carry out send out Ferment, to express 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 the nucleotide sequence of (SEQ ID NO:25, GENBANK accession number NC_021008.1,2130627 to 2132441) with by SEQ The amino acid sequence of the putative protein (SEQ ID NO:26) of ID NO:25 coding is present in GENBANK accession number YP In 007768249.1.
The expression of bifidobacterium longum alpha-Glucosidase BloGlu3
To coding, the DNA sequence dna of whole BloGlu3 protein (SEQ ID NO:26) is optimized with at bacillus subtilis Bacterium is expressed, then Generay Biotech Co. is synthesized (producing SEQ ID NO:27) and be inserted in p3JM plasmid, leading Cause p3JM-BloGlu3.P3JM-BloGlu3 plasmid comprises BloGlu3 sequence (the SEQ ID with Drive Optimization for the aprE promoter NO:27) expression.
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 chloramphenicol On Luria agar plate.Select as by PCR and order-checking confirmed with the bacterium colony being correctly inserted into fragment and make it have MBD culture medium (based on the defined medium of MOPS, is supplemented with other 5mM CaCl2) 250-mL shaking flask in carry out send out Ferment, to express BloGlu3 protein (SEQ ID NO:26).
The sequence of bifidobacterium pseudolongum alpha-Glucosidase BpsGlul
Identify alpha-Glucosidase BpsGlul from bifidobacterium pseudolongum.The amino acid sequence of BpsGlul (SEQ ID NO: 28) it is present in ncbi database (the GENBANK number of logging in WP_022858408.1).
The expression of bifidobacterium pseudolongum alpha-Glucosidase BpsGlul
To coding BpsGlul protein DNA sequence be optimized with in bacillus subtilis express, then by Generay Biotech Co. synthesis (producing SEQ ID NO:29) is simultaneously inserted in p3JM plasmid, causes p3JM-BpsGlul. The amino acid sequence of SEQ ID NO:29 coding SEQ ID NO:30.P3JM-BpsGlul plasmid comprises aprE promoter to drive The expression of the BpsGlul sequence (SEQ ID NO:29) optimizing.
Plasmid p3JM-BpsGlul 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 chloramphenicol On Luria agar plate.Select as by PCR and order-checking confirmed with the bacterium colony being correctly inserted into fragment and make it have MBD culture medium (based on the defined medium of MOPS, is supplemented with other 5mM CaCl2) 250-mL shaking flask in carry out send out Ferment, to express BpsGlul protein (SEQ ID NO:30).
The sequence of bifidobacterium thermophilum alpha-Glucosidase BthGlul
Identify alpha-Glucosidase gene " BthGlul " from bifidobacterium thermophilum RBL67.BthGlul gene (SEQ ID NO:31, GENBANK accession number NC_020546.1,150690 to 152495) nucleotide sequence and compiled by SEQ ID NO:31 The amino acid sequence of the putative protein (SEQ ID NO:32) of code is present in GENBANK accession number YP_007592840.1.
The expression of bifidobacterium thermophilum alpha-Glucosidase BthGlul
To coding, the DNA sequence dna of whole BthGlul protein (SEQ ID NO:32) is optimized with at bacillus subtilis Bacterium is expressed, then Generay Biotech Co. is synthesized (producing SEQ IDNO:33) and be inserted in p3JM plasmid, leading Cause p3JM-BthGlul.P3JM-BthGlul plasmid comprises BthGlul sequence (the SEQ ID with Drive Optimization for the aprE promoter NO:33) expression.
Plasmid p3JM-BthGlul 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 chloramphenicol On Luria agar plate.Select as by PCR and order-checking confirmed with the bacterium colony being correctly inserted into fragment and make it have MBD culture medium (based on the defined medium of MOPS, is supplemented with other 5mM CaCl2) 250-mL shaking flask in carry out send out Ferment, to express BthGlul protein (SEQ ID NO:32).
The sequence of bifidobacterium breve alpha-Glucosidase BbrGlu2
Identify alpha-Glucosidase BbrGlu2 from bifidobacterium breve.The amino acid sequence (SEQ ID NO:34) of BbrGlu2 It is present in ncbi database (the GENBANK number of logging in WP_003827971.1).
The expression of bifidobacterium breve alpha-Glucosidase BbrGlu2
To coding BbrGlu2 protein DNA sequence be optimized with in bacillus subtilis express, then by Generay Biotech Co. synthesis (producing SEQ ID NO:35) is simultaneously inserted in p3JM plasmid, causes p3JM-BbrGlu2. The amino acid sequence of SEQ ID NO:35 coding SEQ ID NO:36.P3JM-BbrGlu2 plasmid comprises aprE promoter to drive The expression of the BbrGlu2 sequence (SEQ ID NO:35) optimizing.
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 chloramphenicol On Luria agar plate.Select as by PCR and order-checking confirmed with the bacterium colony being correctly inserted into fragment and make it have MBD culture medium (based on the defined medium of MOPS, is supplemented with other 5mM CaCl2) 250-mL shaking flask in carry out send out Ferment, to express SEQ ID NO:36.
The sequence of bifidobacterium breve alpha-Glucosidase BbrGlu5
Identify alpha-Glucosidase gene " BbrGlu5 " from bifidobacterium breve ACS-071-V-Sch8b.BbrGlu5 gene The nucleic acid sequence of (SEQ ID NO:37, GENBANK accession number NC_017218.1, the complementary series of 2241075 to 2242895) The amino acid sequence of row and the putative protein (SEQ ID NO:38) being encoded by SEQ IDNO:37 is present in GENBANK accession number In YP_005583701.1.
The expression of bifidobacterium breve alpha-Glucosidase BbrGlu5
To coding, the DNA sequence dna of whole BbrGlu5 protein (SEQ ID NO:38) is optimized with at bacillus subtilis Bacterium is expressed, then Generay Biotech Co. is synthesized (producing SEQ ID NO:39) and be inserted in p3JM plasmid, leading Cause p3JM-BbrGlu5.P3JM-BbrGlu5 plasmid comprises BbrGlu5 sequence (the SEQ ID with Drive Optimization for the aprE promoter NO:39) expression.
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 chloramphenicol On Luria agar plate.Select as by PCR and order-checking confirmed with the bacterium colony being correctly inserted into fragment and make it have MBD culture medium (based on the defined medium of MOPS, is supplemented with other 5mM CaCl2) 250-mL shaking flask in carry out send out Ferment, to express BbrGlu5 protein (SEQ ID NO:38).
Purify alpha-Glucosidase from expressing culture
AclGlul and NcrGlul
Two chromatographic steps are used to purify AclGlul (SEQ ID NO:6) and NcrGlul (SEQ IDNO:12) α-Portugal Both glycosidases.Purifying for each, concentrating the thick zymotic fluid of shaking flask, hereafter adding ammonium sulfate to ultimate density is 2M.Will Solution stowage to through 20mM Tris (pH 8.0), 2M ammonium sulfate pre-equilibration 50-mL phenyl HP post on.With 1M ammonium sulfate, 20mM Tris (pH 8.0) elutes target protein (SEQ ID NO:6 or SEQ ID NO:12) from post.Merge corresponding fraction, use VIVAFLOW 200 ultrafiltration apparatus (Sartorius Stedim) makes it concentrate and buffered liquid exchanges to 20mM Tris (pH 8.0) in (buffer A).Gained solution is put on the 40-mL Q HP post of buffered liquid A pre-equilibration.With NaCl's containing 0.3M Buffer A elutes target protein from post.Then the fraction comprising target protein merged and use 10K AMICON ULTRA-15 equipment concentrates, and is stored in 40% glycerine until using at-20 DEG C.
NfiGlul
Two hydrophobic interaction chromatography steps are used to purify NfiGlul alpha-Glucosidase (SEQ ID NO:9).Concentrate shaking flask Thick zymotic fluid, hereafter add ammonium sulfate to ultimate density be 1M.By solution stowage to through 20mM Tris (pH 8.0), 1M sulphur On the 50-mL phenyl HP post of acid ammonium pre-equilibration.Target protein (SEQ ID NO:9) flowing is made to pass through post.Merge and flow out fraction, Hereafter adding ammonium sulfate to ultimate density is 2M.By solution stowage to through 20mM Tris (pH 8.0), 2M ammonium sulfate pre-equilibration On identical phenyl HP post.Elute target protein with 1M ammonium sulfate, 20mM Tris (pH 8.0) from post.Then mesh will be comprised The fraction of mark protein merges and uses 10K AMICON ULTRA-15 equipment to concentrate, and is stored in 40% glycerine straight at-20 DEG C To use.
TauSec098 knows TauSec099
Via hydrophobic interaction chromatography purify TauSec098 (SEQ ID NO:15) and TauSec099 (SEQ ID NO: 18) both alpha-Glucosidases.Purifying for each, the about 180mL that ammonium sulfate adds to 7-L fermentation tank concentrates thick zymotic fluid In to ultimate density be 1M.Then by this solution stowage to pre-flat through 20mM sodium acetate (pH 5.0), 1M ammonium sulfate (buffer A) On 50-mL HIPREP phenyl-FF agarose column (GE Healthcare) of weighing apparatus.In the identical buffering with three column volumes (CV) After liquid washing, the 75%th, 50% and 0% buffer A is used respectively progressively to elute post, the afterwards MILLIQ with two CV with three CV H2O elutes.Analyze all fractions by SDS-PAGE.Target protein (SEQ ID NO:15 or SEQ ID NO:18) is mainly deposited Being to flow out in fraction, it uses 10KDa AMICON ULTRA-15 equipment to carry out concentrating and buffer fluid exchange to remove excess Ammonium sulfate.It at-80 DEG C, is stored in 40% glycerine the end product more than 90% for the purity until using.
BloGlul, BloGlu2 and BloGlu3
Make BloGlul (SEQ ID NO:20), BloGlu2 (SEQ ID NO:24) and BloGlu3 (SEQ ID NO:26) Alpha-Glucosidase all purifies with three steps.Each is purified, concentrates the thick 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, then centrifuge 30 minutes under 8000xg.Make gained Sediment is resuspended in 20mM Tris (pH 8.0, buffer A).Ammonium sulfate is added in gained solution and to ultimate density be 1M；Then said preparation is loaded into through 20mM Tris (pH 8.0), the 40-mL of 1M ammonium sulfate (buffer B) pre-equilibration HiPrepTMOn phenyl-FF post.After washing, the 75%th, 50% and 0% buffer B and H are used2O respectively with three column volumes by Step wash-out post.All fractions use SDS-PAGE and determination of activity to be analyzed.Merge comprise target protein (SEQ ID NO: 20th, SEQ ID NO:24 or SEQ ID NO:26) fraction, concentrate and followed by through 20mM sodium phosphate (pH 7.0), The HiLoad of 0.15M NaCl pre-equilibrationTM 26/60SuperdexTMOn 75 posts.Then the outflow level of target protein will be comprised Deciliter and and with the concentration of 10K AMICON ULTRA-15 equipment, and be stored in 40% glycerine until using at-20 DEG C.
BpsGlul and BthGlul
Purify BpsGlul (SEQ ID NO:30) and BthGlul (SEQ ID NO:32) alpha-Glucosidase with two steps Both.Each is purified, concentrates the thick zymotic fluid of 1-L DASGIP fermentation tank, hereafter add ammonium sulfate saturated to 60% Degree.Solution is stirred at 4 DEG C 1 hour, then centrifuge 30 minutes under 800xg.Gained sediment is made to be resuspended in 20mM Tris In (pH 8.0, buffer A).Ammonium sulfate is added in gained solution to ultimate density be 1M；Then said preparation is loaded into Through 20mM Tris (pH 8.0), the 40-mL HiPrep of 1M ammonium sulfate (buffer B) pre-equilibrationTMOn phenyl-FF post.In washing After, use the 75%th, 50% and 0% buffer B and H2O respectively progressively elutes post with three column volumes.All fractions use SDS- PAGE and determination of activity are analyzed.Target protein (SEQ ID NO:30 or SEQ ID NO:32) is present in 0% buffer B In the eluate of elution step；Merge this eluate and concentrate with 10K AMICON ULTRA-15 equipment.At-20 DEG C, by pure The end product more than 95% for the degree is stored in 40% glycerine until using.
BbrGlu2 and BbrGlu5
Purify BbrGlu2 (SEQ ID NO:36) and BbrGlu5 (SEQ ID NO:38) alpha-Glucosidase with four steps Both.Each is purified, concentrates the thick zymotic fluid of 1-L DASGIP fermentation tank, hereafter add ammonium sulfate saturated to 60% Degree.Solution is stirred at 4 DEG C 1 hour, then centrifuge 30 minutes under 800xg.Gained sediment is made to be resuspended in 20mM In HEPES (pH 7.0, buffer A).Ammonium sulfate is added in final solution to ultimate density be 1M；Then said preparation is filled It is downloaded to through 20mM HEPES (pH 7.0), the HiPrep of 1M ammonium sulfate pre-equilibrationTMOn phenyl-FF post.With 0.5M ammonium sulfate from post Elute target protein (SEQ ID NO:36 or SEQ IDNO:38).Merge corresponding fraction, with VIVAFLOW 200 ultrafiltration Equipment (SartoriusStedim) makes it concentrate and buffered liquid exchanges in buffer A.Gained solution is put on buffered The HiPrep of liquid A pre-equilibrationTMQ FF 16/10 post.Elute from post by the buffer A of the 0-0.5M NaCl containing linear gradient Go out target protein.Will comprise target protein fraction merge, concentrate and followed by through 20mM HEPES (pH 7.0), The HiLoad of 0.15M NaCl pre-equilibrationTM26/60SuperdexTMOn 75 posts.Then the level deciliter of target protein will be comprised And and concentrate with 10K AMICON ULTRA-15 equipment, and be stored in 40% glycerine until using at-20 DEG C.
Therefore, express and purified other alpha-Glucosidase various.In examples provided below the 11st, the 12nd, 15 and 16 Middle test these alpha-Glucosidases to α-1,5 glucityls-fructose key and α-1,3 and/or α-1, the water of 6 glucityls-glucose key Solve activity.
Embodiment 11
The test hydrolysing activity to various glycosidic bonds for the alpha-Glucosidase
This embodiment disclose test alpha-Glucosidase whether have surmount previously related to this fermentoid (EC3.2.1.20) Hydrolysing activity.The alpha-Glucosidase of embodiment 10 illustrates to α-1,5 glucityls-fructose key and α-1,3 and α-1,6 glucose Base-glucose key has hydrolysing activity.
The substrate specificity of alpha-Glucosidase
(isomaltose, maltose, panose, bright from specific substrates based on the glucose when substrate incubates with alpha-Glucosidase Beading bacterium disaccharides or nigerose) release, analyze the substrate specificity of various alpha-Glucosidases disclosed in embodiment 10.Glucose Rate of release use coupling glucose oxidase/peroxidase (GOX/HRP) method (1980, Anal.Biochem.105: It 389-397) is measured.The quantification of coupling GOX/HRP enzyme of glucose release and peroxide pair produced by glucose response 2, the speed that 2 '-azine group-two 3-ethyl benzo thiazole phenanthroline-6-sulfonic acid (ABTS) aoxidizes.
By 0.5M (pH 5.0) sodium acetate buffer and the 40 μ L of the substrate solution (in water 1%, w/v) with 1mL that make 9mL 0.5M calcium chloride in 15-mL conical pipe, be mixed with each substrate solution.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 dilutions of each alpha-Glucosidase sample and Glucose standards are prepared in MILLIQ water.For aspergillus niger Sugar, tests alpha-Glucosidase sample only with a kind of dosage 10ppm, and reason is that the stoste of substrate solution is limited.By each α- Glucosidase sample (10 μ L) transfers to comprise the precincubation substrate solution of 5 minutes newly micro-under 50 DEG C and 600rpm of 90 μ L In amount titer plate (Corning 3641).Under shake (600rpm), make reaction in THERMOMIXER at 50 DEG C (Eppendorf) carry out in 10 minutes (for isomaltose, maltose, panose and nigerose substrates) or 60 minutes (for Lucrose substrate).Then respectively by the standard glucose of each reactant mixture of 10 μ L and the serial dilution of 10 μ L It is quickly transferred in new microtiter plate (Corning 3641), then correspondingly add the ABTS/GOX/HRP of 90 μ L wherein Solution.Use SOFTMAX PRO plate reading machine (Molecular Devices) to be spaced in quick mensuration at 405nm with 11 seconds to comprise The microtiter plate of reactant mixture 5 minutes.It for each enzyme concentration, is output as reaction rate Vo.Linear regression is used to survey Determine curve VoSlope with enzyme dosage.Formula 1 is used to calculate the specific activity of each alpha-Glucosidase based on glucose standard curve:
Specific activity (unit/mg)=slope (enzyme)/slope (standard) × 1000 (1),
Wherein 1 unit=1 μm ol glucose/minute.
For nigerose, under the enzyme dosage of 10ppm, reaction rate value is directly used in indicative of enzyme activity.
Use preceding method, measure specific to each substrate of each alpha-Glucosidase.Also measured were widow-1,6-glucose Glycosides enzyme (being purchased from Megazyme, see table 4) and the activity to each substrate for the transglucosidase (TG L-2000 sees table 4).Should The result analyzed is provided in table 17.
Table 17
The activity to different substrates for the various alpha-Glucosidases
A, for nigerose, 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 (isomaltose), α-1,3 glucityls-glucose key (nigerose) and α-1, the hydrolysing activity (table 17) of 5 glucityls-fructose key (lucrose).
Therefore, alpha-Glucosidase has and surmounts previously related to EC 3.2.1.20 enzyme hydrolysing activity.Specifically, α-Portugal Glycosidase is to α-1, and 5 glucityls-fructose key and α-1,3 and α-1,6 glucityls-glucose key has hydrolysing activity.
Embodiment 12
Use lucrose and compound sugar in alpha-Glucosidase hydroglucan reaction filtrate
This embodiment describes use alpha-Glucosidase hydrolysis to be present in available from the filtrate of glucan synthetic reaction bright Beading bacterium disaccharides and other compound sugar.Specifically, have studied alpha-Glucosidase disclosed in embodiment 10 (poly-to insoluble glucan α-1,3-glucan) synthetic reaction filtrate in the hydrolysis of lucrose and compound sugar DP2, DP3 and HS (high sugar, DP4+) Impact.
Point analysis of variance of the compound sugar for alpha-glucosidase activity is tested
First the concentrated filtrate of glucan synthetic reaction is prepared according to embodiment 1.
In brief, by chromatographic isolation from concentrated filtrate separating oligomeric sugar, and glycosidic bond feature is analyzed.Use highly acid The chromatographic isolation of cationic ion-exchange resin is for separating the compound sugar fraction of concentrated filtrate.Physical parameter for the post of this separation As follows: FINEX CS11GC, #227 resin；Na+Ionic species；5% divinylbenzene (crosslinked)；0.34mm granularity；1.64m bed Length；0.093m column diameter.
More specifically, make the sugar juice (i.e. concentrated filtrate) concentrating described in table 3 filter and to be diluted to 25g with running water solid Body/100g solution.Adding this sugar juice before 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 feed sugar juice (0.6L) to post, thereafter with the water elution post that flow velocity is 50mL/min.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.Between 11 Oligosaccharide solution is eluted between minute and 21 minutes.A small amount of salt instruction conductibility increase is eluted out simultaneously.Logical Cross HPLC and analyze the compound sugar fraction thus prepared to determine that its product is distributed.In a word, fraction comprise ＞ 89% contain three kinds Or the compound sugar of more kinds of hexose and the appraisable monose less than 1.5% and disaccharides.Use thin film 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 product distribution of the concentration fraction as measured by HPLC is shown in table 18 In.
Table 18
Concentrate the product distribution of compound sugar fraction
The alpha-Glucosidase that glucan oligomer is hydrolyzed by Preliminary screening
It is respectively directed to the work of purifying compound sugar fraction (table 18) 11 kinds of different alpha-Glucosidases of assessment made above Property (embodiment 10) and two kinds of benchmark enzymes be few-1,6-glucosidase (being purchased from Megazyme) and transglucosidase (TG L- 2000) activity.At pH 5.0 and 60 DEG C, in comprising compound sugar substrate (2.9% dry solid) and the solution of 2mM calcium chloride Incubate each alpha-Glucosidase (dosage is 1mg/mL).After incubating 24 hours, will be each by adding the 0.5M NaOH of 50 μ L Individual reaction quencher.
Following compound sugar/monose the content measuring the reaction through quencher.With water by the Sample Dilution 5 of each reaction again with Analyze in HPLC.Agilent 1200 series with AMINEX HPX-42A post (300mm × 7.8mm) is used at 85 DEG C HPLC system carries out HPLC separation.Sample (10mL) is put on HPLC column and under 0.6mL/min flow velocity, uses constant gradient MILLI-Q water separates mutually as flowing.RI-detector is used to detect compound sugar product.Be provided in the following table 19 number Word reflects each DPnAverage peak area percentage (obtained by each sample, duplicate) as the sum of DP1 to DP7 A part.
Table 19
Analysis with alpha-Glucosidase process after glucan filtrate compound sugar
As in table 19 with represented by shade, compared to the control reaction (" blank ") that there is not enzyme, reaction oligomeric Sugar content is generally partial to the sugar of smaller particle size.These results are pointed out, alpha-Glucosidase can be used for hydrolyzing in glucan synthesis instead Compound sugar contained in should and fraction thereof.In addition, based on the key feature (embodiment 3 and 4) of compound sugar and alpha-Glucosidase pair The activity of the various glycosidic bonds in addition to α-Isosorbide-5-Nitrae key (embodiment 11), 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 compound sugar of 6 glucityls-glucose key.The result that table 19 provides is also Showing, compared to bacterium alpha-Glucosidase, fungi alpha-Glucosidase has more preferable hydrolysing activity to soluble oligosaccharide.
The confirmation of the hydrolysing activity of the compound sugar product to glucan synthetic reaction for the alpha-Glucosidase
Having carried out such a reaction, it comprises one or both alpha-Glucosidases and available from poly-α-1, and 3-glucan synthesizes The concentrated 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 is 4ppm.Concentrated filtrate is loaded 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.After incubating 23 hours, by adding the 0.5M of 50 μ L NaOH quencher is reacted.
Following compound sugar/monose the content measuring the reaction through quencher.The Sample Dilution 25 times each being reacted with water Analyze for HPLC.The Agilent 1200 that use has AMINEX HPX-42A post (300mm × 7.8mm) at 85 DEG C is Row HPLC system carries out HPLC separation.Sample (10mL) is put on HPLC column and under 0.6mL/min flow velocity, uses constant gradient MILLI-Q water separates mutually as flowing.RI-detector is used to detect compound sugar product.Be provided in the following table 20 number Word reflects each DPnAverage peak area percentage (obtained by each sample, duplicate) as a total part.Table 20 The result being provided generally confirms the activity (result with regard to being provided in table 19) of some alpha-Glucosidase as discussed above.
Therefore, alpha-Glucosidase can be used for hydrolysis and is present in available from such as poly-α-1 of glucan synthetic reaction, and 3-glucan closes Become the lucrose in the fraction (such as filtrate) of reaction and other compound sugar.
Embodiment 13
Separation of oligomeric/lucrose fraction from gtf-S/MUT3325 reactant
It is 9.5L that sucrose (4.50kg) is dissolved in deionized water to the final total volume through distillation, and under agitation, Gained solution 80 DEG C of heating 5 minutes and is subsequently cooled to 47 DEG C.Under agitation, the gtf-S enzyme comprising 0.6g/L is added 500 grams of CEs of (GTF0459, SEQ ID NO:42) and comprise 10g/L mutant enzyme (MUT3325, SEQ ID NO:47) 15.0mL CE (seeing the conventional method prepared for enzyme).Under agitation, by being slowly added 37 weight %HCl The pH of the mixture of gained is adjusted to pH 5.5 to pH6.0 by 1: 10 (v/v) dilution immediately.Reaction temperature and pH is made to protect respectively Being held in 47 DEG C and pH 5.5-6.0, until analyzing Sucrose conversion ＞ 95% according to HPLC, hereafter reactant mixture being adjusted immediately Joint is to pH 7.0 to 7.5 and is heated to 90 DEG C lasting 20 minutes, is subsequently cooled to 25 DEG C and makes a return journey particle-removing to filter immediately and sink Form sediment.Use following resin and condition, gained filtrate is maintained at 5 DEG C: FINEX CS 11GC before IEX/SEC column chromatography SAC(Ca2+Form), column internal diameter=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.Be incorporated between The post fraction collected between 30 minutes and 67 minutes, is the solid of 66% dissolving and such as institute in conventional method by being concentrated by evaporation State and be analyzed by HPLC.Table 21 illustrates compound sugar and the monosaccharide component of the separation fraction thus prepared.
Table 21
Derive from the analysis of the oligomer/lucrose fraction of gtf-S/MUT3325 reaction
In this embodiment, glucan synthetic reaction is used for producing at least one solubility alpha-glucans product.This is solvable Property product gathered by the glucosyltransferase (GTF0459, SEQ ID NO:42) being present in glucosylation enzyme reaction and α-Portugal Both synergies of glycosylhydrolase (MUT3325, SEQ ID NO:47) produce.This embodiment also illustrates that by glucan synthesis anti- The chromatographic isolation 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 reaction
Sucrose (4.50kg) is dissolved in through distillation deionized water in final total volume be 9.5L, and stirring Under, gained solution is heated 5 minutes at 80 DEG C and is subsequently cooled to 47 DEG C.Under agitation, the gtf-comprising 0.41g/L is added 500 grams of CEs (seeing the conventional method prepared for enzyme) of C enzyme (GTF0088BsT1, SEQ ID NO:45).In stirring Under, by being slowly added 1: 10 (v/v) dilution of 37 weight %HCl, the pH of the mixture of gained is adjusted to pH 5.5 immediately To pH 6.0.Reaction temperature and pH is made to be respectively retained 47 DEG C and pH 5.5-6.0, until analyzing Sucrose conversion according to HPLC Hereafter reactant mixture is adjusted to pH 7.0 to 7.5 and is heated to 90 DEG C lasting 20 minutes, being subsequently cooled to by ＞ 95% immediately 25 DEG C to filter make a return journey particle-removing and precipitation immediately.Use following resin and condition, before IEX/SEC column chromatography, gained is filtered Liquid is maintained at 5 DEG C: FINEX CS 11GC SAC (Ca2+Form), column internal diameter=9.3cm, post bed height 1.58m, T=70 DEG C, stream Speed=50mL/min, linear flow rate=0.44m/h, feed volume=0.6L=171g, feed RI-DS=25.8g/100g, adopt Sample interval=3min.It is incorporated between 34 minutes and 72 minutes the post fraction collected, be 67% dissolving by being concentrated by evaporation Solid and be analyzed by HPLC as described in General Method.Table 22 illustrates the compound sugar of the separation fraction thus prepared And monosaccharide component.
Table 22
Derive from the analysis of the oligomer/lucrose fraction of Gtf-C reaction
In this embodiment, glucan synthetic reaction is used for producing at least one solubility alpha-glucans product.This enforcement Example also illustrates that the chromatographic isolation fraction prepared by the glucan synthetic reaction producing solubility alpha-glucans product.This fraction is therefore For example 1 below 5 and 16, to test the activity of alpha-Glucosidase.
Embodiment 15
With the oligomer/lucrose fraction Preliminary screening α-glucose deriving from Gtf-S/MUT3325 and Gtf-C reaction Glycosides enzyme
This embodiment describes and use alpha-Glucosidase hydrolysis to be present in available from the Portugal producing solubility alpha-glucans product Lucrose in the chromatographic isolation fraction of glycan synthetic reaction and other compound sugar.Specifically, to embodiment 10 disclosure Alpha-Glucosidase the impact of lucrose in the fraction prepared by embodiment 13 and 14 and the hydrolysis of compound sugar is entered Go research.
With deriving from oligomer/leukonid two that gtf-S/MUT3325 (embodiment 13) and gtf-C (embodiment 14) reacts Sugar fraction screens 12 kinds of alpha-Glucosidases and two kinds of benchmark enzymes (widow-1,6-glucosidase and TG altogether as substrate materials L-2000 transglucosidase).The dosage of all enzymes (alpha-Glucosidase and benchmark enzyme) is identical protein concentration.At pH At 5.5 and 47 DEG C, incubate in comprising oligomer/lucrose substrate (10% dry solid) and the solution of 2mM calcium chloride Each alpha-Glucosidase (dosage is 100ppm).After incubating 21 hours, the 0.5M NaOH by adding 50 μ L is anti-by each Answer quencher.
Following compound sugar/monose the content measuring the reaction through quencher.The sample deriving from each reaction is centrifuged And with water, supernatant therein is diluted 25 times for HPLC analysis (conventional method).The percentage reflection being recorded in table 23 is each Individual DPnAverage peak area percentage (being obtained by analyzing each sample, duplicate) as a total part.Result shows, When comparing with bacterium alpha-Glucosidase, fungi alpha-Glucosidase has more preferable hydrolysing activity to glucan oligomer.
As in table 23 with represented by shade, compared to the control reaction (" blank ") that there is not enzyme, reaction oligomeric Sugar 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 glucan conjunction Becoming the compound sugar in reacting and fraction thereof, the chromatogram of the glucan synthetic reaction particularly producing solubility alpha-glucans product is divided From fraction.In addition, based on the key feature (embodiment 13 and 14) and alpha-Glucosidase of compound sugar to except α-Isosorbide-5-Nitrae key (embodiment 11) activity of the various glycosidic bonds outside, it is obvious that alpha-Glucosidase can be used for decomposition and has α-1,5 glucityls-really The compound sugar of sugar key is likely to decomposition and has α-1,3 and α-1, the compound sugar of 6 glucityls-glucose key.The result that table 23 provides It is also shown that compared to bacterium alpha-Glucosidase, fungi alpha-Glucosidase has more preferable hydrolysing activity to soluble oligosaccharide.
Therefore, alpha-Glucosidase can be used for hydrolysis be present in available from glucan synthetic reaction such as synthesizing soluble α-Portugal gather Lucrose in the fraction (such as chromatographic isolation fraction) of the reaction of sugar product and other compound sugar.
Embodiment 16
With the oligomer/lucrose level separating sieve alpha-Glucosidase deriving from Gtf-S/MUT3325 and Gtf-C reaction
This embodiment, according to embodiment 15, describes use alpha-Glucosidase hydrolysis and is present in available from generation solubility α-Portugal Lucrose in the chromatographic isolation fraction of the glucan synthetic reaction of glycan product and other compound sugar.
By analyze the sugar composite assessment causing comprising the reaction of the enzyme of same protein concentration dose forDerive from Oligomer/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, (dosage is 4ppm；For blend, the ratio of two kinds of enzymes is 1: 1 and accumulated dose is 4ppm) and oligomer/lucrose substrate (10%ds) incubate.After incubating 23 hours, carry out quencher reaction by adding the 0.5mM NaOH of 50 μ l.
Following compound sugar/monose the content measuring the reaction through quencher.The sample deriving from each reaction is centrifuged And with water, supernatant therein is diluted 25 times for HPLC analysis (conventional method).It is recorded in the percentage of table 24 (hereafter) Reflect each DPnAverage peak area percentage (being obtained by analyzing each sample, duplicate) as a total part.Result Showing when incubating at 65 DEG C, TauSec098 is effective for hydrolysis DP2 to DP7 oligomer, and TauSec099 is better than TG L-2000 for the hydrolysis of lucrose.TauSec098 and TauSec099 (or TG L- 2000) blend hydrolyzed oligomers effectively and lucrose produce DP1.
Therefore, alpha-Glucosidase can be used for hydrolysis be present in available from glucan synthetic reaction such as synthesizing soluble α-Portugal gather Lucrose in the fraction (such as chromatographic isolation fraction) of the reaction of sugar product and other compound sugar.

Claims (15)

1. one kind makes to comprise at least one α-1,3 or α-1, α-1 of the sugar of 6 glucityls-glucose key, 3 or α-1, and 6 glucityls- The method of glucose key hydrolysis, wherein said sugar is disaccharides or compound sugar, and the method comprise the steps that
Making described sugar contact under suitable conditions with alpha-Glucosidase, the described sugar of wherein said alpha-Glucosidase hydrolysis is at least One α-1,3 or α-1,6 glucityls-glucose 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, the degree of polymerization of wherein said sugar is 3 to 7 before hydrolysis.
4. method according to claim 1, wherein the concentration of the described sugar after described contact procedure is less than described The 50% of the concentration of the described sugar existing before contact.
5. method according to claim 1, wherein said suitable condition includes: (i) glucan synthetic reaction, or (ii) The fraction being obtained by described glucan synthetic reaction；
Wherein said sugar is the accessory substance of glucan synthetic reaction.
6. method according to claim 5, wherein said glucan synthetic reaction produces at least one insoluble α-Portugal and gathers Sugar product.
7. method according to claim 6, wherein said level is divided into the filtrate of described glucan synthetic reaction.
8. method according to claim 5, wherein said glucan synthetic reaction produces at least one solubility α-Portugal and gathers Sugar product, which is:
The product of (i) glucosyltransferase, or
(ii) glucosyltransferase and the synergistic product of alpha-glucans hydrolase, described alpha-glucans hydrolase 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 chromatographic isolation fraction of described glucan synthetic reaction.
10. method according to claim 1, wherein said alpha-Glucosidase is transglucosidase.
11. 1 kinds of compositions being produced by making sugar contact with alpha-Glucosidase,
Wherein said sugar is disaccharides or compound sugar and comprises at least one α-1,3 or α-1,6 glucityls-glucose key,
At least one α-1 of sugar described in wherein said enzyme hydrolysis, 3 or α-1,6 glucityls-glucose key,
And the sugar amount that wherein said composition comprises reduces compared to the sugar amount existing before described contact.
12. compositions according to claim 11, the degree of polymerization of wherein said sugar is 3 to 7 before hydrolysis.
13. compositions according to claim 11, wherein said sugar is in (i) glucan synthetic reaction, or (ii) is by institute State in the fraction that glucan synthetic reaction obtains；
Wherein said sugar is the accessory substance of described glucan synthetic reaction.
14. 1 kinds of enrichments are present in the method for the fructose in the fraction of glucan synthetic reaction, and described method includes:
A () makes to contact under suitable conditions with alpha-Glucosidase available from the fraction of glucan synthetic reaction, wherein said α-Portugal At least one α-1 of contained disaccharides or compound sugar, 3 or α-1,6 glucityls-glucose key in glycosidase hydrolysis fraction；And
(b) from step (a) through hydrolysis fraction separating levulose, denseer than the fructose of the fraction of step (a) to obtain fructose concentration Spend high composition.
15. a fermentation process, described fermentation process includes:
A () makes to contact under suitable conditions with alpha-Glucosidase available from the fraction of glucan synthetic reaction, wherein said α-Portugal At least one α-1 of contained disaccharides or compound sugar, 3 or α-1,6 glucityls-glucose key in the described fraction of glycosidase hydrolysis；
B (), by the fraction of microbial fermentation step (a) to produce 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 glucan synthetic reaction not contacted with described alpha-Glucosidase is fermented Rate is compared, and the product yield of (b) increases.
CN201580010441.2A 2014-02-27 2015-02-26 α glucosidase is used to come enzyme hydrolysis disaccharides and compound sugar Pending CN106068327A (en)

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