CN101705266A - Method for preparing xylitol by micro-organism - Google Patents

Abstract

A method for preparing xylitol by micro-organism pertains to the field of biotechnology, and relates to a method for preparing xylitol by micro-organism. The invention provides a method for preparing xylitol by micro-organism with higher yield. The method comprises the following procedures: 1) modifying the micro-organism, thereby reducing or eliminating oxidation of enzymes on NADH except xylose reductase; 2) culturing the micro-organism in substrate containing xylose and 5 to 50 g/L of sulfite (for example, calcium hydrogen sulfite, sodium sulfite or potassium sulfite); 3) culturing the micro-organism in aerobic growing period and oxygen-limited xylitol generating period; and 4) enriching and extracting xylitol from the substrate. Compared with other biotechnologies, the key point of the preparation method of xylitol is characterized in that the reductase can be recycled, and excessive reductase is not needed, thereby leading to higher conversion rate of xylitol.

Description

The method of preparing xylitol by micro-organism

Technical field

The invention belongs to biological technical field, relate to the method for a kind of preparing xylitol by micro-organism (xylite).

Background technology

Xylitol is five carbon polyols, is naturally occurring sugar alcohol.Because its sugariness is high and its metabolism does not rely on characteristics such as Regular Insulin, anti-dental caries, is diabetes, odontopathy patient's ideal sucrose surrogate.The preparation of Xylitol can have three approach: directly extract from plant (1); (2) chemical method or biological process wood sugar shortening; (3) utilize method of microorganism.Because Xylitol naturally occurring content in plant is too low, directly extract very uneconomical.Xylitol is synthetic through chemical route in the prior art; reduce wood sugar under the high pressure-temperature on nickel catalyzator, average preformance is 50 to 60% of a used wood sugar, this method complex process; cost is very high, and especially the use of nickel catalyzator can cause the problem of environment protection aspect.

Oneself has description to biotechnological means in the prior art, and it provides very high productivity in the process that wood sugar is converted into Xylitol.But shortcoming is to express Xylose reductase consumingly and eliminating simultaneously under the situation of the natural metabolic enzyme of participation Xylitol, and can't enough improve the productivity of Xylitol effectively, so that cost prepares Xylitol by the method route of biotechnology effectively.

Summary of the invention

Purpose of the present invention just is to overcome the prior art above shortcomings, and a kind of method of preparing xylitol by micro-organism with higher yield is provided.

For achieving the above object, the present invention adopts following technical scheme, the present invention includes following operation:

1) thus modified microorganism reduces or eliminates enzyme except that Xylose reductase to the oxidation of NADH;

2) culturing micro-organisms in the substrate that contains wood sugar and 5～50g/L sulphite (for example calcium bisulfite, S-WAT, potassium sulfite);

3) the long-term and limit oxygen Xylitol generation phase culturing micro-organisms at aerobe;

4) enrichment and extraction Xylitol in substrate.

Beneficial effect of the present invention

The present invention uses reproducible raw material and compares with the chemical process of routine, does not have the environmental damage because of using nickel catalyzator to cause.In addition, therefore the characteristics that the intrinsic reaction conditions of biotechnological means self is gentle relatively are easier to and environmentally compatible than chemical process usually.

The present invention compares with the other biological technology, and key is that reductase enzyme can recycle, and need not add excessive reductase enzyme, thereby the transformation efficiency of Xylitol is higher.

Embodiment

Embodiment 1

The preparation yeast strains of expressing the xenogenesis Xylose reductase is described:

The expression of xenogenesis Xylose reductase is the part of prior art in the yeast, for example is described among the WO91/15588.Pichia stipitis (comprises the 10g yeast extract at 100ml YPD in 11 substratum, 20g peptone and 20g glucose, pH=6.5) cell in 30 ℃ of overnight incubation .10ml substratum is through centrifugal agglomerating and separate chromosomal DNA, with reference to Kaiser etc. 1994) test design. this DNA is as the template (Amore etc. 1993) of XYL1 gene 956bp intronless (introne one free) opening code-reading frame pcr amplification. the gained segment is separated in 1% sepharose and is used Genomed " JETQUICK Gel Extraction Spin Kit " to carry out purifying.Plasmid is through corresponding restriction endonuclease processing and through the aforesaid method purifying.Use p425GPD as carrier with the expression in S. cervisiae.This carrier is the multiple copied plasmid and comprises S. cervisiae LEU2 gene (Mumberg etc. 1995) as selectable marker.This carrier is cut to segment and purifying correspondingly through identical restriction endonuclease.The opening code-reading frame of XYL1 gene is pricked and is tied in carrier, identifies recombinant plasmid by plasmid preparation and restriction analysis.In the plasmid (p425GPD one PsXYL1) that uses this method preparation, the transfection of XYLI is easy to be subjected to the control of strong GPD promotor, and guarantees the high expression level of external source xR in the S. cervisiae.Wine brewing yeast strain K0Y50 (MATa; His3 Δ 1; The leu2 Δ; Ura3 Δ 0) transformed by p425GPD one PsXYL1, with reference to the method (1989) of Schietstl and Gietz.Separation has also been analyzed Leucin one prototroph transformant.

Embodiment 2

Replace the inactivation nadh dehydrogenase by gene NDE1 and NDE2 gene:

Be positioned at mitochondrial cytosolic nadh dehydrogenase and XR competition cofactor NADH.Consume in order to reduce cytosolic NADH, replace by suitable dna segment gene and make gene NDE1 and NDE2 inactivation.Replace sheet cracked ends pcr amplification with NDEI initiator codon 5 ' end 40bp homology and terminator codon 3 ' end 40bp homologous.This segment comprises the hiss+ gene of Schizosaccharomyces journey wine and is supplemented with the his3 sudden change (Wach etc. 1997) of S. cervisiae.After the amplification, as above-mentioned this segment of method separation and purification.Use replacement segment transformed saccharomyces cerevisiae bacterial strain KOYSO according to the method for Schiestl and Gietz (1989).Discrete group propylhomoserin prototroph transformant.Use diagnosis PCR to confirm that correct the integration with NDE1 of this segment replaces.This bacterial strain (K0Y50 Δ nde1) transforms through p425GPD one PoXYL1, as described in embodiment 1, and is used to prepare Xylitol.

Make up the NDE2 homology as above-mentioned method and replace segment, in order to preparation nde1/nde2 double-mutant.Use kanMX assembly (Wach etc. 1994) as the selected marker thing this moment.This assembly makes transformant can tolerate G418 (geneticine).After the conversion, K0Y50 Δ nde1 cell places on the substratum that comprises G418.Sex clone PCR test after diagnosing in Hangzhoupro has correctly been inserted the replacement segment and has been made the NDE2 inactivation with definite.Obtained strains (K0Y50 Δ nde1 Δ nde2) transforms through p425GPD one PsXYL1, as described in embodiment 1, and is used to prepare Xylitol.

Embodiment 3

The gene GPD1 of coding phosphoglycerol dehydrogenase and the gene of GPD2 are replaced:

Cytoplasmic phosphoglycerol dehydrogenase GPD1p and GPD2p utilize NADH as cofactor.Their inactivation preferentially causes by using XR wood sugar to be reduced to the oxidation of the NADH of Xylitol pair cell solute.When gene is replaced, Wine brewing yeast strain K0Y50 Δ nde1 and/or KOYS0 Δ nde1 Δ nde2 have been used.

This two bacterial strain is all born 0 sudden change of ura3 Δ.For this splitting action, used and contained and GPD1 and GPD2 homologous segment, as among the embodiment 2 for the description of NDE1.The segment that comprises white candiyeast URA3 gene is as the selected marker thing.The DNA part of this selected marker thing flank is mutually the same.The URA3 of white candiyeast replenishes ura3 sudden change in the S. cervisiae.

The int cell of uridylic metabolism is opposite with the ura3 mutant cell, for 5 one Forodica aid (5 one FOA) sensitivity.After will dividing box and being integrated into genome, on the repetition DNA part, generate spontaneous homologous recombination and therefore lose the URA3 marker.The clone that such reorganization takes place can be by easy separation, because they can tolerate 5 one FOA.

Above-mentioned dna segment is replaced segment method conversion in yeast saccharomyces cerevisiae K0Y50 Δ nde1 and/or K0Y50 Δ nde1 Δ nde2 according to Schiestl and Cietz (1989) through pcr amplification and purifying .CPD1 gene.

Uridylic prototroph transformant is separated on the substratum corresponding the selection.PCR replaces GPD1 by diagnosis.Then, the bacterial strain of this method structure is selected through 5 one FOA tolerance.In the bacterial strain of 5 one FOA tolerance, detect the loss of URA3 marker by diagnosis PCR.At last, as mentioned above, inactivation GPD2 in further working.The bacterial strain of Gou Jianing transforms through p425GPD-PsXYL1 like this, as described in embodiment 1, and is used to prepare Xylitol.

Embodiment 4

The preparation of the bacterial strain by analysis of tetrad (tetrad analysis) desaturase inactivation is in order to prepare the bacterial strain of several desaturase inactivations, each genes involved of inactivation at first respectively, as described in example 2 above.Therefore each mutant of cross-breeding subsequently also forms the robe of deploid bacterial strain, carries out analysis of tetrad.

Double-mutant can be identified in nonparental ditype (NPD) easily.The K0Y50 strain is used for inactivation GPD1 and GPD2, as among the embodiment 2 for the description of NDEI, replaced by gene separately.The transformant of hybridizing opposite match type, obtained strains cause robe to form (s porilate) on corresponding substratum.Tetrad test group atmosphere acid prototroph.Two zero mutant of NPD tetrad GPD1/GPD2 transform through p425GPD one PsXYL1, and are used to prepare Xylitol as the clone of the single robe of Histidine one prototroph and separated.

Claims (2)

1. the method for preparing xylitol by micro-organism is characterized in that the present invention includes following operation:

1) thus modified microorganism reduces or eliminates enzyme except that Xylose reductase to the oxidation of NADH;

2) culturing micro-organisms in the substrate that contains wood sugar and 5～50g/L sulphite;

3) the long-term and limit oxygen Xylitol generation phase culturing micro-organisms at aerobe;

4) enrichment and extraction Xylitol in substrate.

2. the method for preparing xylitol by micro-organism according to claim 1 is characterized in that described sulphite can be calcium bisulfite, S-WAT, a kind of or its mixture in the potassium sulfite.