CN107022497B - The yeast strain breeding and its application in nitrile compounds bioconversion for producing nitrilase - Google Patents
The yeast strain breeding and its application in nitrile compounds bioconversion for producing nitrilase Download PDFInfo
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Abstract
The invention belongs to industrial biotechnology field, in particular to a kind of nitrilase generates the screening technique of saccharomycete and its prepares the application in 3- hydracrylic acid in nitrile compounds bioconversion.The present invention provides a kind of screening techniques of saccharomycete for producing nitrilase, one plant of yeast strain 3H2-2 that can convert 3- hydroxypropionitrile synthesis 3- hydracrylic acid has been screened for the first time, combining form, physio-biochemical characteristics and 18S and ITS sequencing analysis are accredited as Pichia guilliermondii (Meyerozyma guilliermondii).The bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (deposit number CGMCC No.12935).3% glucose is added as energy substance, transformation experiment is carried out to 3- hydroxypropionitrile using the bacterial strain free cell, 500mM 3- hydroxypropionitrile can be converted completely, 3- hydracrylic acid cumulative concentration can reach 216.3mM.Pichia guilliermondii CGMCC 12935 has the characteristics that vitality is strong, nitrile conversion capability is prominent, establishes a kind of new process for the synthesis of 3- hydracrylic acid；This technique has the characteristics that reaction condition is mild, low energy consumption, environmental-friendly.
Description
Technical field
The invention belongs to industrial biotechnology field, in particular to a kind of nitrilase generate saccharomycete screening technique and
It prepares the application in 3- hydracrylic acid in nitrile compounds bioconversion.
Background technique
Nitrilase can convert the itrile group in nitrile substrate to carboxyl to which the chemical combination such as organic acid, amino acid be prepared
Object has important application in fields such as bulk chemical, food additives and medicine intermediates.From Robinson in 1964 et al.
Since the pseudomonas Pseudomonas bacterial strain for being separated to one plant of production ricinine nitrilase from soil for the first time, difference is come
The nitrilase in source has been widely studied, and Japanese scholars produce nitrile using Rhodococcus sp Rhodococcus rhodochrous J1
Organic acid is prepared in hydrolase, a variety of nitrile compounds of hydrolyzable.Since then, the nitrilase producing strains of separate sources are gradually shown in
All reports, including Rhod Rhodococcus, Nocardia Nocardia, acinetobacter Acinetobacter are produced
Alkali bacillus belongs to Alcaligenes, pseudomonas Pseudomonas, Bradyrhizobium Bradyrhizobium, comamonas
Belong to Comamonas, Bacillus Bacillus, Pyrococcus Pyrococcus, Halomonas Halomonas, Arthrobacter
Arthrobacter, thermophilic brood cell bacterium Geobacillus, the various separate sources such as corynebacteria Corynebacterium it is thin
Bacterium；The fungi in the sources sickle-like bacteria Fusarium, aspergillus Aspergillus and Penicillium notatum Penicillium etc..These document reports
The compound obtained using nitrilase include niacin, mandelic acid, acrylic acid, hydroxyacetic acid, iminodiacetic acid and more
Kind unnatural amino acid etc..In these reports, based on the nitrilase of bacterial origin, and Fungal nitrilase then relative reporter
It is less, it is even more especially to be rarely reported from the nitrilase of saccharomycete.
3- hydracrylic acid is three carbon of one kind without chipal compounds, has hydroxyl and carboxyl Liang Ge functional group, each other with lactic acid
Isomer is the precursor of many optically active substances；As a kind of Important Platform compound, it is used for numerous medication chemistries
It is the important monomer of synthetic macromolecule compound and polymer in the production of product.U.S. Department of Energy in 2004 is by 3- hydroxyl third
Acid is classified as one of the chemical products of 12 kinds of the world today most potentiality to be exploited, has important Development volue, in recent years also gradually
It attracts attention.The conventional production methods of 3- hydracrylic acid are mainly carried out by chemical method.Generally utilize potassium cyanide and adjacent halohydrin
Reaction generates 3- hydroxypropionitrile, then is reacted by Reformatsky and generate 3- hydracrylic acid；At high temperature, acrylic acid can occur
Hydration reaction synthesizes 3- hydracrylic acid；In addition, platinum can also be used and a kind of solid acid catalyst ZSM5zeolite is catalyzed respectively
3-HPA and acrylic acid generate 3- hydracrylic acid；From the point of view of summarizing, chemical method synthesis step is relative complex, to equipment requirement
Height, and separation and purification of products process is cumbersome, production cost is accordingly higher.
3- hydracrylic acid is produced using microbial fermentation in the bioanalysis developed in recent years, then can effectively avoid chemical synthesis
Unfavorable factor brought by method, and abundant with raw material sources, environmentally protective, the advantages such as production cost is low.In conjunction with genetic engineering
Means can preferably realize the production of 3- hydracrylic acid.Using the method for engineering strain production 3- hydracrylic acid according to substrate
Difference mainly include two classes: one kind is using cereals carbohydrates glucose as substrate；One kind is sweet with C3compounds
Oil is substrate.And in the research of wild mushroom production 3- hydracrylic acid, such as 1,3-PD, propionic acid, acrylic acid, 3- alanine
It is had been reported that Deng as substrate.But biological fermentation process needs coenzyme to participate in, and yield and product purity be not high, while thallus pair
High concentration substrate tolerance is relatively low.It is using one step of nitrilase catalysis nitriles substance 3- hydroxypropionitrile preparation 3- hydracrylic acid
A kind of substitutability technology, this method step is simple, avoids the influence of intermediate product, and the technique has reaction condition temperature
With low energy consumption, the technical advantages such as environmental-friendly.So far, there is not yet Saccharomyces strain, especially Pichia guilliermondii
Meyerozyma guilliermondii produces the report that nitrilase is used for the synthesis of 3- hydracrylic acid.
Summary of the invention
The present inventor's separation screening from soil sample obtains the Pichia guilliermondii of one plant of production 3- hydracrylic acid
3H2-2, and fermentation research has been carried out to this plant of bacterium.Bacterial strain provided by the invention is to obtain soil sample separation screening from chemical plant to obtain
One saccharomycete, according to morphological feature and 18S and ITS gene sequencing, which belongs to Pichia guilliermondii
(Meyerozyma guilliermondii) 3H2-2, is now deposited in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.12935, the deposit date is
On September 5th, 2016.
Bacterial strain Pichia guilliermondii (Meyerozyma guilliermondii 3H2-2, CGMCC provided by the invention
No.12935 physiology and appearance feature and Molecular Identification) is as follows:
Bacterial strain streak inoculation on solid fermentation culture medium flat plate, grows that bacterium colony is rounded, drops, milky cream
Shape, phage surface is relatively wet, glossy, easy picking.After being cultivated for 24 hours in 30 DEG C of insulating boxs, diameter 1mm or so；Single bacterium after 48h
It falls diameter and reaches 2mm or so；After cultivating 6d, diameter is up to 9mm or so.Bacterium colony center and peripheral portion are without other colors.Aobvious
Micro- microscopic observation, how oval cell is, more cluster growths.Under an electron microscope, difference in size is larger between thallus, length
Between 1.5-5 μm, elliposoidal or rugby shape is presented, it is seen that obviously go out bud scar and birth trace, it was demonstrated that it can carry out budding.
Using fungi universal primer to Pichia guilliermondii (Meyerozyma guilliermondii 3H2-2,
CGMCC No.12935) 18S and ITS gene order carry out PCR amplification, respectively obtain the segment of 1677bp and 610bp size.
By carrying out similarity system design analysis using the existing sequence in BLAST software and database in the website NCBI, the bacterial strain is obtained
18S rDNA sequence and Meyerozyma belong to (KJ126853.2, KC178873 etc.) correlated series it is homologous there are 99% or more
Property, while the related strain sequence with Pichia category (EU784644.1) and Candida category (AY227715.1, AY518523.1)
Homology is finally classified as Meyerozyma and belongs to bacterial strain also 99% or so.It is obtained by the ITS sequence analysis of the bacterial strain, bacterium
There are highest homologies with M.guilliermondii GK8 by strain 3H2-2.Combining form feature and physio-biochemical characteristics, are reflected
It is set to Pichia guilliermondii (Meyerozyma guilliermondii 3H2-2, CGMCC No.12935).
The present invention also provides a kind of screening techniques of 3- hydracrylic acid production bacterium, its feature is as follows:
Primary dcreening operation acquires soil sample, can utilize bottom in the screening and culturing medium separation with 3- hydroxypropionitrile as single nitrogen source
The bacterial strain of object；Secondary screening, first progress seed culture, access fermentation medium with 3% inoculum concentration, take fermentation liquid after culture
Transformation experiment is carried out to 3- hydroxypropionitrile, its enzyme activity is detected using phenol-sodium-hypochlorite process.
Soil sample and its sewage sludge of the soil sample around production nitrile compounds workshop.
The prescreening method is as follows:
1g soil sample and mud sample are taken, is dissolved in 0.9% physiological saline of 10mL, is fullyd shake under the conditions of 30 DEG C
30min is stood；1mL Soil Slurry is taken, (50mL/250mL) is at a temperature of 30 DEG C in triangular flask of the access containing screening and culturing medium
It is cultivated in 120rpm reciprocal shaker；After 48h, 100 μ L muddiness culture solutions is taken to be coated on solid screening and culturing medium plate, it will
Plate is placed in constant temperature incubation carton upside down culture, 30 DEG C of culture 48h；The big and strong single bacterium grown on picking screening and culturing medium plate
It falls, is forwarded to fresh screening and culturing medium scribing line separation, transferred for 3~5 generations.
The constituent of the screening and culturing medium are as follows:
Glucose 5-10g/L；KH2PO40.5-4g/L；MgSO40.05-0.4g/L；FeSO40.01-0.1g/L；
CaCl20.01-0.1g/L；NaCl 0.5-4g/L；3- hydroxypropionitrile 0.5-4g/L.
The constituent of the solid screening and culturing medium are as follows:
Glucose 5-10g/L；KH2PO40.5-4g/L；MgSO40.05-0.4g/L；FeSO40.01-0.1g/L；
CaCl20.01-0.1g/L；NaCl 0.5-4g/L；3- hydroxypropionitrile 0.5-4g/L；15~20g/L of agar powder.
The secondary screening method is as follows:
Fermentation medium group becomes (g/L):
Yeast powder 5-15g/L, peptone 5-20g/L, NaCl 0.5-4g/L, KH2PO41-5g/L, K2HPO41-5g/L,
Glycerol 5-15g/L, 3- hydroxypropionitrile 0.5-4g/L, pH 5.0-8.0.Cultural method are as follows: 250mL triangular flask liquid amount 25-
50mL, inoculum concentration 1-10%, in 25-40 DEG C of culture 20-60h in the shaking table that revolving speed is 100-250r/min；At fermentation liquid
Reason method are as follows: fermentation liquid is centrifuged 2~10min under the conditions of 8000-15000rpm, collects the thallus obtained through culture, is used
The phosphate buffer washing thalline of 0.01-0.5M, pH6.0-8.0 2~4 times；Supernatant, the preservation bacterium under the conditions of 4 DEG C are abandoned in centrifugation
Body redundant detection enzyme activity.
The method of Pichia guilliermondii (Meyerozyma guilliermondii) the 3H2-2 detection enzyme activity is as follows:
Enzyme activity is measured using phenol-sodium-hypochlorite process.Appropriate above-mentioned bacterium is resuspended with the sodium phosphate buffer of 100mM, pH7.2
Body to reaction volume be 0.9mL, by the system at 30 DEG C warm bath 5min, then be added 0.1mL 0.5M 3- hydroxypropionitrile,
Being prepared into Final substrate concentrations is 50mM, and total volume is the enzyme reaction system of 1mL；The every plant of bacterium screened is measured in this experiment the bottom of to
The conversion situation of object, each group do three parallel laboratory tests, in 30 DEG C of shaking table conversion reaction 10min, 12000 × g after reaction
It is centrifuged 10min and terminates reaction, take 10 μ L reaction solution supernatants, be settled to 1mL with ammonium hydroxide is removed, it is molten then to sequentially add 1mL sodium phenate
Liquid, 1.5mL sodium nitroprusside solution, 1.5mL liquor natrii hypochloritis, after cultivating 15min in 27 DEG C of water-baths, detection
OD630nm。
Develop the color liquid making method are as follows: developing solution 1, the phenol sodium solution that 7.8mL 4mM NaOH adds 2.5g phenol to match；It is aobvious
Color liquid 2,0.1mgmL-1Sodium nitroprusside liquid；Developing solution 3,0.02M liquor natrii hypochloritis.
Ammonia Specification Curve of Increasing method are as follows: 10 μ gmL of preparation-1Ammonia titer；Take 0 respectively, 100,200,300,400,
500,600,700,800 μ L ammonia titers are in 9 10mL centrifuge tubes, in order successively addition 1.0mL phenol sodium solution,
1.5mL sodium nitroprusside and 1.5mL liquor natrii hypochloritis, and it is settled to 5mL with ammonium hydroxide is removed, it mixes, and in 27 DEG C of water-baths
15min is reacted in pot；It takes 200 μ L reaction solutions to be placed in 96 orifice plates, its absorption value OD630nm is detected in microplate reader.
Enzyme activity calculation method is as follows:
Enzyme activity (1U) definition: under standard biological conversion reaction conditions, every 1min forms enzyme amount corresponding to 1 μM of ammonia, surveys
It is fixed to be repeated 3 times.
The present invention also provides the application methods of CGMCC No.12935 a kind of, its feature is as follows:
CGMCC No.12935 is accessed into fermentation medium, obtains the fermentation liquid of high activity high-biomass；In 8000-
It is centrifuged 2~10min under the conditions of 15000rpm, the thallus obtained through culture is collected, with 0.01-0.5M, the phosphate of pH6.0-8.0
Buffer washing thalline 2~4 times；Thallus is resuspended in buffer, and is mixed with substrate solution, and 0.5%-10% final concentration is added
Glucose, reaction temperature is maintained at 20-60 DEG C, and speed of agitator is maintained at 50-250r/min, 10~150h of reaction time.It adopts
With the concentration of substrate of the 3- hydracrylic acid concentration and remnants generated in supernatant after high performance liquid chromatography detection conversion.Conversion
Liquid processing method are as follows: conversion fluid 12000rpm is centrifuged 10min, supernatant is used for 3- hydracrylic acid after 0.22 μm of membrane filtration
Content detection.
3- hydracrylic acid is detected using high performance liquid chromatography in the conversion fluid, chromatographic condition are as follows: 3000 type of Dionex
High performance liquid chromatograph, chromatographic column are 18 column of XBridge dC (5.0 μm, 250mm × 4.6mm) of WATERS company, column temperature 30
DEG C, flow velocity 0.8mLmin-1, ultraviolet detection wavelength 210nm, mobile phase is methanol/0.05% phosphoric acid (5/95) isocratic elution.
The advantages of the present invention:
Pichia guilliermondii (the Meyerozyma guilliermondii 3H2- of one plant of production nitrilase is found for the first time
2, CGMCC No.12935) can using nitrile hydrolysis reactor catalysis one step of 3- hydroxypropionitrile generate 3- hydracrylic acid, also cover in season finish it is red
Yeast CGMCC12935 has the characteristics that vitality is strong, nitrile conversion capability is prominent, establishes one kind for the synthesis of 3- hydracrylic acid
New process；This technique has the characteristics that reaction condition is mild, low energy consumption, environmental-friendly.This technique is added in the conversion process
The glucose of 0.5%-10% greatly improves the accumulation of product 3- hydracrylic acid；This technique also has reaction condition mild,
The features such as yield is high, and production cost is low, environmentally friendly.
Detailed description of the invention
Fig. 1 CGMCC No.12935 under 300mM, 400mM and 500mM concentration of substrate and addition different glucose
3- hydracrylic acid change curve is produced afterwards.
Specific embodiment
Embodiment 1
(1) the enrichment isolation process of bacterial strain
The soil sample around nitrile compounds workshop is acquired, when acquiring soil sample, first scalps topsoil with small scoop, acquisition 5~
The soil sample of 15cm depth.The collected soil sample of 1g and mud sample are taken, is dissolved in 0.9% physiological saline of 10mL, in 30 DEG C
Under the conditions of 30min fullys shake, stand；1mL Soil Slurry is taken, (50mL/ in the triangular flask containing screening and culturing medium is accessed
250mL) cultivated in 120rpm reciprocal shaker at a temperature of 30 DEG C；After 48h, 100 μ L muddiness culture solutions is taken to be coated on solid
On screening and culturing medium plate, plate is placed in constant temperature incubation carton upside down culture, 30 DEG C of culture 48h；Picking screening and culturing medium plate
On the big and strong single colonie that grows, be forwarded to fresh screening and culturing medium scribing line separation, transferred for 3~5 generations.
Secondary screening is carried out again, and 88 plants of picking purified bacterial strains carry out seed culture, access fermented and cultured with 3% inoculum concentration
Base cultivates 36h in 120rpm reciprocal shaker at a temperature of 30 DEG C, and culture solution 12000rpm centrifugation 5min is taken to collect thallus,
Using phenol-sodium-hypochlorite process detection bacterial strain to the enzyme activity of substrate 3- hydroxypropionitrile.One plant of relatively best bacterial strain of enzyme activity is obtained,
Number is 3H2-2.
The group of the screening and culturing medium becomes (g/L): glucose 5；KH2PO41；MgSO40.1；FeSO40.02；
CaCl20.02；NaCl 1,3- hydroxypropionitrile 1.
The group of the solid screening and culturing medium becomes (g/L): glucose 5；KH2PO41；MgSO40.1；FeSO40.02；
CaCl20.02；NaCl 1,3- hydroxypropionitrile 1；Agar powder 20.
The fermentation medium (g/L) are as follows: yeast powder 7.5, peptone 15, NaCl 1, KH2PO42, K2HPO42, glycerol
15,3- hydroxypropionitriles 1, pH 7.2.
(2) the form of bacterial strain
Pichia guilliermondii (Meyerozyma guilliermondii 3H2-2, CGMCC No.12935) bacterial strain exists
Streak inoculation is used on screening and culturing medium plate, grows that bacterium colony is rounded, drops, and milky butyrous, phage surface is wetter
Profit, glossy, easy picking.After being cultivated for 24 hours in 30 DEG C of insulating boxs, diameter 1mm or so；Single colonie diameter reaches the left side 2mm after 48h
It is right；After cultivating 6d, diameter is up to 9mm or so.Bacterium colony center and peripheral portion are without other colors.It observes under the microscope, cell
It is mostly oval, more cluster growths.Under an electron microscope, difference in size is larger between thallus, length between 1.5-5 μm,
Elliposoidal or rugby shape is presented, it is seen that obviously go out bud scar and birth trace, it was demonstrated that it can carry out budding.
(3) the Molecular Identification of bacterial strain
The thallus grown in liquid fermentation medium is collected to be extracted after low temperature liquid nitrogen is ground with fungal genomic DNA
Kit extracts total DNA.Amplimer is respectively the universal primer of 18S and the universal primer of ITS sequence.
PCR reaction uses 50 μ L reaction systems,
PCR amplification condition: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 1min, 58 DEG C of annealing 1min, 72 DEG C of extension 90s, totally 30
A circulation, last 72 DEG C extend 10min eventually.The purifying of pcr amplification product is returned by a small amount of glue of Shanghai Sheng Gong biotech company
It receives PCR product purification kit to illustrate to carry out, sequencing is completed by Shanghai Rui Di Biotechnology Co., Ltd.
18S the and ITS gene order obtained after being sequenced carries out BLAST in Genbank and compares its determining kind.Finally
It is named as Pichia guilliermondii (Meyerozyma guilliermondii) 3H2-2, it is micro- which has been deposited in China
Biological inoculum preservation administration committee common micro-organisms center, deposit number CGMCC No.12935.
Embodiment 2
This example explanation has carried out culture to CGMCC No.12935 access fermentation medium and has obtained high activity high-biomass
Thallus and bioconversion 3- hydroxypropionitrile production 3- hydracrylic acid method.
Fermentation medium forms (g/L) are as follows: yeast powder 7.5g, peptone 15g, NaCl 1g, KH2PO42g, K2HPO42g,
Glycerol 15g, 3- hydroxypropionitrile 1g, pH 7.2；
CGMCC No.12935 is accessed in culture medium with 3% inoculum concentration, 30 DEG C, 120rpm reciprocal shaker culture 36h,
12000rpm is centrifuged 5min and collects cultured thallus, with sodium phosphate buffer washing thalline 2~3 times of 100mM, pH7.2, and
Thallus is resuspended in the buffer.
Substrate solution and bacteria suspension are mixed, and make Final substrate concentrations 300mM, control condition of culture at 30 DEG C,
Conversion reaction is carried out in 120rpm reciprocal shaker, substrate conversion is complete after 30h, the 3- hydracrylic acid highest detected at 16h
Cumulative concentration is 45.63mM.
Embodiment 3
Thallus is collected from culture solution by centrifugation, with sodium phosphate buffer washing thalline 2~3 times of 100mM, pH7.2,
Again it suspends and bacteria suspension is made, substrate solution and bacteria suspension are mixed, and make Final substrate concentrations 400mM, control condition of culture
Conversion reaction is carried out in 30 DEG C, 120rpm reciprocal shaker, substrate converts completely after 41h, the 3- hydroxyl detected in 21.5h
Base propionic acid cumulative concentration highest is 52.90mM.
Embodiment 4
Thallus is collected from culture solution by centrifugation, with sodium phosphate buffer washing thalline 2~3 times of 100mM, pH7.2,
Again it suspends and bacteria suspension is made, substrate solution and bacteria suspension are mixed, and make Final substrate concentrations 500mM, control condition of culture
Conversion reaction is carried out in 30 DEG C, 120rpm reciprocal shaker, substrate converts completely after 47.5h, the 3- hydroxyl that when 30h detects
Propionic acid cumulative concentration highest is 64.68mM.
Embodiment 5
CGMCC No.12935 is collected into thallus by centrifugation from culture solution, uses 100mmolL-1, pH7.2 phosphoric acid
Sodium buffer washing thalline 2~3 times suspends again and bacteria suspension is made, and substrate solution and bacteria suspension is mixed, and keep substrate dense eventually
Degree is 300mM, while 3% glucose is added in the transformation system, controls condition of culture at 30 DEG C, 120rpm is reciprocating to be shaken
Conversion reaction is carried out in bed, substrate converts completely after 78.5h, and the 3- hydracrylic acid highest cumulative concentration detected at this time is
158.2mM。
Embodiment 6
CGMCC No.12935 is collected into thallus by centrifugation from culture solution, with the sodium phosphate buffer of 100mM, pH7.2
It liquid washing thalline 2~3 times, suspends again and bacteria suspension is made, substrate solution and bacteria suspension are mixed, and make Final substrate concentrations
400mM, at the same in the transformation system addition 3% glucose, control condition of culture in 30 DEG C, 120rpm reciprocal shaker
Conversion reaction is carried out, completely, the 3- hydracrylic acid highest cumulative concentration detected at this time is 191.5mM for substrate conversion after 100h.
Embodiment 7
CGMCC No.12935 is collected into thallus by centrifugation from culture solution, with the sodium phosphate buffer of 100mM, pH7.2
It liquid washing thalline 2~3 times, suspends again and bacteria suspension is made, substrate solution and bacteria suspension are mixed, and make Final substrate concentrations
500mM, at the same in the transformation system addition 3% glucose, control condition of culture in 30 DEG C, 120rpm reciprocal shaker
Conversion reaction is carried out, completely, the 3- hydracrylic acid highest cumulative concentration detected at this time is 216.3mM for substrate conversion after 100h.
Claims (3)
1. the yeast strain of one plant of production nitrilase, the bacterial strain belong to Pichia guilliermondii (Meyerozyma
Guilliermondii) 3H2-2 is now deposited in the Chinese microorganism strain positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation administration committee common micro-organisms center, deposit number are CGMCC No.12935, and the deposit date is on September 5th, 2016.
2. the culture side of Pichia guilliermondii (Meyerozyma guilliermondii) 3H2-2 described in claim 1
Method, which is characterized in that contain 3- hydroxypropionitrile 0.5-4g/L in the strain fermentation culture medium, other components include but unlimited
In following components, by mass volume ratio are as follows: yeast powder 5-15g/L, peptone 5-20g/L, NaCl 0.5-4g/L, KH2PO4 1-
5g/L, K2HPO41-5g/L, glycerol 5-15g/L, medium pH 5.0-8.0, it is 100-250r/ that condition of culture, which is in revolving speed,
In 25-40 DEG C of culture 20-60h in the shaking table of min.
3. Pichia guilliermondii (Meyerozyma guilliermondii) 3H2-2 converts 3- hydroxypropionitrile and generates 3- hydroxyl
The method of propionic acid, it is characterised in that:
(1) Pichia guilliermondii (Meyerozyma guilliermondii) 3H2-2 microorganism collection: according to claim 2 institute
The producing enzyme cultural method stated carries out the fermented and cultured of Pichia guilliermondii (Meyerozyma guilliermondii) 3H2-2,
After fermentation, it is centrifuged 2~10min under the conditions of 8000-15000rpm, collects the thallus obtained through culture, uses 0.01-
The phosphate buffer washing thalline of 0.5M, pH6.0-8.0 2~4 times；
(2) bioconversion: Pichia guilliermondii (Meyerozyma guilliermondii) 3H2-2 thallus is resuspended in slow
It in fliud flushing, and is mixed with 3- hydroxypropionitrile solution, adds the glucose of 0.5%-10% final concentration, reaction temperature is maintained at 20-
60 DEG C, speed of agitator is maintained at 50-250r/min, 10~150h of reaction time.
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