CN106636051A - Method for improving thermal stability of trehalose synthase - Google Patents

Method for improving thermal stability of trehalose synthase Download PDF

Info

Publication number
CN106636051A
CN106636051A CN201610955878.XA CN201610955878A CN106636051A CN 106636051 A CN106636051 A CN 106636051A CN 201610955878 A CN201610955878 A CN 201610955878A CN 106636051 A CN106636051 A CN 106636051A
Authority
CN
China
Prior art keywords
trehalose
heat shock
synthetase
shock protein
trehalose synthetase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610955878.XA
Other languages
Chinese (zh)
Inventor
江凌
杨鑫
黄和
唐苏苏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Tech University
Original Assignee
Nanjing Tech University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Tech University filed Critical Nanjing Tech University
Publication of CN106636051A publication Critical patent/CN106636051A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99016—Maltose alpha-D-glucosyltransferase (5.4.99.16)

Abstract

The invention provides a method for improving the thermal stability of trehalose synthase. The method is mainly characterized by comprising the steps of co-expressing a heat shock protein gene and a trehalose synthase gene in escherichia coli, and exogenously adding trehalose into a system which expresses the trehalose synthase. The trehalose synthase obtained by synthesis based on the method is higher in thermal stability, so that the trehalose synthase is higher in environmental adaptability and higher in catalysis efficiency.

Description

One kind improves the heat-staple method of trehalose synthetase
Technical field
The invention belongs to biological technical field or technical field of enzyme engineering, are related to a kind of expression of trehalose synthetase Journey, and in particular to the trehalose synthetase, and the method for improving its heat endurance are expressed in microorganism.
Background technology
Trehalose synthetase（EC 5.4.99.16）It is a kind of new mutase, base effect in the pool is turned by α by intramolecular, α- The bonded step of maltose one of Isosorbide-5-Nitrae glucosides is converted into α, and α -1, the bonded trehalose of 1 glucosides, process is simple, it is easy to regulate and control is fitted Close industry's enlarging production.The step enzymatic process of trehalose synthetase one neither consumes anakinetomer（Such as UDP, diphosphonic acid urine core Glycosides）, phosphoric acid is also independent of, and the current production technology of substrate maltose is ripe, it is cheap and easy to get, have larger compared with finished product trehalose Price space.The trehalose synthetase molecular weight having now found that concentrates on 60-80 kDa, and the maltose conversion ratio of report exists 50%-70% or so, wherein conversion ratio higher trehalose synthetase gene is more from extreme microorganism and Thermophilic Bacteria, also embodies The resistance of trehalose.
Trehalose is a kind of non-reducing disaccharide of generally existing in nature, used as a kind of natural non-specific biological Protective agent enjoys attracting attention for people.With trehalose range of application expanding day, the market demand is continuously increased.Trehalose is chemically Matter is highly stable, and 1,1 glycosidic bond determines that trehalose sheet, as irreducibility, hydrolytic resistance, shows with protein interaction , with higher heat endurance and wider pH stability ranges, there is no Mei Lade with protein or amino acid anti-in chemical inertness Should.In nature, trehalose can adjust infiltration as carbon source/energy, be the constituent of bacteria cell wall, dry-run protection Agent, antifreezing agent, immunogenicity（The cord factor of mycobacterium）And plant growth regulator.Trehalose is to multiple biological activities thing Matter has protective effect, can form guarantor in cell surface in high temperature, high and cold, hyperosmosis and under being dried the mal-conditions such as dehydration Cuticula, effective protection protein molecule consistency inactivation, in scientific circles the good reputation of " sugar of life " is have.
The quality control system of protein, the i.e. synthesis of protein homeostasis, including protein, folding, unfolding and turnover. Intracellular, molecular chaperones not only can recognize and help the folding of new synthetic proteins, and to the aggregation of many misfolded proteins- Degraded has regulating and controlling effect.Intracellular most of native protein mass-energy spontaneously form more stable natural structure（Or part, generation Thank to factor auxiliary stable）, but the polypeptide chain of 10%-20% newly synthesis needs the help of molecular chaperones correctly to fold.Molecule companion Companion's network and protease system are the two big quality control systems for ensureing protein normal function.Molecular chaperones is widely present in life In object, protected protein matter intracellular synthesizes and transport, prevents its from producing aggregation；Or stress various ambient pressures, aid in it to synthesize Correct protein.Drummond and its colleague use the protein in isotope marks yeast cells, high-temperature process cell（Cell It is not lethal）, and then quick freeze, obtains the real-time denaturation map picture of cellular endogenous albumen and by the poly- of mass spectral analysis protein Collection.While research institute speculates that high temperature causes albuminous degeneration, stimulate cell overexpression molecular chaperones heat shock protein, assist denaturation The renaturation of albumen, the complex of protein denaturation aggregation may remodeling proteins route of synthesis, suppress great majority translating New albumen, it is to avoid the denaturation aggregation of new synthetic proteins.It is flat between rigidity and catalytic center flexibility in protein overall structure Weighing apparatus, has together decided on the heat endurance of enzyme.（Edward W.J. WALLACE, et al. Reversible, specific, active aggregates of endogenous proteins assemble upon heat stress [J]. Cell, 2015, 162(6): 1286-1298.）.
This seminar constructs one plant of colibacillus engineering that can produce trehalose synthetase in early-stage Study, Chinese invention patent and patentable is applied in 2012.05.22, patent publication No. is CN102690795A, invention name Referred to as " a kind of grey streptomyces chromogenes trehalose synthetase and its encoding gene and application ".
At present, the building-up process of trehalose synthetase is transformed by protein engineering, further improves urging for enzyme Change efficiency, have broad prospects.
The content of the invention
The technical problem to be solved is to improve trehalose synthetase catalysis maltose to generate trehalose reaction The catalytic efficiency of middle enzyme, to reduce catalyst cost.
In order to solve the technical problem of the present invention, the technical scheme that the present invention is provided is：
One kind improves the heat-staple method of trehalose synthetase, wherein, be co-expressed heat shock protein gene and sea in Escherichia coli Algae sugar synthase gene, and also include, the external source addition trehalose in the system of expression trehalose synthetase.
Method of the present invention, wherein, the heat shock protein gene is GroEL/S genes（GENE BANK ID94866/ID948655）, DnaK/J genes（GENE BANK ID944750/ID944753）, TF genes（GENE BANK ID945081）, GrpE genes（GENE BANK ID947097）In one or more.
Method of the present invention, wherein, the addition concentration of the trehalose is 0.25-1mol/L.
Method of the present invention, wherein, the heat shock protein and trehalose synthetase are being expressed by abduction delivering During, abduction delivering heat shock protein successively, trehalose synthetase；And external source addition is extra large after trehalose synthetase abduction delivering Algae sugar.
Method of the present invention, wherein, the heat shock protein by arabinose abduction delivering, by grape Glyco inhabiting heat shock The expression of albumen, the induced concentration of arabinose is 0.5-4 mg/mL, and the inhibition concentration of glucose is 0.5-2 mg/mL；Marine alga Sugared synzyme is by IPTG abduction deliverings.
Method of the present invention, wherein, described in abduction delivering during heat shock protein, Escherichia coli OD600Value is in 0.2- Between 0.4；Described in abduction delivering during trehalose synthetase, OD600Value is that, in 0.6-0.8, the time of abduction delivering is 8-12h.
Method of the present invention, wherein, the trehalose synthetase gene is Tres genes.
In a preferred scheme, by grey streptomyces chromogenes trehalose synthetase pET28a-Tres plasmids, with heat shock egg White matter grain pG-KJE8 (DnaK-DnaJ-GrpE GroES-GroEL), pGro7 (GroES-GroEL), pKJE7 (DnaK- DnaJ-GrpE), pG-TF2 (GroES-GroEL-tig), pTF16 (tig) are using heat shock method cotransformation to e. coli bl21 (DE3) coexpression in.
Wherein, trehalose synthetase pET28a-Tres plasmid resistances are kan, and working concentration is 30-50 ug/mL, is used Concentration is the IPTG of 0.1-1 mmol/L in OD600Induce when reaching 0.6-0.8；Heat shock protein plasmid resistance is cmr, is worked Concentration is 20 ug/mL, and concentration is 0.5-4 mg/mL L-arabinoses and/or 1-10 ng/mL tetracyclines in OD600Reach The expression of induced heat shock protein during 0.2-0.4, concentration is the expression of 0.5-2 mg/mL grape Glyco inhabiting heat shock proteins.Tool Body, the bacterial strain with coexpression ability is transferred in new LB liquid medium with 1.5% inoculum concentration.Work as Escherichia coli 37 DEG C of 1 h of culture of engineering bacteria or so, OD6000.2-0.4 is reached, containing pG-KJE8 (DnaK-DnaJ-GrpE GroES- GroEL L-arabinose and tetracycline) are used as derivant, pGro7 (GroES-GroEL), pKJE7 (DnaK-DnaJ- GrpE), pTF16 (tig) uses L-arabinose as derivant, and pG-TF2 (GroES-GroEL-tig) uses tetracycline As derivant.The expression of grape Glyco inhabiting arabinose operon, as control.37 DEG C are cultivated 2 h or so, OD600Reach 0.6-0.8, adds IPTG to induce the expression of trehalose synthetase.
Meanwhile, any time period in colibacillary incubation adds trehalose, preferably in trehalose synthetase Add after trehalose after abduction delivering, make concentration of the trehalose in system be 0.25-1mol/L.
The beneficial effects of the present invention is：
（1）By the measure in incubation, the catalysis effect of the trehalose synthetase after hot conditions process is effectively increased Rate, is conducive to the process of its catalysis maltose trehalose synthesis.
（2）Through transformation, trehalose synthetase heat resisting temperature is improved, and can resist the severe degree of environment, and then Reduce the requirement to production environment.
（2）The know-why of the present invention may is that：Trehalose as a kind of chemical molecular companion, by maintaining cell Osmotic pressure, the natural structure of stable protein improves the ability of the extraneous various pressure of protein resistant, the mode of action similar to point Sub- companion, is independently of the system that another kind of auxiliary protein of chaperone systems is correctly folded.After trehalose addition system, Dielectric viscosity and water activity can substantially be changed.Trehalose not only can assisting molecule companion's stable protein structure, reduce egg The false folding of white matter and aggregation, and as the companion of general lasting presence, formed in direct auxiliary protein folding process Flexible channel domain, increases the flexibility of non-native conformation, alleviates the false folding that protein is produced under pressure condition.
（3）Another know-why of the present invention may is that heat shock protein can recognize and help the folding of new synthetic proteins It is folded, and the aggregation-degraded to misfolded protein has regulating and controlling effect.The maturation of intracellular new polypeptide chain be higher temperature and compared with In high protein concentration and very crowded environment, carry out with high fidelity at a terrific speed, molecular chaperones protection intracellular The synthesis of protein and transport, prevent it from producing aggregation；Stress various ambient pressures, auxiliary protein correctly folds.
Description of the drawings
Fig. 1 be trehalose synthetase without trehalose and addition 0.2 M trehaloses under the conditions of 60 DEG C insulation enzyme activity with The change of time.
Fig. 2 is that trehalose synthetase is expressed and is co-expressed under the conditions of high temperature induction with trehalose synthetase and heat shock protein Trehalose synthetase enzyme activity changes.
Fig. 3 is chemical molecular companion（Trehalose）The lower trehalose synthetase enzyme of 50 DEG C of inductions is acted synergistically on heat shock protein Situation living.
Specific embodiment
With reference to embodiment, the invention will be further described.Listed embodiment is only used as explaination, and indicates this The spirit and scope of invention are not limited to the details in this and its modification case.
Embodiment 1
This example demonstrates that the structure screening process of the source of each biomaterial and bacterial strain
Expressive host used is E.coli BL21 (DE3) in the present embodiment, is this Laboratories Accession.Trehalose used is closed Into enzyme gene from grey streptomyces chromogenes（Streptomyces griseochromogenes）, laboratory preservation, mutually inside the Pass Hold patent applied for and authorize.Contain 572 amino acid from grey streptomyces chromogenes trehalose synthetase gene, molecular weight is about For 66 kDa, predict protein prediction protein contains 22.9% alpha-helix, and 34.09% exposure is in a solvent.
Molecular chaperones plasmid used in the present embodiment is purchased from the molecular chaperones plasmid kit of TaKaRa, is respectively pG- KJE8 (DnaK-DnaJ-GrpE GroES-GroEL), pGro7 (GroES-GroEL), pKJE7 (DnaK-DnaJ- GrpE), pG-TF2 (GroES-GroEL-tig), pTF16 (tig).The plasmid can be in Escherichia coli with pET series plasmids Middle co expression, with plasmid compatibility.
In the present embodiment, being built using the method for two kinds of plasmids of heat shock method cotransformation can be while overexpression trehalose synthetase With the bacterial strain of molecular chaperone protein.
Heat shock method conversion plasmid method is as follows：50 μ l E.coli BL21 (DE3) competent cells are taken, thawed on ice takes In a kind of addition competent cell in five kinds of molecular chaperones plasmids of 1 μ l pET28a-Tres and 1 μ l, gently rotate and mix. The min of ice bath 30,42oThe s of 1 min of C heat shocks 30, the min of ice bath 2.Add 900 μ l LB culture mediums, 37oC cultivates 45 min. Take 200 μ l nutrient solutions and be uniformly coated on Kan+And Cmr+On dual anti-flat board, incubated overnight screening positive clone.Can obtain simultaneously The expression bacterial strain of expression molecular chaperones and destination protein.
Embodiment 2
This example demonstrates that the effect of the concrete steps of external source addition trehalose and raising trehalose synthetase heat resistance.
Trehalose used by the present embodiment be food-grade, active principle content 98%, white crystalline powder.
Step one：The grey streptomyces chromogenes trehalose synthetase great expression in Escherichia coli of induction, using ni-sepharose purification, Obtain pure trehalose synthetase and be dissolved in PBS（Na2HPO4, NaH2PO4, pH 7.0）In solution.
Step 2：20 milliliters of trehalose synthetase enzyme liquids are taken, 0 M is added respectively, 0.2 M trehaloses after dissolving, are put respectively In 60 DEG C of water-baths, the rmp of rotating speed 100.Ensure enzyme thermally equivalent.
Step 3：0 min is incubated, 5 min, 10 min, 15 min, 20 min, 25 min take 200 ul enzyme liquids and add 800 Ul maltose substrates（Concentration：0.15 M, PBS dissolve）In, 30 min are reacted under the conditions of 30 DEG C and surveys trehalose synthetase enzyme activity. （Note：The trehalose synthetase enzyme activity of addition Trehalosc protection removes the trehalose in enzyme liquid, surveys enzyme activity for trehalose Synthesis enzymatic.）
Trehalose synthetase enzyme activity determination method：Using high performance liquid chromatograph（HPLC）To carry out trehalose synthetase conversion wheat Product analysis after bud sugar, chromatographic column is Shodex Sugar SZ5532 posts（6.0 mm ID × 150 mm）；Detector： Shodex RI101；Column temperature：60 DEG C, pressure：30 × 105 Pa；Response sample dilutes 5 times, the ul of sample introduction 10；Mobile phase is second Nitrile：Water（75：25）；The mL/min of flow velocity 1.0.Enzyme activity unit is defined：Under optimum condition, 1 min converts 1 nmol maltose The amount of trehalose synthetase for needed for trehalose is 1 enzyme-activity unit（U）.
Result after measuring and calculating is as shown in figure 1, result shows that the addition of trehalose can improve trehalose synthetase in high temperature The time that lower enzyme activity keeps.
Embodiment 3
This example demonstrates that be co-expressed in trehalose synthetase and heat shock protein Escherichia coli can improve under the conditions of high temperature induction The amount of soluble expression of trehalose synthetase.
Heat shock protein used by the present embodiment is pGro7 (GroES-GroEL) plasmid, using final concentration of 0.5 mg/mL The expression of L-arabinose induced heat shock protein, using the expression of final concentration of 0.5 mg/mL grapes Glyco inhabiting heat shock protein.Portugal Grape Glyco inhabiting heat shock protein is expressed, and the concentration of glucose of 0.5 mg/mL does not interfere with the expression of trehalose synthetase.
Inducing temperature chooses 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, the zymotic fluid of same OD is taken respectively, and thalline is collected by centrifugation, and surpasses Sound is crushed, and according to the step 3 enzyme activity determination method of embodiment 2 enzyme activity is determined.
Result after measuring and calculating is as shown in Fig. 2 result shows heat shock protein with coexpression in trehalose synthetase Escherichia coli Improve identical cell concentration under the conditions of trehalose synthetase high temperature induction and obtain higher enzyme activity.
Embodiment 4
This example demonstrates that chemical molecular companion（Trehalose）Act synergistically on heat shock protein and induce under high temperature trehalose synthesis Enzyme can improve the catalytic efficiency of trehalose synthetase under the conditions of high temperature induction with coexpression in heat shock protein Escherichia coli
Heat shock protein and co expression in trehalose synthetase Escherichia coli are carried out according to embodiment 3, chemical molecular companion is cooperateed with （Trehalose）Trehalose synthetase enzyme activity under the conditions of to improve high temperature induction.
Add chemical molecular companion when Escherichia coli cultivate 4 h respectively（Trehalose）.Inducing temperature is 50 DEG C, and external source adds Plus trehalose concentration is 0 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M.Determine trehalose under identical cell concentration to close Into enzyme enzyme activity.
Chemical molecular companion（Trehalose）Addition improve Escherichia coli cell concentration under the high temperature conditions.Trehalose By utilization under Escherichia coli high temperature, determine and trehalose composition is not detected by bacterial cell disruption liquid.Eliminate external source addition marine alga Sugar is to the impact during trehalose synthetase enzyme activity determination.
The result of the present embodiment：The expression of L-arabinose induced heat shock protein, cultivates the marine alga for adding 0.15 M after 4 h Sugar, with respect to enzyme activity highest, cell concentration also reaches highest to trehalose synthetase.The expression of grape Glyco inhabiting heat shock protein, grape Sugar improves cell concentration, but the addition of trehalose does not improve cell concentration, and external source adds the trehalose of 0.05 M, marine alga Sugared synzyme enzyme activity is higher.As a result show, chemical molecular companion enzyme activity under the conditions of trehalose synthetase high temperature induction is improved is There is optimum concentration, concentration is too high will not to improve on the contrary cell concentration, will not also improve relative enzyme activity.

Claims (7)

1. it is a kind of to improve the heat-staple method of trehalose synthetase, it is characterised in that be co-expressed heat shock protein in Escherichia coli Gene and trehalose synthetase gene, and also include, the external source addition trehalose in the system of expression trehalose synthetase.
2. the method for claim 1, it is characterised in that the heat shock protein gene is GroEL/S genes, DnaK/J bases Cause, TF genes, one or more in GrpE genes.
3. the method for claim 1, it is characterised in that the addition concentration of the trehalose is 0.25-1mol/L.
4. the method for claim 1, it is characterised in that the heat shock protein and trehalose synthetase are by inducing table Reach, and during expression, successively abduction delivering heat shock protein, trehalose synthetase；And in trehalose synthetase abduction delivering Afterwards external source adds trehalose.
5. method as claimed in claim 4, it is characterised in that the heat shock protein by arabinose abduction delivering, by grape The expression of Glyco inhabiting heat shock protein, the induced concentration of arabinose is 0.5-4 mg/mL, and the inhibition concentration of glucose is 0.5-2 mg/mL；Trehalose synthetase is by IPTG abduction deliverings.
6. method as claimed in claim 5, it is characterised in that described in abduction delivering during heat shock protein, Escherichia coli OD600Value It is between 0.2-0.4；Described in abduction delivering during trehalose synthetase, OD600Value is the time of abduction delivering in 0.6-0.8 For 8-12h.
7. the method for claim 1, it is characterised in that the trehalose synthetase gene is Tres genes.
CN201610955878.XA 2016-04-11 2016-10-27 Method for improving thermal stability of trehalose synthase Pending CN106636051A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2016102196612 2016-04-11
CN201610219661 2016-04-11

Publications (1)

Publication Number Publication Date
CN106636051A true CN106636051A (en) 2017-05-10

Family

ID=58821584

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610955878.XA Pending CN106636051A (en) 2016-04-11 2016-10-27 Method for improving thermal stability of trehalose synthase

Country Status (1)

Country Link
CN (1) CN106636051A (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1177638A (en) * 1996-07-25 1998-04-01 理化学研究所 Method for increasing activity of enzyme under high temperature
CN1746299A (en) * 2004-09-07 2006-03-15 中国科学院微生物研究所 A kind of thermally-stabilised TreP and encoding gene and application
CN101801404A (en) * 2007-06-29 2010-08-11 先进科技及再生医学有限责任公司 Protein formulations for use at elevated temperatures
CN102690795A (en) * 2012-05-22 2012-09-26 南京工业大学 Trehalose synthase of streptomyces griseochromogenes and coding gene and application of trehalose synthase
CN104593400A (en) * 2015-01-12 2015-05-06 北京化工大学 Method for improving expression level of trehalose synthase gene by molecular chaperone co-expression
CN104651332A (en) * 2015-02-15 2015-05-27 北京化工大学 Method for improving enzyme activity of trehalose synthase by C-terminal fragments of thermophilic bacteria trehalose synthase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1177638A (en) * 1996-07-25 1998-04-01 理化学研究所 Method for increasing activity of enzyme under high temperature
CN1746299A (en) * 2004-09-07 2006-03-15 中国科学院微生物研究所 A kind of thermally-stabilised TreP and encoding gene and application
CN101801404A (en) * 2007-06-29 2010-08-11 先进科技及再生医学有限责任公司 Protein formulations for use at elevated temperatures
CN102690795A (en) * 2012-05-22 2012-09-26 南京工业大学 Trehalose synthase of streptomyces griseochromogenes and coding gene and application of trehalose synthase
CN104593400A (en) * 2015-01-12 2015-05-06 北京化工大学 Method for improving expression level of trehalose synthase gene by molecular chaperone co-expression
CN104651332A (en) * 2015-02-15 2015-05-27 北京化工大学 Method for improving enzyme activity of trehalose synthase by C-terminal fragments of thermophilic bacteria trehalose synthase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
杨鑫 等: "灰产色链霉菌海藻糖合成酶的自诱导表达及两阶段温度控制发酵的优化", 《食品与发酵工业》 *

Similar Documents

Publication Publication Date Title
Migliardini et al. Biomimetic CO2 capture using a highly thermostable bacterial α-carbonic anhydrase immobilized on a polyurethane foam
Del Prete et al. Biochemical characterization of the γ-carbonic anhydrase from the oral pathogen Porphyromonas gingivalis, PgiCA
Krinsky et al. A simple and rapid method for preparing a cell-free bacterial lysate for protein synthesis
CN104152505B (en) A kind of method utilizing recombinant bacterial strain conversion to prepare 4HIL
Comini et al. Trypanothione synthesis in Crithidia revisited
García-Fraga et al. Optimized expression conditions for enhancing production of two recombinant chitinolytic enzymes from different prokaryote domains
Hung et al. Characterization of a salt-tolerant xylanase from Thermoanaerobacterium saccharolyticum NTOU1
Novak et al. Characterisation of an l-haloacid dehalogenase from the marine psychrophile Psychromonas ingrahamii with potential industrial application
Gupta et al. Characterization of a glycoside hydrolase family 1 β-galactosidase from hot spring metagenome with transglycosylation activity
CN105331642A (en) Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase
Premkumar et al. An unusual halotolerant α-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli
Agarwal et al. Characterization of a novel amylosucrase gene from the metagenome of a thermal aquatic habitat, and its use in turanose production from sucrose biomass
Hernalsteens et al. Partial purification and characterization of extracellular fructofuranosidase with transfructosylating activity from Candida sp.
Yu et al. Development of a cold-adapted Pseudoalteromonas expression system for the Pseudoalteromonas proteins intractable for the Escherichia coli system
Guo et al. Overproduction, purification, and property analysis of an extracellular recombinant fructosyltransferase
Zou et al. Magnesium ions increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis
CN102676557A (en) Encoding gene of type I pullulanase as well as recombinant expression and application thereof
Chang et al. Simultaneous production of trehalose, bioethanol, and high-protein product from rice by an enzymatic process
CN106834391A (en) A kind of formula and method based on artificial multienzyme biochemical reaction network synthetic protein
Yao et al. Food-grade γ-aminobutyric acid production by immobilized glutamate decarboxylase from Lactobacillus plantarum in rice vinegar and monosodium glutamate system
Xiong et al. Efficient production of γ-GABA using recombinant E. coli expressing glutamate decarboxylase (GAD) derived from eukaryote Saccharomyces cerevisiae
Ademakinwa et al. Some biochemical, catalytic, thermodynamic and kinetic properties of purified fructosyltransferase from wild and improved mutant-type Aureobasidium pullulans NAC8
Xiao et al. Genome mining for new α-amylase and glucoamylase encoding sequences and high level expression of a glucoamylase from Talaromyces stipitatus for potential raw starch hydrolysis
Zhang et al. Expression and characterization of recombinant sucrose phosphorylase
Wang et al. Cloning and expression of a trehalose synthase from Pseudomonas putida KT2440 for the scale-up production of trehalose from maltose

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20170510