CN105802951A

CN105802951A - Immobilized lipase as well as preparation method and application thereof

Info

Publication number: CN105802951A
Application number: CN201410857223.XA
Authority: CN
Inventors: 王明启; 王刚; 曾阿娜; 许骏
Original assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Current assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Priority date: 2014-12-30
Filing date: 2014-12-30
Publication date: 2016-07-27
Anticipated expiration: 2034-12-30
Also published as: CN105802951B

Abstract

The invention discloses immobilized lipase. The immobilized lipase contains a solid carrier and lipase fusion protein which is bound with the solid carrier and contains a cellulose binding domain (CBD). The invention also discloses a method for producing the immobilized lipase and an application of the immobilized lipase in the field of oil processing.

Description

Immobilized-lipase and its production and use

Invention field

The invention belongs to enzyme engineering and oils and fats or field of food industry, in particular it relates to immobilized-lipase and its production and use.

Background of invention

Lipase is the Acyl-hydrolase that a class is special, it is possible to the chemical reaction such as catalysis Ester hydrolysis, Lipase absobed, ester exchange and stereoisomer fractionation on oil-water interface, is often applied to the fields such as food, daily use chemicals, bioenergy.Resolvase is fixed on insoluble solid carrier, is conducive to improving the stability in use of enzyme.

Research currently, with respect to immobilized-lipase has had many progress.Such as, silica gel or mesopore silicon oxide, magnetic granule, nano fibrous membrane, the mode fixed fat enzyme that resin etc. directly adsorbs are adopted.Useful finishing cationic polymer (such as polymine) removes Electrostatic Absorption lipase, and is consolidated with cross-linking agent (such as glutaraldehyde).Also having and adopt polyacrylate, polyurethane, organosilicon etc. blendes together, with thick enzyme powder or enzymatic solution, the scheme that pulpous state is coated in substrate as binding agent.

Up to now, lipase method and the product of fixing transformation are not found.

Summary of the invention

First aspect, it is provided that immobilized-lipase, comprises solid carrier and the lipase fusion protein containing cellulose binding domain (Cellulosebindingdomain, CBD) in connection.

In some embodiments, the lipase fusion protein in described immobilized-lipase comprises following aminoacid sequence or is made up of following aminoacid sequence:

The aminoacid sequence of (a) lipase or its variant, described variant is the sequence that the aminoacid sequence by described lipase obtains after replacing, lacking and/or add at least one aminoacid, preferably, the aminoacid of described replacement, disappearance and/or interpolation is conserved amino acid, it is highly preferred that described replacement, disappearance and/or add occurs at the N end of described aminoacid sequence and/or C end；And

B () merges aminoacid sequence or its variant of the cellulose binding domain being connected with sequence (a) described, described variant is the sequence obtained after replacing, lacking and/or add at least one aminoacid by described CBD sequence, preferably, described replacement, disappearance and/or interpolation occur between conserved amino acid, it is highly preferred that described replacement, disappearance and/or add occurs at the N end of described aminoacid sequence and/or C end.

In some embodiments, described solid carrier is containing cellulosic carrier, it is preferable that containing the solid carrier of gossypin, it is more preferred to, described solid carrier is selected from cotton and the material containing microcrystalline Cellulose.

Second aspect, it is provided that the method producing immobilized-lipase, including

Solid carrier is provided,

By the lipase fusion protein containing CBD and base contact.

In some embodiments, the lipase fusion protein in described method comprises following aminoacid sequence or is made up of following aminoacid sequence:

B () merges, with sequence (a) described, the cellulose binding domain (Cellulosebindingdomain being connected, CBD) aminoacid sequence or its variant, described variant is the sequence obtained after replacing, lacking and/or add at least one aminoacid by described CBD sequence, preferably, described replacement, disappearance and/or interpolation occur between conserved amino acid, it is highly preferred that described replacement, disappearance and/or add occurs at the N end of described aminoacid sequence and/or C end.

In some embodiments, the solid carrier used in described method is containing cellulosic carrier, it is preferable that containing the solid carrier of gossypin, it is more preferred to, described solid carrier is selected from cotton and the material containing microcrystalline Cellulose.

In some embodiments, described method provides the step of solid carrier include washing solid carrier with containing surfactant packages, optionally, also comprise the step with solid carrier described in acid treatment, it is preferable that after acid treatment, also include the step regulating pH.

The third aspect, it is provided that the immobilized-lipase of the disclosure is application in producing diesel oil.

Fourth aspect, it is provided that the method for hydrolysis lipid, including

The immobilized-lipase of the disclosure is provided, and

Described immobilized-lipase is contacted with lipid, so that lipid hydrolysis.

5th aspect, it is provided that esterification process, including

The immobilized-lipase of the disclosure is provided, and

Described immobilized-lipase is contacted with fatty acid and lower alcohol, so that described fatty acid esterification.

6th aspect, additionally provides the application of the immobilized enzyme of the disclosure, for instance merge lipase application in dairy processing industry, household cleaning products, oil chemistry and medical field (treatment obesity and atherosclerosis etc.).

Brief Description Of Drawings

Figure 1A shows rhizomucor miehei (Rhizomucormieheilipase, the RML) lipase (CBD of cellulose binding domain (CBD)^new-RML) express.Wherein, swimming lane 1: before derivant adds, thalline sampling detects, swimming lane 2: bacterial cell disruption liquid supernatant after induction, swimming lane 3: bacterial cell disruption liquid precipitate after induction.

Figure 1B shows antarctic candida (CandidaantarcticalipaseB, the CALB) lipase (CBD of cellulose binding domain^new-CALB) express.Wherein swimming lane 1: empty carrier (without genes of interest) transformant fermentation liquid, swimming lane 2: genes of interest transformant fermentation liquid.

Fig. 2 A-2B shows microcrystalline Cellulose lipase (RML and CBD^new-RML) immobilization detection.

Fig. 2 A shows RML microcrystalline Cellulose immobilization: swimming lane 1: crude enzyme liquid, swimming lane 2: flow through liquid after crude enzyme liquid immobilization, swimming lane 3: immobilized enzyme cleaning mixture (the Tris-Cl buffer solution containing 0.8MNaCl), swimming lane 4: immobilized enzyme degeneration liquid (the Tris-Cl buffer degeneration with containing 10M carbamide).

Fig. 2 B shows CBD^new-RML microcrystalline Cellulose immobilization: swimming lane 1: crude enzyme liquid, swimming lane 2: flow through liquid after crude enzyme liquid immobilization, swimming lane 3-5: immobilized enzyme cleaning mixture (the Tris-Cl buffer solution containing 0.8MNaCl), swimming lane 6: immobilized enzyme degeneration liquid (the Tris-Cl buffer degeneration with containing 10M carbamide).

Fig. 2 C shows microcrystalline Cellulose lipase (CBD^new-CALB) immobilization detection, wherein swimming lane 1: enzyme fermentation liquid, swimming lane 2: effluent after fermentation liquid immobilization, swimming lane 3: immobilized enzyme cleaning mixture (Tris-Cl buffer solution), swimming lane 4: immobilized enzyme degeneration liquid (the Tris-Cl buffer degeneration with containing 10M carbamide).

Fig. 3 A-3C shows lipase (RML and CBD^new-RML) cotton immobilization detection.

Fig. 3 A shows RML immobilization on treated cotton: swimming lane 1: crude enzyme liquid, swimming lane 2: flow through liquid after crude enzyme liquid immobilization, swimming lane 3: immobilized enzyme cleaning mixture (the Tris-Cl buffer solution containing 0.8MNaCl), swimming lane 4: immobilized enzyme degeneration liquid (the Tris-Cl buffer degeneration with containing 10M carbamide).

Fig. 3 B shows CBD-RML immobilization on undressed cotton: swimming lane 1: crude enzyme liquid, swimming lane 2: flow through liquid after crude enzyme liquid immobilization, swimming lane 3: immobilized enzyme cleaning mixture (the Tris-Cl buffer solution containing 0.8MNaCl), swimming lane 4: immobilized enzyme degeneration liquid (the Tris-Cl buffer degeneration with containing 10M carbamide).

Fig. 3 C shows CBD^new-RML lipase immobilization on treated cotton: swimming lane 1: crude enzyme liquid, swimming lane 2: flow through liquid after crude enzyme liquid immobilization, swimming lane 3-5: immobilized enzyme cleaning mixture (the Tris-Cl buffer solution containing 0.8MNaCl), swimming lane 6: immobilized enzyme degeneration liquid (the Tris-Cl buffer degeneration with containing 10M carbamide).

Fig. 3 D shows CBD^new-CALB lipase immobilization detection on treated cotton, wherein, swimming lane 1: enzyme fermentation liquid, swimming lane 2: effluent after fermentation liquid immobilization, swimming lane 3: immobilized enzyme cleaning mixture (Tris-Cl buffer solution), swimming lane 4: immobilized enzyme degeneration liquid (the Tris-Cl buffer degeneration with containing 10M carbamide).

Fig. 4 A shows microcrystalline Cellulose and cotton immobilization CBD^new-RML lipase load capacity detects, wherein swimming lane 1-5: concentration is the BSA solution of 50,100,200,500 and 1000 μ g/mL, swimming lane 6: microcrystalline Cellulose immobilized enzyme degeneration liquid, swimming lane 7: cotton immobilized enzyme degeneration liquid.

Fig. 4 B microcrystalline Cellulose and cotton immobilization CBD^new-CALB lipase load capacity detects, wherein swimming lane 1-5: concentration is the BSA solution of 50,100,200,500 and 1000 μ g/mL, swimming lane 6: microcrystalline Cellulose immobilized enzyme degeneration liquid, swimming lane 7: cotton immobilized enzyme degeneration liquid.

Fig. 5 A shows microcrystalline Cellulose and cotton immobilization CBD^new-RML lipase Ester hydrolysis viability examination, vertical coordinate is the Ester hydrolysis vigor that every gram of immobilized enzyme shows.

Fig. 5 B-5C shows microcrystalline Cellulose and cotton immobilization CBD^newThe ability detection that palm oil fatty acid (PFAD) and methanol are esterified by-CALB lipase.Fig. 5 B shows palm oil fatty acid (PFAD) the methanol esterification rate curve of microcrystalline Cellulose and cotton immobilized-lipase, and abscissa is the water content relative to PFAD volume, and vertical coordinate is conversion ratio；Fig. 5 C shows the cotton immobilized enzyme example reaction thing state of 30% water content: left is this example reaction product, and the right side is the cotton product as negative control of the same water content of non-immobilized enzyme.General curdled appearance, it is believed that generate without ester, when having ester PFAM to generate, owing to its melting point is low, is generally oily, as shown in the left diagram.

Fig. 6 A-6B shows that merging lipase CBD1-RML and CBD3-RML immobilization on treated cotton detects respectively, wherein swimming lane 1 is crude enzyme liquid, swimming lane 2 flows through liquid for after crude enzyme liquid immobilization, swimming lane 3 is immobilized enzyme cleaning mixture (the Tris-Cl buffer solution containing 0.8MNaCl), and swimming lane 4 is immobilized enzyme degeneration liquid (the Tris-Cl buffer degeneration with containing 10M carbamide).

Detailed description of the invention

First aspect, it is provided that immobilized-lipase, it comprises solid carrier and the lipase fusion protein containing cellulose binding domain (Cellulosebindingdomain, CBD) in connection.

In some embodiments, described enzyme is lipase.In some embodiments, lipase is selected from animal, plant and microbial lipase or its combination.

In plant, what fatty enzyme was more is the seed of oil crop, such as castor bean, Semen Brassicae campestris etc.；Animal body include lipase more be pancreas and the fatty tissue of higher mammal；Lipase content in antibacterial, fungus and yeast more horn of plenty.Owing to microbe species is many, hereditary variation soon, easily occurs in breeding, there is more wider array of action pH than animals and plants, operative temperature scope and Substratspezifitaet, and microbe-derived lipase is typically all the exoenzyme of secreted, being suitable for industrialized great production and obtain high-purity sample, therefore microbial lipase is the important sources of industrial lipase.

It is therefore preferred that lipase used herein from produce lipase microorganism.Such as, described microorganism can be rhizomucor miehei (Rhizomucormiehei), dredge the thermophilic hyphomycete of cotton like (Thermomyceslanuginosus), Rhizopus delemar (Rhizopusdelemar), Rhizopus oryzae (Rhizopusoryzae), aspergillus oryzae (Aspergillusoryzea), antarctic candida (Candidaantarctic), Aspergillus niger (Aspergillusniger), fold candida mycoderma (Candidarugosa), Candida cylindracea (Candidacylindracea) and Rhodopseudomonas (Pseudononassp.) etc..In a preferred embodiment, lipase used herein is rhizomucor miehei lipase (RML) or candida antarctica lipase B (CandidaantarcticalipaseB, CALB).

In some embodiments, cellulose binding domain disclosed in the present application (Cellulosebindingdomain, CBD) is be present in relative molecular mass in fiber-like dhdps enzyme (such as cellulase) 0.4 × 10⁴～2.0 × 10⁴The fragment that Da does not wait, it can be inserted into and separate cellulosic crystal region, is adsorbed onto cellulose chain surface by the accumulation force of the hydrophobic plane of aromatic amino acid (hydrophobic interaction) and aromatic rings and glucose ring.

In some embodiments, described cellulose binding domain is from the cellulase of the microorganism of animal, plant or such as fungus and antibacterial.In some embodiments, described cellulose binding domain is from the cellulase of fungus and antibacterial.In some embodiments, described cellulose binding domain is from the cellulose binding domain in the exoglucanase cellulase of the microorganism of above-mentioned animal, plant or such as fungus and antibacterial, endoglucanase and/or beta glucan glycosides enzyme.In some embodiments, described cellulose binding domain is from Bacterial cellulose enzyme, for instance the cellulose binding domain in folded Coccus (Sarcina) cellulase of acetic acid Pseudomonas (Acetobacter), Agrobacterium (Agrobacterium), rhizobium (Rhizobiou) and/or bar.The example of typical Bacterial cellulose enzyme has Acetobacter gluconicum (Glucoacetobacterxylinum), Bacteroides succinogenes (B.succinogenes), excrement alkali fiber Zymomonas mobilis cellulase or exoglucanase cellulase, endoglucanase and/or beta glucan glycosides enzyme.In some embodiments, described cellulose binding domain is from the cellulose binding domain of fungal cellulase, such as, the cellulose binding domain of Gossampinus (Trichoderma), aspergillus (Aspergillus) and Penicillium (Penicillium) cellulase.The example of typical fungal cellulase has trichoderma reesei exoglucanase, Trichoderma harzianum EG II.

In some embodiments, the source of the cellulose binding domain in described lipase fusion protein can the source of connected lipase identical or different, as long as the two is capable of connecting forms fusion protein.

In some embodiments, the selection of described lipase is according to the needs of its application, its source to be selected.In the specific embodiments that some lipid hydrolysis react, the lipase of the particular source of optional deflection hydrolysis function, such as rhizomucor miehei lipase (RML), the thin thermophilic hyphomycete of cotton like (Thermomyceslanuginosus, TL) etc.；In the specific embodiments of some esterifications, the lipase of the particular source of optional deflection esterification, for instance, candida antarctica lipase B (CandidaantarcticalipaseB, CALB), TL mutant (CalleratransL, Novi believes) etc..

In some embodiments, described enzyme variants or CBD Variant amino acid sequences have homology or the homogeneity of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, more than 99% with its natural acid sequence.In preferred embodiments, enzyme variants or CBD variant have homology or the homogeneity of more than 99% with its native sequences.In some embodiments, the variant of described enzyme or CBD can be the native sequences of the enzyme of other kinds or the CBD belonging to same species with this enzyme or CBD, because kind difference causes having above-mentioned different sequence or sequence iden between sequence.

" homology " as herein described or " homogeneity " are defined as after sequence alignment and introducing room, the percentage ratio of aminoacid or residue identical in nucleotide sequence variants, if it is desired, reach the homology of largest percentage.It is known in the art for the method and computer program of comparison, such as utilize Blast disclosed in NCBI (Basiclocalalignmentsearchtool) to be analyzed and comparison, wherein can use BLASTP, BLASTN, BLASTX, TBLASTN etc..

In some embodiments, on the natural acid sequence basis of described enzyme or CBD, replace, lack or add 1-30, it is preferred to 1-20, more preferably the polypeptide variants obtained after 1-10 amino acid residue remains able to merge.In preferred embodiments, above-mentioned variant differs about 1,2,3,4,5,6,7,8,9 or 10 amino acid whose replacements, disappearance and/or interpolation with its natural acid sequence.In a more preferred embodiment, above-mentioned variant differs about 1,2,3,4 or 5 amino acid whose replacements, disappearance or interpolation with natural acid sequence.In a more preferred embodiment, above-mentioned variant be by the C end of natural acid sequence and/or N end regions be truncated or add about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,25 or more aminoacid and obtain.

In preferred embodiments, the sequence of above-mentioned variant is to comprise the sequence that one or several conservative amino acid replaces in its natural acid sequence, and the sequence after being wherein substituted has the function similar with described native sequences.

Some aminoacid replacement being referred to as " conserved amino acid replacement " can frequently occur in protein, but does not change conformation or the function of this protein, and this is the rule set up in protein chemistry.

Conserved amino acid replacement in the application includes but not limited to replace any another one of these aliphatic amino acids by any one in glycine (G), alanine (A), isoleucine (I), valine (V) and leucine (L)；Replacing threonine (T) with serine (S), vice versa；Replacing glutamic acid (E) with aspartic acid (D), vice versa；Replacing agedoite (N) with glutamine (Q), vice versa；Replacing arginine (R) with lysine (K), vice versa；Any another one in these aromatic amino acids is replaced with phenylalanine (F), tyrosine (Y) and tryptophan (W)；And replace cysteine (C) with methionine (M), vice versa.Other replacement can also be considered to be conservative, and this depends on specific amino acid environment and its effect in protein three-dimensional structure.Such as, glycine (G) and alanine (A) often can exchange, and can exchange such as alanine (A) and valine (V).The methionine (M) of relative hydrophobic can often exchange with leucine and isoleucine, and sometimes exchanges with valine.Lysine (K) and arginine (R) are through following location swap of being everlasting: wherein the key character of amino acid residue is its electric charge, and the different pK of both amino acid residues inconspicuous.

In some embodiments, cellulose binding domain polypeptide or variant sequence thereof are selected from SEQIDNO:4, SEQIDNO:14, and SEQIDNO:19.

In some embodiments, described solid carrier is containing cellulosic carrier, it is preferable that containing the solid carrier of plant cellulose, for instance selected from gossypin, bamboo cellulose, flaxen fiber element, hemicellulose and lignin or its combination in any.In some embodiments, described solid carrier is selected from cotton, the material containing gossypin, the material containing microcrystalline Cellulose or its combination in any.In some embodiments, the cotton of use includes but not limited to cotton cloth, cotton towel, duck, diablement fort and/or napped cotton fabric etc..

Second aspect, the application provides the method producing immobilized-lipase, including

Solid carrier is provided,

Lipase fusion protein containing CBD is contacted with solid carrier.

In some embodiments, described solid carrier is containing cellulosic carrier, it is preferable that containing the solid carrier of plant cellulose, for instance selected from gossypin, bamboo cellulose, flaxen fiber element, hemicellulose and lignin or its combination in any.In some embodiments, described solid carrier is selected from cotton, the material containing gossypin, the material containing microcrystalline Cellulose or its combination in any.In some embodiments, the cotton of use includes but not limited to cotton cloth, cotton towel, duck, diablement fort and/or napped cotton fabric.

In some embodiments, described method provide the step of solid carrier to include by the process step washing solid carrier containing surfactant packages；Optionally, the step with solid carrier described in acid treatment is also comprised.

In some embodiments, containing the reagent that surfactant packages is containing higher fatty acids (such as 10～18 carbon) salt, for instance include but not limited to detergent, liquid detergent, cleaning agent, suds and/or liquid detergent etc..

In some embodiments, described method also comprises the step with solid carrier described in acid treatment.Use solid carrier described in acid treatment, the waxiness on solid carrier, pectin can be removed further, thus being more beneficial for the combination of the lipase fusion protein containing cellulose binding domain (Cellulosebindingdomain, CBD) and solid carrier.Therefore, in some embodiments, described acid is for being capable of the effect above but the described cellulosic acid of inconspicuous destruction, for instance can be weak acid, diluted acid or organic acid, or can not be the strong acid of such as concentrated sulphuric acid or concentrated hydrochloric acid.In some specific embodiments, can be used for the acid of described method and such as may include but be not limited to phosphoric acid, acetic acid, dilute hydrochloric acid, HF, carbonic acid, sulfonic acid, chloroform and formic acid etc., in preferred embodiments, described acid is phosphoric acid, in a more preferred embodiment, described acid is 10-95% (W/V) phosphoric acid, it is preferable that, 30-90% (W/V) phosphoric acid, more preferably 50-90% (W/V) phosphoric acid or 60-85% (W/V) phosphoric acid.Such as, 60%, 65%, 70%, 75%, 80% or 85% (W/V) phosphoric acid.In some embodiments, described acid is 0.05-5mol/l hydrochloric acid, it is preferable that 0.05-3mol/l hydrochloric acid, more preferably 0.1-3mol/l hydrochloric acid, it is particularly preferred that 0.5-1mol/l hydrochloric acid.Such as, 0.1mol/l hydrochloric acid, 0.2mol/l hydrochloric acid, 0.5mol/l hydrochloric acid, 0.8mol/l hydrochloric acid, 1mol/l hydrochloric acid, 1.5mol/l hydrochloric acid, 2mol/l hydrochloric acid, 2.5mol/l hydrochloric acid, 3mol/l hydrochloric acid.

In some embodiments, described method also includes, and regulates the step of the pH of the solid carrier after described acid treatment, to remove or substantially to remove the acid of residual or to regulate solid carrier to suitable pH.In some specific embodiments, the solution for the applicable use of the pH of the solid carrier after regulating described acid treatment such as includes but not limited to aqueous slkali, saline solution, buffer, water etc..In some embodiments, described aqueous slkali and saline solution include but not limited to the solution such as sodium carbonate, sodium bicarbonate, sodium acetate, sodium phosphate, ammonia, aluminium hydroxide, magnesium hydroxide, zinc hydroxide, hydroxide two ferrum, hydroxide three-iron.In preferred embodiments, described alkali and salt are sodium carbonate, sodium bicarbonate, sodium hydroxide, for instance, 0.1%-30% (W/V) sodium bicarbonate, it is preferable that 0.5%-10% (W/V) sodium bicarbonate_,, more preferably 0.5%-5% (W/V) sodium bicarbonate, for instance, 0.5%, 1%, 2% (W/V) sodium bicarbonate；0.1%-30% (W/V) sodium carbonate, it is preferable that 0.5%-10% (W/V) sodium carbonate_,, more preferably 0.5%-5% (W/V) sodium carbonate, for instance, 0.5%, 1%, 2% (W/V) sodium carbonate；0.1%-30% (W/V) sodium hydroxide, it is preferable that 0.5%-10% (W/V) sodium hydroxide, more preferably 0.5%-5% (W/V) sodium hydroxide, for instance, 0.5%, 1%, 2% (W/V) sodium hydroxide.

In some embodiments, after each process step in the step of described offer solid carrier, optionally with clear water or deionized water or buffer solution or flushing solid carrier.Optionally, described solid carrier puts into lower alcohol after processing, for instance include but not limited to preserve in ethanol, isopropanol, it is preferable that 10-95% alcoholic solution, for instance the alcoholic solution of 10,20,30,40,50,60,70,80 and 90%.In some embodiments, described the step that the lipase fusion protein containing CBD contacts with solid carrier is carried out in buffer.

In some embodiments, the buffer used in described the application method is such as but not limited to phosphate buffer (PBS), Tris-HCl, citrate buffer solution, acetate buffer, borate buffer, Arginine buffer etc..

In some embodiments, the CBD lipase fusion protein that contains in described method is the lipase fusion protein described in the application first aspect and embodiment thereof.

In some embodiments, can be prepared by any suitable method that those skilled in the art are known containing CBD lipase fusion protein, for instance produced by recombinant technique, or chemosynthesis.

The recombination method producing fusion protein is known in the art.Such as by making the nucleotide sequence of encoding said fusion protein or nucleic acid molecules carry out gene expression in suitable host cell.The need to, it is possible to use whole label (eventualtag) to carry out fusion protein described in isolated or purified.

In some embodiments, the polynucleotide of described coding CBD can be operatively connected with the DNA sequence of codase, obtains the nucleic acid molecules of above-mentioned encoding fusion protein.

What can utilize in known in the art and obtainable multiple mature technology any prepares, manipulates and/or expresses polynucleotide or nucleic acid molecules.Such as, the polynucleotide sequence of the fusion protein of code book application may be used in recombinant DNA molecules to instruct fusion protein to express in suitable host cell.Due to the degeneracy that genetic codon is intrinsic, other DNA sequence encoding substantially the same or functionally equivalent aminoacid sequence can be used in the application, and these sequences may be used for clone and express given albumen.

In certain embodiments, the polynucleotide of the application or nucleic acid molecules are produced by synthetic, for instance directly chemosynthesis or enzymatic synthesis.In alternative embodiments, above-mentioned polynucleotide or nucleic acid molecules are produced by recombinant technique.In some specific embodiment, by the method for over-lap PCR (overlap-PCR) by encode above-mentioned CBD polypeptide nucleotide sequence with encode relevant enzyme nucleotide sequence tandem sequence repeats together with.

In certain embodiments, it is possible to by conventional method preferably as dideoxy chain termination (Sangeretal.PNAS, 1977,74:5463-5467) measures the sequence of polynucleotide or the nucleic acid molecules obtained.This kind of polynucleotide sequence measures and also can complete with the sequencing kit bought.In order to obtain the cDNA sequence of total length, order-checking need to be repeatedly performed.Sometimes for the cDNA sequence measuring multiple clones, the cDNA sequence of total length just can be spliced into.

In some embodiments, this application provides expression vector, it comprises the nucleic acid molecules encoding fusion protein described herein.

" expression vector " described in the disclosure is the nucleic acid construct recombinantly or synthetically produced, and it has a series of permission specific nucleic acid specified nucleic acid elements at host cell transcription.Expression vector in the application can be such as pET30a, pET-24a (+), the plasmid vector of pIRES2-EGFP, pcDNA3.1, pCI-neo, pDC516, pVAC, pcDNA4.0, pGEM-T, pDC315, or such as adenovirus, adeno-associated virus, retrovirus, semliki forest virus (sFv) carrier viral vector.

In preferred embodiments, the expression vector being used for cloning nucleic acid molecules described herein is plasmid vector.In a more preferred embodiment, described plasmid vector is pET30a.

In certain embodiments, method well-known to those having ordinary skill in the art is utilized to build the expression vector of the recombinant nucleotide sequence comprising encoding fusion protein and suitable transcribe/translational control element.These methods include (Sambroook, etal.MolecularCloning, aLaboratoryManual, coldSpringHarborLaboratory.NewYork, 1989) such as recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology.Nucleotide sequence is operably connected in the suitable promoter in expression vector, to instruct mRNA to synthesize.The representative example of these promoteres includes: colibacillary lac or trp promoter；The PL promoter of bacteriophage lambda；Eukaryotic promoter includes the promoter that CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, the LTRs of retrovirus retrovirus and some other known controlled gene are expressed in prokaryotic cell or eukaryotic cell or its virus.

In particular embodiments, by T4 ligase (Fermentas), recombinant nucleotide sequence is connected in carrier, for instance by RML-CBD^newRecombination sequence is connected in carrier PET30a.

In addition, expression vector preferably comprises one or more selected marker, to provide the phenotypic character of the host cell for selecting conversion, the dihydrofolate reductase cultivated such as eukaryotic cell, neomycin resistance and green fluorescent protein (GFP), or for colibacillary tetracycline or amicillin resistance etc..

In some embodiments, this application provides the host cell of expression vector.

In certain embodiments, the nucleic acid molecules of encoding fusion protein or the expression vector containing this nucleic acid molecules are transformed or transduced into host cell, it is thus achieved that containing the genetically engineered host cell of this nucleic acid molecules or expression vector.

Host cell used herein can be any one host cell well known to those skilled in the art, including prokaryotic cell, eukaryotic cell, for instance bacterial cell, fungal cell, yeast cells, mammalian cell, insect cell or plant cell etc..Exemplary bacterial cell includes any kind in Escherichia, Bacillus, streptomyces, Salmonella, Rhodopseudomonas and staphylococcus, including such as escherichia coli, Lactococcus, bacillus subtilis, wax printing fabric, Salmonella typhimurium, pseudomonas fluorescens.Select suitable host in the limit of power of those skilled in the art.

Any technology known in the art can be utilized to import in host cell by expression vector, including the gene transfer of conversion, transduction, transfection, viral infection, particle gun or Ti-mediation.Concrete method includes (Davis, L., Dibner, M., Battey, I., BasicMethodsinMolecularBiology, (1986)) such as calcium phosphate transfection, the transfection of DEAE-glucosan mediation, fat transfection or electroporations.Exemplarily, when host is prokaryote such as escherichia coli, competent cell can be gathered in the crops at exponential growth after date, use CaCl well known in the art₂Method converts.

In particular embodiments, the host cell used by the present invention is escherichia coli.In preferred embodiments, the expression vector conversion carrying recombination of polynucleotide sequence is carried out abduction delivering to escherichia coli (Escherichiacoli, E.coli) BL21.

In particular embodiments, it is provided that the method for fusion protein of preparation the application, comprising:

1) by the cloned nucleic acid molecule of encoding fusion protein on expression vector,

2) expression vector is converted or transduction is to suitable host cell,

3) in suitable culture medium, host cell is cultivated,

4) fusion protein described in separation also purification from described host cell or culture medium.

Suitable host cell refers to the host cell being suitable to expression vector or purpose expression of nucleic acid.Suitable culture medium refers to be suitable to host cell growth or it is carried out the culture medium of abduction delivering.

In preferred embodiments, the CBD polypeptide portion in fusion protein and enzyme part are connected by connexon.Connexon is also known as joint sequence (Linker).Joint sequence is designed as the common technology in fusion protein preparation, is namely coupled together by different genes of interest by nucleotide sequence one section suitable so that it is expressing in suitable organism becomes a peptide chain, and the aminoacid wherein playing interconnection function is called connexon.Those skilled in the art can select suitable connexon according to the size of fusion protein molecule and characteristic.

In certain embodiments, according to host cell used, it is possible to select various conventional medium.Cultivate when being suitable to host cell growth.Preferably, the host cell of through engineering approaches can be cultivated in being modified the conventional nutrient culture being suitable to activate promoter, to screen transformant or the polynucleotide of the amplification present invention.Convert suitable host cell and when after host cell growth to suitable cell density, the promoter selected with suitable method (such as temperature transition or chemical induction) induction, cell is further cultured for a period of time, to allow it to produce desired polypeptides or albumen.

In certain embodiments, by harvested by centrifugation host cell, by either physically or chemically smudge cells, and it is preserved for being further purified by the crude extract obtained.Microbial cell for protein expression can be crushed by any method easily, including freeze-thaw cycle, ultrasonic, Mechanical Crushing or use lysis agent.These methods are well known to those skilled in the art.

In certain embodiments, the recombinant polypeptide that host cell produces can be coated in cell or expresses on cell membrane or be secreted into extracellular.If it is required, its physics, chemistry separation by various separation methods with other characteristic and the albumen of purification of Recombinant can be utilized.Such as, the albumen of expression can be reclaimed and purification by following methods well known in the art from recombinant cell culture thing: the combination that the renaturation of routine processes, protein precipitant processes (salting-out method), centrifugal, bacterium, ultrasonic Treatment, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods are broken in infiltration.In particular embodiments, Ni is passed through²⁺Agarose gel chromatography post carrys out purified fusion protein.

In one specific embodiment, provide the method preparing RML-CBD or CBD-CALB fusion protein, comprising: the recombination of polynucleotide by coding RML-CBD or CBD-CALB is cloned in expression vector pET30a (antibacterial carrier respectively, and pAOX1 (yeast carrier RML-CBD), CBD-CALB) in, and pET30a, the pAOX1 with this polynucleotide sequence converted respectively to e. coli bl21 and Pichia sp., carry out abduction delivering, then separate and purification RML-CBD or CBD-CALB fusion protein from e. coli bl21 or Pichia sp..

In some embodiments, immobilized-lipase disclosed in the present application is utilized to make palm oil fatty acid (PFAD), soya fatty acid or oleic acid and methanol or ethanol generation esterification, thus producing diesel oil.

Fourth aspect, it is provided that the method for hydrolysis lipid, including

The immobilized-lipase of the disclosure is provided, and

Described immobilized-lipase is contacted with lipid, so that lipid hydrolysis.

In some embodiments, it is possible to by the lipid of described immobilized lipase enzyme hydrolysis selected from p-nitrophenyl palmitate (pNPP), tributyrin, Fructus Canarii albi wet goods.

5th aspect, it is provided that esterification process, including

The immobilized-lipase of the disclosure is provided, and

In some embodiments, the fatty acid in described method is C16-C22 fatty acid；Optionally palm oil fatty acid, oleic acid, soya fatty acid, described lower alcohol is selected from methanol, ethanol, propanol, isopropanol, butanol and isobutanol.

Additionally, immobilized enzyme disclosed in the present application may be used in dairy processing industry, household cleaning products, oil chemistry (such as producing biodiesel) and medical field (such as treatment obesity and atherosclerosis etc.).

Word disclosed herein " includes ", " comprising " and " containing " means " including but not limited to ", and is not intended to get rid of other parts, additive, component or step.

It should be understood that in the feature described in particular aspects disclosed herein, embodiment, characteristic, component or step, be applicable to described herein any other aspect, embodiment or embodiment, it is possible to combination in any or omission, unless contradiction with it.

Immobilized enzyme disclosed in the present application, preparation method and application thereof possess one or more of advantage:

I. with silica gel on the market, silicon oxide particle in the immobilized-lipase such as macroporous resin (as Novi believes immobilized enzyme Novozym435) and document, the immobilized-lipase such as magnetic granule is compared, and immobilized enzyme the adopted immobilization carrier cost of the application is low.

Ii. the firmness between enzyme and the carrier in the immobilized enzyme of the application is higher.

Iii. the immobilized enzyme of the application is high-purity enzyme product.

Iv. the load capacity of the immobilized enzyme of the application is high.

Chemical reagent or the binding agents such as v. the immobilized enzyme of the application can without polymine, glutaraldehyde, polyacrylate, polyurethane, organosilicon.

Vi. the enzyme of the application is directly and the combination of cellulose chain, does not involve the crosslinking to enzyme, bonding, mixed slurry process, it is to avoid the damage to enzyme.

Vii. the enzyme of the application is immobilized while, completes the purification to enzyme.

Above disclosure generally describes the embodiment of the application, by the embodiment of the further example the application of the following examples.These embodiments are described merely for the purpose of illustration, rather than the scope of the embodiment of restriction the application.

Embodiment

The nucleotide sequence of rhizomucor miehei (Rhizomucormiehei, RML) the lipase RML lipase used in embodiment is such as shown in SEQIDNO:1；It is not connected with the CBD nucleotide sequence of connexon such as (GenBankNo.AAA23089) shown in SEQIDNO:2, through optimizing the CBD containing connexon (referred to herein as CBD^new) nucleotide sequence and aminoacid sequence respectively as shown in SEQIDNO:3 and SEQIDNO:4；Amplimer sequence used is: RML-F-BamHI (SEQIDNO:5)；RML-R(SEQIDNO:6)；CBD-F(SEQIDNO:7)；CBD-R-SalI(SEQIDNO:8).Above-mentioned CBD^newSequence is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

The nucleic acid of candida antarctica lipase B (CandidaantarcticalipaseB, the CALB) lipase used and aminoacid sequence are such as shown in SEQIDNO:9；Corresponding amplimer sequence is: CALB-F-HindIII (SEQIDNO:10)；CALB-R(SEQIDNO:11)；CBD-F(SEQIDNO:12)；CBD-R-EcoRI(SEQIDNO:13).

Derive from the aminoacid sequence of the CBD (herein referred as CBD1) of trichoderma reesei exocellulase and nucleotide sequence respectively as shown in SEQIDNO:14 and 15.Amplimer sequence used by preparation CBD1-RML is: RML-F-BamHI (SEQIDNO:5)；RML-1-R(SEQIDNO:16)；CBD-1-F(SEQIDNO:17)；CBD-1-R-SalI(SEQIDNO:18).

Derive from the aminoacid sequence of the CBD (herein referred as CBD3) of Trichoderma harzianum EG II (THEGII) and nucleotide sequence respectively as shown in SEQIDNO:19 and 20.Amplimer sequence used by preparation CBD3-RML is: CBD-3-F-BamHI (SEQIDNO:21)；CBD-3-R(SEQIDNO:22)；RML-3-F(SEQIDNO:23)；RML-3-R-SalI(SEQIDNO:24).

The embodiment 1A lipase (CBD-RML) containing immobilized molecules label is expressed

With primestar exo+ polymerase (Takara company) to RML (by the raw work synthesis in Shanghai) and CBD^newNucleotide sequence (the CBD sequence containing connexon (linker)) carries out pcr amplification respectively, with RML and CBD^newPCR primer be template, by over-lap PCR (overlap-PCR) (Cao Yang etc., the foundation of Overlap extension PCR method and application.Hebei medicine volume 11 phase November the 27th in 2005) method by RML and CBD^newSequence tandem sequence repeats together, RML and CBD^newNaturally occurring connexon (linker) sequence in CBD is had to connect between sequence.Then with T4 ligase (Fermentas) by RML-CBD^newRecombination sequence is connected in carrier PET30a (purchased from the raw work in Shanghai).

By the carrier heat shock method (42 DEG C after connection, 90 seconds) proceed to bacillus coli DH 5 alpha competent cell (purchased from the raw work in Shanghai), and by its solid LB media flat board (1.5% agarose the antibiotics ampicillin containing 50 μ g/mL, yeast powder 5%, peptone 10%, NaCl5%) in 37 DEG C of overnight incubation, then continue in 5mLLB culture medium (yeast powder 5%, peptone 10%, NaCl5%) in 37 DEG C, 200rpm shaken cultivation.Extract plasmid with Qigen plasmid extraction kit, serve the raw work order-checking in sea.The correct plasmid of sequencing result is required expression vector.

Choose the correct expression vector of sequence to convert to escherichia coli (Escherichiacoli, E.coli) BL21.In LB culture medium, amplification culture is to OD₆₀₀When value is 0.6～0.8, adds isopropyl-beta D-thio galactopyranoside (IPTG) derivant (1mM) and induce, continue 16 DEG C, 150rpm incubated overnight, collect thalline.

Thalline Tris-HCl (pH8.0) buffer collected is suspended, ultrasonic disruption；Or adopting lysate Eddy diffusion thalline, ice bath crushes 20min.Then by broken liquid under the rotating speed of 10000 revs/min (rpm), centrifugal 15min.The supernatant obtained after centrifugal is crude enzyme liquid.

Utilizing polyacrylamid gel electrophoresis (SDS-PAGE) to detect sample, as shown in Figure 1A, it will be seen that add after derivant, destination protein is able to great expression to result, and greatly in the middle of supernatant.

The embodiment 1B lipase (CBD-CALB) containing immobilized molecules label is expressed

With primerstar exo+ polymerase (Takara company) to CALB and CBD^newSequence carries out pcr amplification respectively, with the PCR primer of CALB and CBD for template, by the method for overlap-PCR by CALB and CBD sequence tandem sequence repeats together, has naturally occurring Linker in CBD to connect between CALB and CBD sequence.Then with T4 ligase (Fermentas), CALB-CBD recombination sequence is connected in carrier pAOX1.Extract plasmid, order-checking.After carrier SalI single endonuclease digestion linearisation correct for order-checking, according to the standard transformation methods of Pichia sp., electroporated Pichia sp. competent cell, it is applied to Selective agar medium MDS and screens flat board, in 28 DEG C of overnight incubation.

Picking yeast list colony inoculation in 5mLYPD culture medium, 28 DEG C, 200rpm incubated overnight.Being seeded in the BMGY culture medium of 50mL, 28 DEG C, 220rpm cultivates, and collects thalline.With the resuspended washing thalline of sterilized water 2 times, then with the resuspended thalline of BMMY culture medium.The methanol of 2% is added in BMMY culture medium, 28 DEG C, 220rpm abduction delivering.Live every 24h sampling and measuring enzyme, and add 0.5mL methanol in 50mL culture medium.After induction 3d, fermentation liquid concentration is collected.Result is as shown in Figure 1B.

Embodiment 2A microcrystalline Cellulose lipase immobilization

Take microcrystalline Cellulose (Avicel, 11365-1KG, Sigma) 100mg to centrifuge tube, add 200 μ LTris-HCl (pH8.0) buffer and soak 10min, be centrifuged and abandon supernatant.

Adding 500 μ LCBD-RML cellular lysate supernatant or crude enzyme liquids, 4 DEG C are swayed and hatch 30min, centrifugal abandon supernatant.

With the Tris-HCl buffer solution containing 0.8MNaCl.

SDS-PAGE detects each outflow or washing sample, and immobilized enzyme is with the 150 μ L Tris-HCl buffer degeneration containing 10M carbamide and detects.

Compareing with the RML without CBD sequence, repeat the above steps, result is as seen in figs. 2a-2b.

From result shown in Fig. 2 A-2B it will be seen that the RML lipase being not introduced into CBD label can not be immobilized, and the CBD-RML lipase introducing CBD label can by microcrystalline Cellulose purification and immobilization.

Embodiment 2B microcrystalline Cellulose lipase immobilization

Single step purification and immobilized-lipase effect from fermentation liquid, using Sigma product microcrystalline Cellulose Avicel (11365-1KG) of pure cellulose composition as positive control, take its powder 70mg to centrifuge tube, add 200 μ LTris-Cl (pH6.0) buffer and soak 10min, be centrifuged and abandon supernatant.

Adding 1mLCBD-CALB fermentation liquid or crude enzyme liquid, 4 DEG C are swayed and hatch 30min, centrifugal abandon supernatant.

Use Tris-Cl buffer solution.

SDS-PAGE detects each outflow or washing sample, and immobilized enzyme is with the 250 μ L Tris-Cl buffer degeneration containing 10M carbamide and detects, and result is as shown in Figure 2 C.

From result shown in Fig. 2 C it will be seen that the CBD-CALB lipase introducing CBD label can by microcrystalline Cellulose purification and immobilization.

Embodiment 3A cotton lipase immobilization

Cotton processes:

The pure cotton canvas that will buy on market, is cut into about 1cm²Fritter, claiming its quality is 41.6 ± 5.5mg.Boil 1h with 10% commercial laundry liquid, clear water wash, followed by 85% (W/V) phosphatase 24 DEG C process 1h, after rinse with water, then with 1% (W/V) NaHCO₃Neutralize, and be washed till pH5-7 with clear water, be finally placed in 20% alcoholic solution 4 DEG C of preservations.

Take cotton fritter 2 to centrifuge tube, add 200 μ LTris-HCl (pH8.0) buffer and soak 10min, abandon supernatant.

Adding 500 μ l crude enzyme liquids, cellular lysate supernatant, 4 DEG C are swayed and hatch 30min, abandon supernatant.

With the Tris-HCl buffer solution containing 0.8MNaCl.

SDS-PAGE detects each outflow or washing sample, and immobilized enzyme is with the 250 μ L Tris-HCl buffer degeneration containing 10M carbamide and detects.

Compareing with the RML without CBD sequence, repeat the above steps, repeat CBD-RML immobilization step with the cotton without any process simultaneously, as negative control, result is as shown in figs. 3 a-3 c.

Still can not by cotton immobilization it will be seen that be not introduced into the RML lipase of CBD label, but undressed cotton can not the immobilization CBD-RML lipase containing CBD label.

Can be seen that high ion concentration cleaning mixture still by CBD-RML lipase eluting, can not illustrate that its degree of being firmly combined with is very high simultaneously；The electrophoretic band of the enzyme sample that degeneration is got off from carrier proves, its purity is also very high.

Embodiment 3B cotton lipase immobilization

Cotton processes:

The pure cotton canvas that will buy on market, is cut into about 1cm²Fritter,.Boil 1h with 10% suds, clear water wash, followed by 70% (W/V) phosphatase 24 DEG C process 1h, after rinse with water, then with 1% (W/V) KHCO₃Neutralize, and be washed till pH5-7 with clear water, be finally placed in 20% aqueous isopropanol 4 DEG C of preservations.

Taking above-mentioned process cloth 105 DEG C heating 4h to constant weight, be down to room temperature and claim its weight, every cotton block weight is about 35mg.

Take 2 pieces of cotton to centrifuge tube, add 200 μ LTris-Cl (pH6.0) buffer and soak 10min, abandon supernatant.

Adding 1mLCBD-CALB lipase fermentation liquid, 4 DEG C are swayed and hatch 30min, abandon supernatant.

Use Tris-Cl buffer solution.

SDS-PAGE detects each outflow or washing sample, and immobilized enzyme is with the 250 μ L Tris-Cl buffer degeneration containing 10M carbamide and detects, and result is as shown in Figure 3 D.

Cotton after above-mentioned process all can reach purification similar for Avicel and immobilization effect.

Embodiment 4A microcrystalline Cellulose and cotton immobilized-lipase load capacity

With the bovine serum albumin (BSA) of 1mg/mL concentration for standard sample, being diluted to 500,200,100 and 50 μ g/mL respectively, the common sample preparation of urea-denatured liquid with microcrystalline Cellulose and cotton electrophoresis detection, result is as shown in Figure 4 A.

AlphaImagerHP software analysis band, with the product of band area and density for abscissa, with concentration for transverse and longitudinal mark, obtains standard curve equation y=8 × 10^-5*x-732.62(R², and calculate the CBD-RML lipase load capacity of gained microcrystalline Cellulose and cotton and be respectively as follows: 2.28mg/g base material, 3.97mg/g base material=0.9856).Above lipase is because being connected to CBD, and its load capacity should be regarded as the load capacity of pure enzyme.

Therefore, contrasting the thick enzyme load capacity (because it does not have immobilization specificity) that macroporous resin in the market is about 1mg/g, the microcrystalline Cellulose of the disclosure and the enzyme load capacity of cotton immobilization product seem more considerable.

Embodiment 4B microcrystalline Cellulose and cotton immobilized-lipase load capacity

With the bovine serum albumin (BSA) of 1mg/mL concentration for standard sample, being diluted to 500,200,100 and 50 μ g/mL respectively, the common sample preparation of urea-denatured liquid with microcrystalline Cellulose and cotton electrophoresis detection, result is as shown in Figure 4 B.

AlphaImagerHP software analysis band, with the product of band area and density for abscissa, with concentration for transverse and longitudinal mark, obtains standard curve equation y=8 × 10^-5*x-732.62(R², and calculate the CBD-CALB lipase load capacity of gained microcrystalline Cellulose and cotton and be each about 1mg/g base material=0.9856).Above lipase is because being connected to CBD, and its load capacity should be regarded as the load capacity of pure enzyme.

Embodiment 5A microcrystalline Cellulose and cotton immobilized-lipase Ester hydrolysis vigor

According to the value of calculation of gained in above-described embodiment, each immobilized enzyme and resolvase molar concentration Tris-Cl buffer are regulated to 5 μ g/ml, pNPP method is adopted to detect its Ester hydrolysis vigor, sample hydrolysis substrate is p-nitrophenyl palmitate (pNPP), reaction condition is temperature 40 DEG C, pH value 8.0.

The concrete grammar that Nitrobenzol cetylate (pNPP) method measures lipase activity is as follows.

With p-nitrophenyl cetylate (pNPP) for substrate, carry out the calculating of enzyme activity with the enzyme liquid of unit volume growing amount of enzymolysis generation paranitrophenol (pNP) within the unit interval.Concrete grammar is as follows: be pre-configured with substrate and buffer, substrate: 6mg/mLpNPP (isopropanol dissolving), buffer: 0.05MTris (pH8.0,0.1% arabic gum).Substrate and buffer are made into reaction mixture with 1:9 (v/v).Take two 2mL centrifuge tubes, respectively control tube and sample cell.Add 400 μ L reaction mixtures respectively to two centrifuge tubes, at the pre-temperature bath 5min of suitable reaction temperature (such as 35 DEG C).In sample cell, add the dilution enzyme liquid of 20 μ L, after mix homogeneously, continue temperature bath 15min.Add 1.5mL ethanol to above-mentioned two centrifuge tubes and terminate reaction, and add the dilution enzyme liquid of same amount to control tube.With the centrifugal 2min of 12000rpm, take supernatant, survey the light absorption value at 405nm place.

Enzyme activity unit is defined as: namely 1 unit refers to that catalysis per minute discharges the enzyme amount needed for the pNP of 1 μm of ol under standard laboratory conditions.According to standard curve gained enzyme activity computing formula it is:

A=-([A1-A0] × 0.7885-0.0118) × V1 × n/ (V2 × t).

Wherein, A: sample enzyme is lived (U/mL), A1: the OD405 of sample enzyme liquid, A0: OD405, the V1 of comparison enzyme liquid: the volume (mL) of overall reaction liquid, n: the extension rate of enzyme liquid, V2: the volume (mL) of enzyme liquid, t: response time (min).

The Ester hydrolysis vigor (using gained enzyme activity unit divided by the quality of immobilization base material) of final calculated microcrystalline Cellulose and cotton immobilized-lipase is as shown in Figure 5, respectively 352.61 ± 18.26U/g microcrystalline Cellulose (Avicel) and 589.23 ± 13.35U/g cotton (Cotton), both gaps are probably derived from the difference of load capacity.

As shown in embodiment 4A, enzyme amount respectively 2.28mg and the 3.97mg of every gram of microcrystalline Cellulose and the load of cotton institute, being converted into the RML of the same amount vigor shown is 75.31U and 131.13U (being 33.03 ± 3.27U/mg through being hydrolyzed specific enzyme activity with the RML of immobilized enzyme synchronism detection gained).

Can being drawn by above-mentioned analysis, compared with original lipase RML, by introducing CBD label and being immobilized to the mode on microcrystalline Cellulose and cotton, enzyme can't being made to live and reduce, even show activity increases.

Embodiment 5B microcrystalline Cellulose and the esterification ability detection of cotton immobilized-lipase

Step is as follows:

Palm oil fatty acid (PFAD) heating and melting, takes in 400 μ L to 1.5mL centrifuge tubes, 50 DEG C, and 14000rpm lowers the temperature.

Calculate gained load capacity according to above-described embodiment, add containing the CBD-CALB microcrystalline Cellulose that enzyme amount is 100 μ g or cotton immobilized-lipase sample, methanol 50 μ L, add a methanol, coreaction 4h every 1h.

Then centrifugal 2min under 12000rpm, takes 200 μ L of supernatant (zero, weighs, general 0.15～0.17g) to triangular flask.Dissolve with 5mL ethanol, add one or two phenolphthalein and give instruction agent.

Titration determination acid value (or claim neutralization number, acid number, acidity, represent the milligram number neutralizing the potassium hydroxide (KOH) needed for 1 gram of chemical substance).Acid value=V*C*M/ reactant quality, such as initial acid value 10*0.05*56/0.15=186.

Conversion ratio (esterification yield)=(original acid value-initial acid value)/original acid value.

Result is as shown in Figure 5 B, abscissa is the water content relative to PFAD volume, vertical coordinate is conversion ratio, it can be seen that, at 30% water content place, cotton (Cotton) immobilization product can give expression to most high ester voltinism can (nearly 90%), and microcrystalline Cellulose (Avicel) sample containing same enzyme amount to show the highest esterification performance needed for water content be 80%, and esterification yield is less than 70%.This is likely to need not the too much water yield to be just fully contacted with substrate with Cotton immobilized enzyme sample, and pulverous Avicel sample needs the more water yield to keep dispersity relevant, because more water content is unfavorable for the carrying out one of the product of esterification (water be) of esterification.Fig. 5 C illustrates reactive state during the highest esterification ability of cotton, and the melting point of esterification products palm oil fatty acid methyl ester (PFAM) is relatively low, so end-product shows as oily, as shown in the left diagram.

Other CBD sequence cotton immobilization effects of embodiment 6

Prepare CBD1-RML and CBD3-RML respectively according to the method for embodiment 1A and merge lipase.Method described in embodiment 3A processes cotton detection fusion enzyme immobilizatio effect.Result is as shown in Figure 6 A and 6 B.With CBD^newThe result of-RML is similar, and CBD1-RML and CBD3-RML lipase can not be eluted by high ion concentration cleaning mixture from cotton, illustrates that its degree of being firmly combined with is very high；The electrophoretic band of the enzyme sample that degeneration is got off from carrier proves, its purity is also very high.

Claims

1. immobilized-lipase, comprise solid carrier and in connection containing cellulose binding domain (Cellulosebindingdomain, CBD) lipase fusion protein, or be made up of solid carrier and the lipase fusion protein containing cellulose binding domain in connection.

2. the immobilized-lipase described in claim 1, wherein said lipase fusion protein comprises following aminoacid sequence or is made up of following aminoacid sequence:

3. the immobilized-lipase described in any one of claim 1-2, wherein said solid carrier is containing cellulosic carrier, preferably comprise the solid carrier of plant cellulose, for instance gossypin, bamboo cellulose, flaxen fiber element, hemicellulose and lignin or its combination in any.

4. the immobilized-lipase described in any one of claim 1-3, wherein said solid carrier is selected from cotton, the material containing gossypin and the material containing microcrystalline Cellulose.

5. the method producing immobilized-lipase,

Solid carrier is provided,

By the lipase fusion protein containing cellulose binding domain (Cellulosebindingdomain, CBD) and base contact.

6. the method described in claim 5, wherein said lipase fusion protein comprises following aminoacid sequence or is made up of following aminoacid sequence:

7. the method described in any one of claim 5-6, wherein said solid carrier is containing cellulosic carrier, it is preferable that containing the solid carrier of plant cellulose.

8. the method described in any one of claim 5-7, wherein said solid carrier is selected from cotton and the material containing microcrystalline Cellulose.

9. the method described in any one of claim 5-8, wherein provides the step of solid carrier to include with the reagent wash solid carrier containing surfactant, optionally, also comprise the step with solid carrier described in acid treatment, preferably, after acid treatment, also include the step regulating pH.

10. the immobilized-lipase of any one of claim 1-4 application in producing diesel oil, it is preferable that include utilizing described immobilized-lipase by palm oil fatty acid (PFAD) methanol esterification.