CN107699583A - A kind of intracellular serine protease ISPr encoding gene and its recombination expression - Google Patents

A kind of intracellular serine protease ISPr encoding gene and its recombination expression Download PDF

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Publication number
CN107699583A
CN107699583A CN201711158779.XA CN201711158779A CN107699583A CN 107699583 A CN107699583 A CN 107699583A CN 201711158779 A CN201711158779 A CN 201711158779A CN 107699583 A CN107699583 A CN 107699583A
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ispr
protease
serine protease
expression
intracellular serine
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田永强
侯燕燕
田杰伟
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Sichuan University
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Sichuan University
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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21106—Hepsin (3.4.21.106)

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  • Enzymes And Modification Thereof (AREA)

Abstract

The invention discloses a kind of intracellular serine protease gene and its application, belong to biological technical field.ISPr full length genes of the present invention are the genomic DNAs by extracting bacillus LCB10, are obtained by round pcr.Full length gene 954bp, encode 317 amino acid.The present invention carries out protease property representation to realize that ISPr genes realize heterologous clone, expression, purifying in Escherichia coli first.The protease has good sodium chloride tolerance, and this will have broad prospects in leather loses hair or feathers industry and food industry applications.

Description

A kind of intracellular serine protease ISPr encoding gene and its recombination expression
Technical field
The present invention relates to the recombination expression of intracellular serine protease ISPr a kind of and its technical field of application.
Background technology
Protease is a kind of important industrial enzyme preparation, wherein, alkali protease accounts for 25%, is that one kind can be in alkaline ring Catalytic proteins are hydrolyzed to the protease of amino acid under border.Alkali protease is widely used in enzyme detergent industry, food adds The industries such as work, feed, leather industry, silk scouring, medicine and environmental protection.
The alkali protease research in China there is problems：（1）Microbial resources underexploitation, protease species compared with It is few；（2）Sphere of action is narrower；（3）It is enzyme preparation single varieties, expensive；（4）Application surface is wide etc. not enough.Alkali protease Market is currently still the state that supply falls short of demand, and current research emphasis is still breeding high-yield alkali protease bacterial strain, to it Training systern and relevant nature research are carried out, lifts the alkali protease quality and yield in China.
With the continuous intensification that people are recognized the huge applications potentiality of protease, the research of protease also begins to gradually turn To the high efficient expression in heterologous host, to be more beneficial for industrial expansion.It is false to have bacillus licheniformis, verdigris at present The protease gene in the sources such as monad, aspergillus fumigatus, realized respectively in Escherichia coli, bacillus subtilis, Pichia pastoris Heterogenous expression.Although the Escherichia coli host preferred as one, successful expression multiple protein, it is as expressive host One of bottleneck, when being overexpression protease, toxic action easily is produced to thalline and easily forms inclusion body.Therefore, Obtaining an albumen enzyme engineering bacteria that high efficient expression can be carried out under the conditions of non-cryogenic becomes most important.
The content of the invention
Present invention firstly provides a kind of gene ISPr for encoding intracellular serine protease, is with from the salt-soda soil in Xinjiang The high proteinase yield bacterial strain LCB10 filtered out in soil is starting strain, extracts its total genomic dna, designs primer and combines Its complete genome sequencing, ISPr genetic fragments are amplified by PCR, full length gene 954bp, encode 317aa.
The gene of acquisition is also connected by the present invention with carrier PET30a, builds recombinant expression carrier PET30a-ispr, conversion E. coli bl21 (DE3), culture gained recombination bacillus coli, successful expression protease gene after induction.Will expression Bacterial strain collect thalline, broken centrifugation obtains supernatant, and the pure enzyme preparation for purifying to obtain by anion-exchange chromatography has egg White enzymatic activity.
New serine protease its molecular weight obtained by the present invention is about 35kD, optimum temperature 40oC, it is most suitable PH9.0, the protease remain to keep more than 85% prolease activity under 7% sodium chloride concentration, and this will be in leather industry With there is good application prospect in food industry.
Brief description of the drawings
Fig. 1 is the electrophoretogram of ISPr genes；M1, plasmid marker；1, recombinant plasmid PET30a-ispr；2, PET30a- Ispr plasmid double digestions；3, the PCR fragment of full length gene；M2, linear marker.
Fig. 2 is the albumen expression of enzymes SDS-PAGE of recombination bacillus coli；M, standard protein；1, the thalline before induction；2, lure Thalline after leading；3, bacteria break supernatant；4, break bacterium precipitation；5, the protease of purifying.
Fig. 3, Fig. 4, Fig. 5 are respectively the protease ISPr purified the property such as temperature, pH, the tolerance of salinity.
Embodiment
Embodiment one：The assay method of proteinase activity
The protease for taking 100 μ l to purify is added in 200 μ l 50mM Glycine-NaOH cushioning liquid, adds 100 μ l Casein solution（2%）, 40o10min is reacted in C water-baths, adds 200 μ l trichloroacetic acids（6.56%）Terminating reaction, 10000rpm centrifuges 10min.The μ l of supernatant 200 are taken after centrifugation, add 1ml sodium carbonate（4.24%）, 200 μ l forint phenol reagents, survey Determine 660nm absorbance.Blank adds isometric enzyme liquid to be used as blank control again to add after TCA.
Embodiment two：The acquisition of ISPr genes
Bacillus LCB10 genomic DNA is extracted, carries out whole genome sequence sequencing（Completed by U.S. lucky biology）, with reference to The ORF reading frames arrived, the access of pertinent literature and the comparison of database are carried out, have selected a new intracellular protein enzyme gene ISPr, there is 82% amino acid sequence similarity with the ISP-1 genes in the known sources of Bacillus subtilis 168.
Embodiment three:The expression of ISPr genes
Design primer amplification ISPr genes, sense primer (5'-GGAATTCCATATGCAAATGCGATTAATTCC-3'), downstream Primer (5'-CCGCTCGAGAAGGGAAAGAAGTTTTGCTT-3').Obtained gene restriction enzyme will be expandedNdeI WithXhoIBe connected to after double digestion on PET30a plasmid vectors, the recombinant plasmid transformed of structure into Escherichia coli DH α, containing Have and select positive recombinant, sequence verification on the LB solid medium flat boards of kalamycin resistance.The recombinant plasmid that will be obtained PET30a-ispr converts BL2（DE3）, obtain gene engineering expression bacterium.Picking individual colonies extremely contain the liquid of 50 μ g/ml kanamycins In body culture medium, 37oC is incubated overnight.Next day is taken in culture 1ml to the 100ml containing kanamycins LB culture mediums, 37oC It is 0.6 or so to cultivate to the OD values of thalline, adds 100 μ l IPTG（1M）Fiber differentiation 4h.Protease table is made through SDS-PAGE Up to the analysis of amount.
Example IV：Protease ISPr purifying
For recombinant bacterial strain after induced expression, thalline is collected by centrifugation in 8000rpm, adds broken born of the same parents' buffer solution（50mM Tris-HCl , 50mM NaCl, pH 7.5,1mg/ml lysozymes）In, do the purifying of further protease.With Hitrap Q FF anion Exchange column is purified, and is used（50mM Tris-HCl, 50mM NaCl, pH 7.5）Pillar is balanced, it is linearly terraced with NaCl containing 1M Degree elution destination protein, carries out SDS-PAGE identifications.
Embodiment five：Protease ISPr zymologic property research
Respectively in different temperatures（20oC,30oC,40oC,50oC,60oC,70oC,75oC）Determine protease activity.Measure is in different pH Under the conditions of proteinase activity（Citrate pH4.0-5.0, sodium phosphate pH6.0-7.0, Tris-HCl pH8.0-9.0, carbonic acid Salt pH10.0, Glycine-NaOH pH11.0）.Determine the sodium chloride tolerance of protease（Various concentrations 1%-15%）.
Sequence table
<110>Tian Yongqiang
Hou Yanyan
Tian Jiewei
<120> ISPr sequence
<141> 2017-11-17
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 0
<212> DNA
<213> Bacillus sp. LCB10
<400> 1

Claims (2)

1. a kind of intracellular serine protease ISPr encoding gene and its Recombinant protein expression.
2. intracellular serine protease ISPr as claimed in claim 1,954 nucleotides compositions of the protease-based reason, are compiled Code 317 amino acid, the protease amino acid sequence similitude reported with other be 81%, the ISPr genes connect into PET30a carriers,E.coliRealize high efficient expression in BL21 (DE3), it is purified after protease characteristics be：Optimum temperature 40oC, optimal pH 10.0, the enzyme have good NaCl tolerances, will have in leather loses hair or feathers industry and food industry fine Application prospect.
CN201711158779.XA 2017-11-20 2017-11-20 A kind of intracellular serine protease ISPr encoding gene and its recombination expression Pending CN107699583A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114790461A (en) * 2021-01-26 2022-07-26 四川大学 Heterologous expression of extracellular serine protease SptA and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
K. J. SASTRY等: "Characterization of Bacillus subtilis mutants with a temperature-sensitive intracellular protease", 《JOURNAL OF BACTERIOLOGY》 *
LOUISE BAND 等: "Construction and properties of an intracellular serine protease mutant of Bacillus subtilis", 《JOURNAL OF BACTERIOLOGY》 *
YOSHINAO KOIDE 等: "Cloning and sequencing of the major intracellular serine protease gene of Bacillus subtilis", 《JOURNAL OF BACTERIOLOGY》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114790461A (en) * 2021-01-26 2022-07-26 四川大学 Heterologous expression of extracellular serine protease SptA and application thereof

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