CN109295039A

CN109295039A - A kind of heat resistant xylanase mutant and its Pichia yeast engineering

Info

Publication number: CN109295039A
Application number: CN201811182854.0A
Authority: CN
Inventors: 钱娟娟; 刘文龙; 盛花开; 王兴吉; 郭庆文; 王克芬
Original assignee: Shandong Longkete Enzyme Preparation Co Ltd
Current assignee: Shandong Longkete Enzyme Preparation Co Ltd
Priority date: 2018-10-11
Filing date: 2018-10-11
Publication date: 2019-02-01
Anticipated expiration: 2038-10-11
Also published as: CN109295039B

Abstract

The present invention relates to field of biotechnology, and in particular to a kind of heat resistant xylanase mutant and its encoding gene, Pichia yeast engineering.The xylanase mutant is from one plant of Geobacillus stearothermophilus (Geobacillus stearothermophilus) GS-1 through ultraviolet mutagenesis；The mutant has enzyme activity high, and the good feature of thermal stability, shake flask fermentation liquid enzyme activity is up to 3565U/mL；In 80 DEG C of heat preservation 10min enzyme activity residuals 99%, 85% or more is still remained in 100 DEG C of heat preservation 10min enzyme activity.

Description

A kind of heat resistant xylanase mutant and its Pichia yeast engineering

Technical field:

The present invention relates to field of biotechnology, and in particular to a kind of heat resistant xylanase mutant and its encoding gene are finished Red Yeast engineering bacteria.

Background technique:

Xylan is that one kind is widely present in the intracorporal hemicellulose of plant, accounts for about the 35% of dry cell weight.Xylan Thoroughly degradation needs the common participation of a variety of enzymes, and that play a major role is inscribe β-Isosorbide-5-Nitrae-D- zytase (EC 3.2.1.8), abbreviation zytase has important application value, is widely used in making in chemical bleaching, feed, slurrying In the industries such as paper, food, bioconversion, medicine, have broad application prospects.Zytase is widely present in microorganism, Since the research work for carrying out xylanase gene since the end of the seventies in last century, there is hundreds of from bacterium and fungi Xylanase gene be cloned and carried out the expression of homologous and heterologous recombination in protokaryon and eucaryote.But since paper pulp floats The techniques such as white, feed granulating, food processing are required to higher temperature, therefore the research and development of heat resistant xylanase has obtained very The favor of more researchers.

Summary of the invention:

The purpose of the present invention is to provide xylanase mutants and its Pichia yeast engineering that a kind of heat resistance improves Bacterial strain.The xylanase mutant is from one plant of Geobacillus stearothermophilus (Geobacillus through ultraviolet mutagenesis Xylanase mutant encoding gene X-2 is cloned simultaneously construction recombination plasmid by stearothermophilus) GS-1 from GS-1 Afterwards, it is expressed in Pichia pastoris GS115, to obtain Pichia yeast engineering bacterial strain.

It uses and such as gives a definition in the present invention:

1. the nomenclature of amino acid and DNA nucleic acid sequence

Using the generally acknowledged IUPAC nomenclature of amino acid residue, with three-letter codes form.DNA nucleic acid sequence is using generally acknowledged IUPAC nomenclature.

2. the mark of xylanase mutant

The amino acid being mutated in mutant is indicated using " amino acid of Original amino acid position replacement ".Such as Lys48Arg indicates that the amino acid of position 48 is substituted for Arg by the Lys of wild type, and the number of position corresponds to SEQ ID No.2 The amino acid sequence of middle wild-type xylanase is numbered；It is same that mutant is indicated using " base of original base positions replacement " The base of middle mutation, the number of position correspond to the nucleotides sequence column number of wild-type xylanase in SEQ ID No.1.

In the present invention, X-1 indicates that the encoding gene of wild-type xylanase, X-2 indicate the volume of xylanase mutant Code gene, information such as following table.

The amino acid sequence of the xylanase mutant is as shown in sequence table SEQ ID No.4；

The nucleotides sequence of the xylanase mutant encoding gene X-2 is classified as shown in SEQ ID No.3；

Expression vector for expressing the zytase is plasmid pPIC9, micro- life for expression vector conversion Object host cell is Pichia pastoris GS115；

The Pichia yeast engineering is that zytase is mutated encoding gene X-2 connection expression vector pPIC9, obtains weight Group plasmid pPIC9-X-2, and in Pichia pastoris GS115 obtained by expression；

Experimental procedure of the invention is specific as follows:

1, it by design primer, expands to obtain the encoding gene X-2 of xylanase mutant using PCR method；

2, the encoding gene X-2 of xylanase mutant is subjected to digestion, is connected to expression vector pPIC9, is recombinated Carrier；

3, recombinant vector is transformed into Pichia pastoris GS115, obtains the production bacterial strain GS115/ of novel xylanase pPIC 9-X-2；

It 4, is that production strain fermentation produces zytase with GS115/pPIC 9-X-2.

The zymologic property of the xylanase mutant is as follows:

(1) pH:pH4-8.5 enzyme activity is stablized, most suitable action pH 6.5.

(2) temperature: optimum temperature is 70 DEG C.

(3) heat resistance: the zytase still remains 85% or more in 100 DEG C of heat preservation 10min enzyme activity.

The utility model has the advantages that

1, the invention discloses a kind of completely new xylanase mutant, which has enzyme activity high, and thermal stability is good The characteristics of.For the mutant shake flask fermentation liquid enzyme activity up to 3565U/mL, wild type enzyme activity is 524U/mL；

2, the zytase that the present invention obtains is in 80 DEG C of heat preservation 10min enzyme activity residuals 99%, in 100 DEG C of heat preservation 10min enzymes Work still remains 85% or more.

Detailed description of the invention:

Fig. 1 bacterium colony PCR identifies electrophoretogram；

Wherein, M Marker；1 carries out colony PCR product electrophoresis for bacterium solution；

Fig. 2 protein expression electrophoretogram；

Wherein, M Marker；1 is P-0 fermented liquid supernatant electrophoresis；2 be P-X-2 fermented liquid supernatant electrophoresis；

Fig. 3 zytase optimal pH curve；

Fig. 4 zytase optimum temperature curve；

Fig. 5 zytase temperature stability curve.

Specific embodiment:

More detailed explanation is made to the present invention by specific embodiment, is illustrative only, and not as to this hair The restriction of bright practical range.For those skilled in the art, the improvement that can be also made on the basis of the principle of the invention, these improvement Also it should be regarded as the scope of protection of the invention.Do not make the experimental methods of molecular biology illustrated in the present embodiment, can refer to " point Sub- cloning experimentation guide ".

The acquisition of 1 xylanase mutant gene X-2 of embodiment

The Geobacillus stearothermophilus GS that laboratory saves, obtaining one plant through Uv-induced screening has heat-resisting xylan The bacterial strain GS-1 of enzymatic activity designs PCR primer according to the nucleotide sequence of its mutated gene X-2, and restriction enzyme site is added in the end 5' Restriction enzyme site NotI is added in the end EcoRI, 3', obtains mutated gene X-2 by PCR, nucleotides sequence is classified as SEQ ID No.3. Primer sequence is as follows:

X-2-F 5'-AATGAATTCATGAACAGTCCCCTCCCCT-3'(SEQ ID NO.5)

X-2-R 5'-TTGCGGCCGCTCAGACACTCACTGCCCTC-3'(SEQ ID NO.6)

The building of 2 recombinant vector pPIC9-X-2 of embodiment

EcoRI and NotI digestion, recovery product, by X-2 after the recovery and pPIC9 are carried out to X-2 and plasmid pPIC9 respectively It mixing, is connected under the conditions of 16 DEG C with T4 ligase overnight in proportion, connection product converts bacillus coli DH 5 alpha competent cell, Converted product is coated on LB (mycin of benzyl containing ammonia) solid plate, 37 DEG C of inversion overnight incubations, and picking single bacterium drops down onto the training of LB liquid Base, 37 DEG C of cultures are supported, bacterium solution carries out bacterium colony PCR, and electroresis appraisal result such as Fig. 1, sequencing result display sequence is correct, sequence size 996bp.It is stand-by to extract recombinant plasmid pPIC9-X-2.

3 recombinant plasmid transformed Pichia pastoris of embodiment

1. the preparation of Pichia pastoris GS115 competent cell

1) picking Pichia pastoris plate single colonie, is inoculated in the YPD culture medium of 5mL, and 30 DEG C, 220r/min shaken overnight；

2) bacterium solution being incubated overnight for taking 0.5mL, is inoculated in the YPD culture medium of 50mL Fresh, and 30 DEG C, 220r/min Shaken cultivation makes OD600 value reach 1.3-1.5；

3) it takes above-mentioned culture solution in 4 DEG C, 3000r/min, is centrifuged 5min；

4) supernatant is abandoned, the sterile water that 50mL is pre-chilled on ice is added, thallus is resuspended in oscillation；

5) 3000r/min 4 DEG C, is centrifuged 5min, abandons supernatant, blots tube wall residual liquid, and the nothing that 25mL is pre-chilled on ice is added Thallus is resuspended in bacterium water, oscillation；

6) 3000r/min 4 DEG C, is centrifuged 5min, abandons supernatant, blots tube wall residual liquid, is added what 10mL was pre-chilled on ice Thallus is resuspended in the sterile sorbitol solution of 1mol/L；

7) 3000r/min 4 DEG C, is centrifuged 5min, abandons supernatant, blots tube wall residual liquid, is added what 1mL was pre-chilled on ice The sterilizing sorbitol solution (being previously added glycerol to final concentration 15%) of 1mol/L, oscillation mix.

8) 100 μ L/ pipes of packing are to sterile EP tube, and (freshly prepared competent cell effect is more for -70 DEG C of refrigerator frost preservations It is good).

2. the conversion of linearization plasmid

It extracts obtained recombinant plasmid pPIC9-X-2 and carries out single endonuclease digestion with NdeI, obtain linearization plasmid.Take fresh preparation (or -70 DEG C freeze) competent cell be placed in ice bath.

1) 100 μ L competent cells are moved in a new sterile EP tube, 10 μ L linearization plasmids is added, featheriness is mixed Even, suction is transferred in the Electroporation Transformation cup of 0.2cm type；

2) conversion cup is placed in ice bath 5-10 minutes, keeps low temperature.

3) Electroporation Transformation electric shock condition: 1500V, 200 Ω, 25 μ F, discharge time 5ms or so are primary to shock by electricity.

4) after shocking by electricity, the sorbitol solution of the 1mol/L of 4 DEG C of 1mL pre-coolings is added in electroporated cup at once, uses liquid relief Rifle piping and druming uniformly, is placed in ice bath；

5) sterile working is coated with MD culture medium (1.34%YNB on superclean bench；4×10^-5% biotin；2% grape Sugared plate), 100-200 μ L/ plate, 30 DEG C of inversions of the plate coated are cultivated 3-4 days；

6) screening obtains recombinant bacterium on MD plate, obtains aim sequence through bacterium colony PCR, it is purpose base that sequencing, which compares display, Because of the nucleotide sequence of X-2, i.e. obtained strains are the recombinant bacterium containing pPIC9-X-2, are named as P-X-2.

Same method constructs to obtain the recombinant bacterium containing empty plasmid, is named as P-0, the recombinant bacterium containing original gene, It is named as P-X-1.

The inducing expression of saccharomycete of the embodiment 4 containing recombinant plasmid pPIC9-X-2

BMGY culture medium prescription: 1% yeast extract, 2% peptone, 0.1mol/L pH6.0 phosphate buffer, 1.34%YNB, 4 × 10^-5% biotin, 1% glycerol.

BMMY culture medium prescription: 1% yeast extract, 2% peptone, 0.1mol/L pH6.0 phosphate buffer, 1.34%YNB, 4 × 10^-5% biotin, 0.5% methanol.

Recombinant bacterium P-0, P-X-1, P-X-2 are inoculated in respectively in the triangular flask equipped with 30mL BMGY culture medium, 30 DEG C, It is 10 or so that 220r/min, which is cultivated to OD600, and thalline were collected by centrifugation, is resuspended thallus with the BMMY induced medium of 35mL, and 30 DEG C, continues to cultivate 48h under the conditions of 220r/min, the xylanase activity in supernatant is measured after fermentation liquid centrifugation, it is as a result as follows Table, protein electrophoresis result such as Fig. 2, albumen size about 38.5kDa.

Bacterial strain	Xylanase activity/UmL^-1
		P-0	0
P-X-1	524
		P-X-2	3565

The zymologic property of 5 zytase of embodiment

It is measured using the DNS method of improvement, the definition of enzyme activity: under the conditions of 37 DEG C of temperature, 5.5 pH, discharging 1 μ per minute Enzyme amount needed for mol reduced sugar is defined as 1 enzyme-activity unit (U).

(1) most suitable action pH

Using 4 gained P-X-2 fermented liquid supernatant liquid of embodiment as sample, on the basis of the zytase highest enzyme activity measured, It under the conditions of 37 DEG C, is reacted in the buffer of pH 2.5-11 respectively, measures the enzyme activity under condition of different pH.It can by Fig. 3 Know, which stablizes in pH range 4-8.5 enzyme activity, can all react under the conditions of meta-acid meta-alkali, most suitable action pH 6.5.

(2) optimum temperature

Using 4 gained P-X-2 fermented liquid supernatant liquid of embodiment as sample, on the basis of the zytase highest enzyme activity measured, In the buffer of pH5.5, measure enzyme activity under the conditions of 30 DEG C -100 DEG C respectively, calculate opposite enzyme activity, as a result as shown in figure 4, The produced zytase optimum temperature of P-X-2 is 70 DEG C.

(3) thermal stability

Using 4 gained P-X-1 and P-X-2 fermented liquid supernatant liquid of embodiment as sample, with the xylanase activity not dealt with For 100% benchmark, under pH5.5 buffer conditions, sample respectively count by the isothermal holding 10min at 50 DEG C -100 DEG C, survey enzyme activity Remaining enzyme activity is calculated, from figure 5 it can be seen that at 80 DEG C, xylanase mutant relative activity residue 99% or so, at 100 DEG C, Enzyme activity still retains 85% or more, and compared with the produced zytase of recombinant bacterium P-X-1 of original gene X-1 building, thermal stability has Large increase illustrates that the produced zytase of recombinant bacterium P-X-2 has good heat resistance, can be widely used for the row such as food, feed Industry.

Sequence table

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Claims

1. a kind of xylanase mutant, which is characterized in that the amino acid sequence of the xylanase mutant such as sequence table SEQ Shown in ID No.4.

2. a kind of xylanase mutant described in claim 1, which is characterized in that the xylanase mutant encoding gene The nucleotides sequence of X-2 is classified as shown in SEQ ID No.3.

3. including the plasmid or recombinant bacterium of xylanase mutant as claimed in claim 1 or 2.

4. plasmid as claimed in claim 3, which is characterized in that expression vector is plasmid pPIC9.

5. recombinant bacterium as claimed in claim 3, which is characterized in that host cell is Pichia pastoris GS115.

6. the application of zytase as claimed in claim 1 or 2.

7. the application of recombinant bacterium described in claim 5.

8. the application of recombinant bacterium as claimed in claim 7, which is characterized in that the method that the recombinant bacterium is used to produce zytase It is as follows: recombinant bacterium to be inoculated in the triangular flask equipped with 30mL BMGY culture medium, 30 DEG C, 220r/min is cultivated to OD₆₀₀It is 10 Left and right, thalline were collected by centrifugation, thallus is resuspended with the BMMY induced medium of 35mL, and at 30 DEG C, continue under the conditions of 220r/min Cultivate 48h.