CN103114049B - Recombined pichia pastoris strain for commonly expressing glucamylase and alpha-amylase and construction method thereof as well as mixed enzyme preparation - Google Patents

Abstract

The invention provides a recombinant Pichia pastoris strain co-expressing glucoamylase and α-amylase and its construction method, and a mixed enzyme preparation prepared by the recombinant Pichia pastoris strain. The recombinant Pichia strain contains both a glucoamylase gene and an α-amylase gene from Mucor micromyces, the gene sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. Compared with the strain carrying only the glucoamylase gene, the activity of glucoamylase of the mixed enzyme preparation prepared by the recombinant strain increased by 79%, and the activity of starch liquefying enzyme increased by 183% compared to the strain carrying only the α-amylase gene. The optimum action temperature and optimum action pH of the two enzymes are similar, and they have a synergistic promotion effect in the process of starch hydrolysis, so they have great application potential and advantages.

Description

A recombinant Pichia pastoris strain co-expressing glucoamylase and α-amylase and its construction method, and mixed enzyme preparation

technical field

The invention relates to the biological field, in particular to a recombinant Pichia pastoris strain co-expressing glucoamylase and α-amylase and a construction method thereof, and a mixed enzyme preparation prepared by the recombinant Pichia pastoris strain.

technical background

As an important storage material of plants, starch is one of the most abundant substances in the world. Amylase refers to a class of glycosidases that can hydrolyze starch to produce low molecular weight polysaccharides or monosaccharides. Amylase is widely used in starch hydrolysis or its fermentation industry, such as ethanol production, starch sugar production, beer brewing, etc. The most important amylases in current industrial applications are glucoamylase, α-amylase, pullulanase and so on.

Glucoamylase (EC3.2.1.3), also known as glucoamylase, is an exonuclease that acts on the non-reducing end of maltooligosaccharides to cut α-1,4 glycosidic bonds to generate glucose; it can also act on α -1,6 glycosidic bonds, but the rate of hydrolysis is much lower than that of α-1,4 glycosidic bonds. α-amylase (E.C.3.2.1.1), also known as liquefying enzyme, acts on starch, glycogen and other polysaccharides, hydrolyzes the α-1,4 glycosidic bonds in the molecule in a random manner, and produces maltooligosaccharides and A small amount of glucose. Since α-amylase hydrolyzes starch to form more non-reducing ends, which provide more substrates for glucoamylase, there is a synergistic effect between the two in the process of hydrolyzing starch, which can promote the production of glucose.

For many steps in the starch hydrolysis industry, such as starch saccharification and maltose production, it is obviously more advantageous to operate at a slightly higher temperature, such as a higher reaction rate and a lower risk of bacterial contamination. However, the currently widely used glucoamylase and liquefaction enzyme generally have the defect of being unstable and easily inactivated under high temperature conditions, which greatly increases the production cost. Although there are also many research reports to find new heat-resistant glucoamylases and α-amylases through strain screening, and to further obtain their expression genes by gene cloning technology, so far there are few new heat-resistant glucoamylases or α-amylases. Enzymes are really used in industrialization mainly because most new enzymes have defects of either too low activity or too low expression level. As more and more high-efficiency heterologous expression systems are developed, improving the expression level of new glucoamylase or α-amylase through heterologous expression has become one of the effective methods for the industrial application of new heat-resistant enzyme preparations.

Although there is also a report in the prior art to disclose the simultaneous expression of Aspergillus niger glucoamylase and barley α-amylase in Saccharomyces cerevisiae, this research is to endow the bacterial species with the ability to directly degrade starch to produce alcohol. Saccharomyces cerevisiae strains have the activity of amylolytic enzymes, but they are only used in the brewing industry. Therefore, a heterologous expression system that can efficiently express glucoamylase and α-amylase at the same time, and can be used in the field of starch liquefaction and saccharification, will have greater application potential and value. However, so far no one has expressed glucoamylase and α-amylase in Pichia pastoris for related research, or prepared a mixed enzyme preparation of these two enzymes.

Contents of the invention

The purpose of the present invention is to construct a novel recombinant Pichia strain co-expressing glucoamylase and α-amylase, its construction method, and a mixed enzyme preparation prepared by the recombinant Pichia strain.

The present invention provides a recombinant Pichia strain that co-expresses glucoamylase and α-amylase, and the recombinant Pichia strain contains both the glucoamylase gene and the α-amylase gene from Mucor micromus, and its gene sequence Shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

The recombinant Pichia strain can simultaneously secrete and express glucoamylase and alpha-amylase.

The present invention also provides a method for constructing the above-mentioned recombinant Pichia strain. The steps of the construction method are as follows: 1) Using the genome of Mucor micromyces as a template, the glucoamylase (Gla) gene is cloned, and its sequence is as follows: As shown in SEQ ID NO.1, pPIC9K was used as a vector to construct a recombinant expression vector pPIC9KGla, which was transformed into Pichia pastoris KM71 to obtain recombinant Pichia pastoris KM71/9KGla; 2) Using the genome of Mucor micromyces as a template, α-starch was obtained by cloning The enzyme (Amy) gene, whose sequence is shown in SEQ ID NO.2, uses pPICZα as a carrier to construct a recombinant expression vector pPICZαAmy; 3) transfer the recombinant expression vector pPICZαAmy into recombinant Pichia pastoris KM71/9KGla, and pass antibiotic selection , to obtain the recombinant Pichia yeast strain KM71/9KGla-ZαAmy containing both glucoamylase gene and α-amylase gene.

In step 1), the sequences of the upstream and downstream primers of the glucoamylase (Gla) gene cloned from the Mucorella pumila genome are respectively shown in SEQ ID NO.3 and SEQ ID NO.4.

In step 2), the sequences of the upstream and downstream primers of the α-amylase (Amy) gene cloned from the genome of Mucor pumila are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

The present invention also provides a mixed enzyme preparation prepared from a recombinant Pichia pastoris strain co-expressing glucoamylase and α-amylase, including glucoamylase and α-amylase.

The optimal pH of the mixed enzyme preparation is 4-5, preferably 5.

The optimum action temperature of the mixed enzyme preparation is 60-80°C, preferably 70°C.

The glucoamylase involved in the present invention is obtained by cloning from Mucor micromyces, constructing the Pichia pastoris recombinant expression vector pPIC9KGla, transferring it into Pichia pastoris KM71, and inducing expression through shaking flask culture, and the highest glucoamylase activity reaches 1237U/ml , the protein expression level reached 0.762mg/ml.

The α-amylase gene involved in the present invention was obtained by cloning from Mucor micromyces in our laboratory, and the Pichia pastoris recombinant expression vector pPICZαAmy was constructed, which was transferred into Pichia pastoris KM71, and induced and expressed by shaking flask culture. The highest α-amylase The enzyme activity reached 2927U/ml, and the protein expression level reached 0.236mg/ml.

The recombinant Pichia pastoris expression system KM71/9KGla-ZαAmy carrying Mucor pumila glucoamylase and α-amylase genes involved in the present invention is induced and expressed by shake flask culture, and the protein expression level reaches 0.939mg/ml; the highest glucose The amylase activity reaches 2218U/ml, which is 79% higher than that of the recombinant Pichia pastoris carrying only the glucoamylase gene; Increased by 183.1%.

The application advantages of the recombinant Pichia pastoris strain co-expressing Mucor pumila glucoamylase and α-amylase obtained in the present invention are:

(1) Compared with the recombinant Pichia pastoris carrying the mucorpa glucoamylase gene, although the protein expression level increased by 23.2%, the glucoamylase activity increased by 79%; For the recombinant Pichia pastoris with amylase gene, the enzyme activity of α-amylase also increased by 183%.

(2) The glucoamylase or α-amylase involved in the present invention is all derived from Mucor micromyces, and its biochemical properties, including the optimum temperature and optimum pH, are similar, so it is beneficial to hydrolyze starch under the same conditions. collective effect.

(3) In the process of starch hydrolysis, compared with glucoamylase acting alone, it can improve the hydrolysis efficiency of starch and reduce the occurrence of reverse reaction; compared with α-amylase acting alone, the proportion of product glucose is increased.

The mixed enzyme preparation of the two enzymes prepared by using the recombinant Pichia pastoris strain co-expressing Mucor pumilus glucoamylase and α-amylase provided by the present invention is conducive to improving the hydrolysis efficiency of starch and reducing costs, so it has better application prospect.

Description of drawings

Fig. 1 shows the result of screening on the YPDS flat plate containing antibiotic Zeocin respectively of recombinant Pichia pastoris strain KM71/9KGla and KM71/9KGla-ZαAmy;

Figure 2 shows the verification of amylase activity produced by recombinant Pichia pastoris strains KM71/9KGla, KM71/ZαAmy, and KM71/9KGla-ZαAmy respectively;

Figure 3 shows the glucoamylase activity enzyme activity curves of recombinant Pichia pastoris strains KM71/9KGla and KM71/9KGla-ZαAmy induced expression respectively through shake flask culture;

Figure 4 shows the α-amylase activity enzyme activity curves of recombinant Pichia pastoris strains KM71/ZαAmy and KM71/9KGla-ZαAmy induced by shake flask culture respectively;

Figure 5 shows the SDS-PAGE electrophoresis patterns of the proteins induced and expressed by the recombinant Pichia pastoris strains KM71/9KGla, KM71/ZαAmy, and KM71/9KGla-ZαAmy respectively through shake flask culture;

Fig. 6 shows the relational graph of the enzyme activity and action pH of grape amylase and α-amylase in the mixed enzyme preparation;

Fig. 7 shows the relation graph of the enzyme activity of grape amylase and α-amylase and action temperature in the mixed enzyme preparation;

Figure 8 shows the thin-layer chromatograms of the products of mixed enzyme preparation, α-amylase and glucoamylase hydrolyzing soluble starch.

Detailed ways

The following examples are only used to illustrate the present invention but not to limit the protection scope of the present invention.

The test methods described in the following examples, unless otherwise specified, are conventional methods; the reagents or consumables, unless otherwise specified, can be obtained through commercial channels.

Embodiment 1. Construction and Transformation of Recombinant Expression Vector pPIC9KGla

1.1 Primer design

R.pGAf (SEQ ID NO.3): ATGCGTTATGCAACCCCGC

R.pGTr (SEQ ID NO. 4): GGCGTTATTTATTACCCTCTTTTGACC

R.pGEf (SEQ ID NO. 5): G GAATTC ATGCGTTATGCAACCCCGC

R.pGNr (SEQ ID NO. 6): TT GCGGCCGC TTACCCTCTTTTGACCA

1.2 Amplification of Mucor pumila glucoamylase cDNA sequence

Using the first strand of cDNA of Rhizomucor pusillus (purchased from the General Microbiology Center CGMCC of China Microbiological Culture Collection Management Committee in Beijing, strain number 3.204) as a template, PCR amplification was carried out with R.pGAf/R.pGTr primers , cloned to obtain the glucoamylase (Gla) gene, the sequence of which is shown in SEQ ID NO.1. PCR reaction conditions: 94°C for 5min; 94°C for 30s, 60°C for 30s, 72°C for 2min, 20 cycles (annealing temperature decreased by 0.5°C for each cycle); 94°C for 30s, 54°C for 30s, 72°C for 2min, 15 cycles; 72°C for 10min, and stored at 4°C. The PCR product was subjected to agarose gel electrophoresis, and after recovery, it was connected to pMD19-T Simple Vector, and the positive clones were verified by colony PCR and sequenced.

1.3 Construction of recombinant expression vector pPIC9KGla

Using the pMD19-T SimpleVector with the glucose starch cDNA sequence from Mucor micromyces as a template, the primers R.pGEf/R.pGNr were used for PCR amplification; the PCR product was digested with EcoRI and NotI (purchased from Fermentas), Then it was connected with the yeast expression vector pPIC9K (sourced from Invitrogen) which was also digested by EcoR I and NotⅠ, transformed into Escherichia coli DH5α (sourced from Tiangen Biochemical), and the positive clones were verified by PCR and sequenced; if the sequencing results showed no frameshift and other bases mutation, it indicates that the constructed recombinant expression vector pPIC9KGla is available.

1.4 Preparation of Pichia Competent Cells

(1) Pichia pastoris KM71 was inoculated into YPD liquid medium and cultured at 30°C to OD1-2A (600nm) for preparing competent cells;

(2) Centrifuge the culture solution at 6000rpm at 4°C for 3min to collect the bacteria;

(3) Resuspend the bacteria with 8ml buffer solution (100mM LiAc, 10mM DTT, 0.6M sorbitol, 10mM pH7.5 Tris-HCl), and let stand at room temperature for 30min;

(4) 6000rpm, centrifuge at 4°C for 5min, and collect the bacteria;

(5) Wash the cells with 2-3ml of 1M sorbitol, repeat twice;

(6) Resuspend the cells with an appropriate amount of 1M sorbitol to make the cell concentration 10 ¹⁰ cells/ml, and put them on ice until use.

1.5 Transformation of recombinant expression vector pPIC9KGla

(1) Linearize 100 ul of the recombinant plasmid pPIC9KGla with SacI, concentrate it with Takara nucleic acid co-precipitation agent, and dissolve the plasmid with 10 ul of sterile ultrapure water; prepare two copies at the same time;

(2) Mix 10ul of the above plasmid with 100ul of yeast competent cells KM71, transfer to a pre-cooled electrode cup, perform electric shock, quickly add 1ml of pre-cooled 1M sorbitol, transfer to a 30°C incubator and let it stand for 1h ; coated MD plate; 30 ℃ incubator culture.

1.6 Screening of high copy transformants

The transformants grown on the MD plate were washed with sterile water, and coated with YPDS plates containing different concentrations of G418 antibiotics, and the Pichia transformant KM71/9KGla containing a high-copy glucoamylase gene was screened.

Example 2. Construction and Transformation of Recombinant Expression Vector pPICZαAmy

2.1 Primer design

R.pA-f (SEQ ID NO. 7): ATGAAATTCAGCATCTCTCTCTCGG

R.pA-r (SEQ ID NO. 8): TTAAGCAGAGGTGAAGATAGCGGA

R.pAEf (SEQ ID NO. 9): GAATTCAGCCCTTTGCCCCAACAGCA

R.pANr (SEQ ID NO. 10): TT GCGGCCGC TTAAGCAGAGGTGAAGATAG

2.2 Amplification of Mucor pumila α-amylase cDNA sequence

Using the first-strand cDNA of Mucor pumilus as a template, PCR amplification was performed with primers R.pA-f/R.pA-r, and the α-amylase (Amy) gene was cloned, and its sequence is shown in SEQ ID NO.2 . PCR reaction conditions: 94°C for 5min; 94°C for 30s, 65°C for 30s, 72°C for 1min30s, 30 cycles (annealing temperature decreased by 0.5°C for each cycle); 94°C for 30s, 50°C for 30s, 72°C for 1min30s, 10 cycles; 72°C for 10min, and stored at 4°C. The PCR product was subjected to agarose gel electrophoresis, recovered and connected to pMD19-T Simple Vector, transformed into Escherichia coli DH5α, and the positive clones obtained by colony PCR verification were sent to BGI for sequencing.

2.3 Construction of recombinant expression vector pPICZαAmy

Using the pMD19-T Simple Vector with the cDNA sequence of the mucormyces α-amylase as the template, the primers R.pAEf/R.pANr were used for PCR amplification; Digested from Fermentas), then ligated with the yeast expression vector pPICZα (sourced from Invitrogen) which was also digested with EcoRI and NotⅠ, transformed into Escherichia coli DH5α (sourced from Tiangen Biochemical), and verified the positive clone to obtain the recombinant plasmid pPICZαAmy.

2.4 Preparation of Pichia Competent Cells

Pichia pastoris KM71 and recombinant Pichia pastoris KM71/9KGla were respectively inoculated in YPD liquid medium, cultured at 30°C to OD1-2A (600nm), and competent cells were prepared according to the method described in Example 1.3.

2.5 Transformation of recombinant expression vector pPICZαAmy

(1) Linearize pPICZαAmy with SacⅠ and transform Pichia KM71 and recombinant Pichia KM71/9KGla respectively to obtain recombinant Pichia strains KM71/ZαAmy and KM71/9KGla-ZαAmy;

(2) The above-mentioned recombinant strain KM71/9KGla-ZαAmy was coated on a YPDS plate containing the antibiotic Zeocin to screen positive transformants. At the same time, the competent cells KM71/9KGla that were not transferred to the recombinant plasmid ZαAmy were coated on the same plate as a screening control.

The results are shown in Figure 1: the recombinant strain KM71/9KGla-ZαAmy transformant obtained by transforming ZαAmy grew a single colony on the YPDS plate containing the antibiotic Zeocin, while the recombinant strain KM71/9KGla without transforming ZαAmy could not grow on the same plate . The result indicated that the recombinant Pichia pastoris strain KM71/9KGla-ZαAmy was successfully constructed.

Example 3. Verification of amylase activity produced by recombinant Pichia pastoris transformants

(1) Single colonies of recombinant Pichia pastoris strains KM71/9KGla, KM71/ZαAmy, and KM71/9KGla-ZαAmy transformants were picked and inoculated on BMMY plates containing 2% soluble starch, and cultured in a 30°C incubator. Add 200ul filter-sterilized absolute ethanol dropwise to the lower layer;

(2) After 2-3 days, use iodine solution to develop color, and observe the size of the hydrolysis circle around the transformant to judge whether the transformant successfully expresses Mucor pumila amylase.

As a result, as shown in Figure 2, hydrolysis circles appeared around the above-mentioned recombinant Pichia transformants, indicating that the above-mentioned recombinant Pichia strains KM71/9KGla, KM71/ZαAmy, and KM71/9KGla-ZαAmy can all secrete and express amylase.

Example 4. Induced expression of recombinant Pichia pastoris strains

4.1 Pick a single colony of recombinant Pichia pastoris strains KM71/9KGla, KM71/ZαAmy, and KM71/9KGla-ZαAmy to inoculate a 50ml centrifuge tube containing 3ml of YPD medium, and place it at 30°C and culture overnight on a shaker at 200rpm;

4.2 Put all 3ml of seed culture solution into a 500ml shake flask containing 150ml of BMGY medium, culture at 30°C, 225rpm for 24h;

4.3 Collect the bacteria by centrifugation, resuspend the bacteria with 30ml BMMY medium, pay attention to control the amount of the bacteria here, the wet weight is 3.5g, transfer to a 250ml shaker flask, place at 30°C, 230rpm shaker for induction fermentation culture , add 150ul filter-sterilized anhydrous methanol per 24ml;

4.4 From the 48th hour, take samples every 24 hours, centrifuge, and measure the glucoamylase (glucoamylase) activity or α-amylase (liquefaction enzyme) activity of the supernatant.

The method for measuring the enzyme activity of glucoamylase (glucoamylase) in the present invention is as follows: add 0.5ml of 1.0% (w/v) soluble starch solution to a 20ml stoppered test tube, preheat at 60°C for 10min, and then add 0.5ml Appropriately diluted enzyme solution, react for 10 minutes; add 1.0ml DNS chromogen, boil water bath for 5 minutes, cool in running water bath, dilute with 10ml distilled water, measure absorbance at 540nm; definition of enzyme activity unit: one glucoamylase (glucoamylase) Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze soluble starch to release 1 μmol of glucose equivalent reducing sugar within 1 min under given conditions.

The method for measuring the enzyme activity of recombinant α-amylase (liquefied enzyme) in the present invention is as follows: add 5ml of 0.5% (w/v) soluble starch solution to a 20ml stoppered test tube, preheat it for 10min, and then add it to dilute to an appropriate multiple 0.5ml of enzyme solution, reacted for 5min, and added 5ml of pre-cooled 0.1M HCl to terminate the reaction. 0.5ml of the reaction solution was added to a test tube filled with 9.3ml of distilled water, and 0.2ml of I ₂ -KI (I ₂ , 0.08% (w/v); KI, 0.8% (w/v)) solution was added to develop color. Shake well to stabilize the blue solution for 10 min, then measure the absorbance at a wavelength of 660 nm on a 721 spectrophotometer. Enzyme activity unit is defined as: One α-amylase (liquefying enzyme) enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze 1 mg of starch within 5 minutes under given conditions.

The results are shown in Figure 3, the glucoamylase activity of the recombinant Pichia pastoris strains KM71/9KGla and KM71/9KGla-ZαAmy all reached the maximum at 96h, and the highest glucoamylase activity of the recombinant strain KM71/9KGla-ZαAmy reached 2218U/ml, while the highest enzyme activity of the recombinant strain KM71/9KGla that only carries the glucoamylase gene is 1237U/ml, therefore, the highest glucoamylase activity of the recombinant bacterial strain KM71/9KGla-ZαAmy has improved compared with the recombinant bacterial strain KM71/9KGla 79%;

The results are shown in Figure 4, the α-amylase activity of the recombinant Pichia pastoris strains KM71/ZαAmy and KM71/9KGla-ZαAmy reached the maximum at 96 hours, and the highest α-amylase activity of the recombinant strain KM71/9KGla-ZαAmy reached 8285U/ml, while the highest enzyme activity of the recombinant strain KM71/ZαAmy carrying only the α-amylase gene was 2927U/ml, therefore, the highest α-amylase activity of the recombinant strain KM71/9KGla-ZαAmy was compared with that of the recombinant strain KM71/ZαAmy Increased by 183%.

4.5 SDS-PAGE protein electrophoresis analysis was carried out on the fermentation supernatant at 96 hours respectively. The results are shown in Figure 5: where, M represents the protein marker, and the first and second lanes represent the Pichia transformed with empty vector Zα or empty vector 9K Yeast KM71, the 3rd, 4th and 5th lanes represent recombinant strains KM71/ZαAmy, KM71/9KGla and KM71/9KGla-ZαAmy, respectively. It can be seen from observation that Pichia pastoris KM71 transformed with empty vector Zα or empty vector 9K has no strains. Bands appeared, and the recombinant Pichia pastoris KM71/ZαAmy, KM71/9KGla and KM71/9KGLa-ZαAmy transformed with glucoamylase gene or α-amylase gene all showed clear bands at the expected positions, thus indicating successful expression. Example 5. Determination of protein concentration

5.1 Establish the standard curve of bovine serum albumin: add 200ug/ml BSA standard protein solution 0μl, 10μl, 20μl, 30μl, 40μl, 50ul and 75ul in turn to the test tube, make up 200ul with double distilled water; add 2ml Bradford Working solution, oscillate and mix well, place at room temperature for 5min, measure absorbance at 595nm; make three parallels for each sample, take the standard protein content as the abscissa, and A595 as the ordinate to prepare a standard curve;

5.2 Determination of sample protein content: dilute the sample by n and m times, and prepare a solution with a concentration of about 0.025-0.075mg/ml; take 200ul of the diluted sample and add it to a test tube, add 2ml of Bradford working solution, oscillate evenly, and place it at room temperature for 5min. Absorbance was measured at 595nm; each sample was made in triplicate. Subtract the blank from the measured absorbance value, calculate the protein concentration from the corresponding protein content of the standard curve difference, and calculate the protein concentration according to the respective dilution factors. After subtracting the average value, it is the protein concentration of the sample.

After measurement, the protein expression level of the glucoamylase of the recombinant bacterial strain KM71/9KGla constructed by the present invention is 0.762mg/ml, and the protein expression level of the α-amylase of the recombinant bacterial strain KM71/ZαAmy is 0.236mg/ml, while carrying The protein expression level of the recombinant Pichia pastoris expression system KM71/9KGla-ZαAmy of Mucor pumilus glucoamylase and α-amylase genes reached 0.939 mg/ml, which was significantly improved.

Example 6. Determination of enzymatic properties

6.1 Determination of optimum pH

At 50°C, at pH 3.0 (glycine-hydrochloric acid), 4.0, 5.0, 6.0 (citric acid-sodium citrate), 7.0 (potassium dihydrogen phosphate-dipotassium hydrogen phosphate), 8.0 (Tris-hydrochloric acid) The enzyme activities of Mucor micromus glucoamylase and α-amylase were measured respectively, and the relative enzyme activities under other temperature conditions were calculated with the highest enzyme activity as 100%.

As a result, as shown in Figure 6, the optimal pH of the recombinant glucoamylase is 4, and the optimal pH of the recombinant α-amylase is 5, which are relatively close to each other, so that it is convenient for the mixed enzyme containing these two enzymes at the same time. The application of the formulation provides favorable pH conditions.

6.2 Determination of optimum temperature

Under the condition of pH 5.0 (100mM citric acid-sodium citrate buffer), the glucoamylase and α- For the enzyme activity of amylase, take the highest enzyme activity as 100%, and calculate the relative enzyme activity under other temperature conditions.

The results are shown in Fig. 7, the optimum action temperature of recombinant glucoamylase and α-amylase is 60-80°C, preferably 70°C.

Embodiment 7. Thin-layer chromatography analysis analyzes the product of glucoamylase hydrolysis soluble starch

Prepare a 1% (w/v) soluble starch substrate solution with pH 5.0 buffer, add 0.2ml of appropriately diluted enzyme solution to 1ml of the substrate solution, mix well, and place it in a 50°C water bath for reaction. After reacting for 48 hours, take it out and boil it in a boiling water bath for 15 minutes to inactivate the enzyme, centrifuge at the maximum speed, filter with a 0.22 μm filter, and perform thin-layer chromatography.

Use a capillary tube for spotting, each sample is about 2-3 μl, and the diameter of the spotting circle should be as small as possible when spotting. Develop in a 250ml airtight glass bottle (chloroform: glacial acetic acid: water (30:35:5)), continue to develop for 30 minutes after the solvent line runs off the edge of the silica gel plate, take it out, and dry it.

Spray the staining agent (α-naphthol-sulfuric acid reagent: 21ml of 15% α-naphthol ethanol solution, add 13ml of concentrated sulfuric acid, add 81ml of ethanol, mix with 8ml of water, put it in a brown bottle, and prepare it freshly) spray on the silica gel plate Put it on, dry it, place it in an oven at 100°C for 10 minutes, and take it out for observation.

The results are shown in Figure 8, wherein, M represents glucose, maltose, maltotriose and maltotetraose successively from top to bottom, sample 1 represents the mixed enzyme preparation prepared by the present invention, sample 2 represents α-amylase, and sample 3 Represents glucoamylase, and it can be known by observing the spectrogram that the mixed enzyme preparation prepared by the present invention is more conducive to the hydrolysis of soluble starch due to the synergistic effect of α-amylase and glucoamylase in the process of hydrolyzing soluble starch.

Claims (9)

1. the recombinant pichia yeast strain of a coexpression glucoamylase and α-amylase, it is characterized in that, described recombinant pichia yeast strain is simultaneously containing from the glucose amylase gene Gla of mucor pusillus and alpha-amylase gene Amy, and its gene order is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2; Wherein, the step of the construction process of described recombinant pichia yeast strain is as follows:

1) with mucor pusillus genome for template, clone obtain glucoamylase Gla gene, its sequence is as shown in SEQ ID NO.1, take pPIC9K as carrier, build recombinant expression vector pPIC9KGla, proceed to pichia spp KM71, obtain recombinant yeast pichia pastoris KM71/9KGla;

2) with mucor pusillus genome for template, clone obtains α-amylase Amy gene, and its sequence, as shown in SEQ ID NO.2, with pPICZ α for carrier, builds recombinant expression vector pPICZ α Amy;

3) described recombinant expression vector pPICZ α Amy is proceeded to recombinant yeast pichia pastoris KM71/9KGla, by antibiotic-screening, obtain the recombinant pichia yeast strain KM71/9KGla-Z α Amy simultaneously containing glucose amylase gene and alpha-amylase gene.

2. recombinant pichia yeast strain according to claim 1, is characterized in that, described recombinant pichia yeast strain can simultaneously secreting, expressing glucoamylase and α-amylase.

3. recombinant pichia yeast strain according to claim 1, it is characterized in that, step 1) in from the upstream and downstream primer sequence of glucoamylase Gla gene described in mucor pusillus genomic clone respectively as shown in SEQ ID NO.3 and SEQ ID NO.4.

4. recombinant pichia yeast strain according to claim 1, is characterized in that, step 2) in from the upstream and downstream primer sequence of α-amylase Amy gene described in mucor pusillus genomic clone respectively as shown in SEQ ID NO.7 and SEQ ID NO.8.

5. a mixing enzyme preparation, it is characterized in that, the coexpression glucoamylase of described mixing enzyme preparation according to any one of claim 1-4 and the recombinant pichia yeast strain preparation of α-amylase, comprise glucoamylase and the α-amylase of the expression of described recombinant pichia yeast strain.

6. mixing enzyme preparation according to claim 5, is characterized in that, the suitableeest action pH of described mixing enzyme preparation is 4-5.

7. mixing enzyme preparation according to claim 6, is characterized in that, the suitableeest action pH of described mixing enzyme preparation is 5.

8. mixing enzyme preparation according to claim 5, is characterized in that, the optimum temperature of described mixing enzyme preparation is 60-80 DEG C.

9. mixing enzyme preparation according to claim 8, is characterized in that, the optimum temperature of described mixing enzyme preparation is 70 DEG C.