CN1322110C - Yeast gene engineering bacteria and heat resistant alkali resistant xylanase preparation and application method - Google Patents

Abstract

The present invention relates to yeast genetic engineering bacteria which are constructed by a genetic engineering means and can express heat resistance and alkali resistance xylanase with high efficiency. Heat resistance and alkali resistance xylanase is prepared through cultivation, separation and purification, and consequently, xylooligosaccharide can be economically converted and prepared with high efficiency. The enzyme activity of the heat resistance and alkali resistance xylanase prepared by the yeast genetic engineering bacteria GS115/HB705 of the present invention can reach 426 IU/ml, heat resistance can reach 80 DEG C, and alkali resistance can reach pH10.6. The xylooligosaccharide produced by the heat resistance and alkali resistance xylanase has the advantages of high purity and good benefit.

Description

A kind of yeast genetically engineered bacteria, heat-resistant and alkali-resistant xylanase preparation and application method

technical field

The present invention relates to yeast genetically engineered bacteria, in particular to a yeast genetically engineered bacterium that efficiently expresses heat-resistant and alkali-resistant xylanase, a heat-resistant and alkali-resistant xylanase preparation, and a heat-resistant and alkali-resistant xylanase hydrolysis preparation Xylooligosaccharide method.

Background technique

Plants are the main renewable organic resources in nature, and their main components are cellulose, hemicellulose and lignin. Among them, hemicellulose accounts for about 35% of the dry weight of plants, and is the most abundant polysaccharide in nature except cellulose, and hemicellulose is mainly composed of xylan and other polysaccharides. As the main enzyme for xylan degradation, β-1,4-endoxylanase degrades xylan into xylan oligosaccharides or xylose.

Now, xylanase has been used in production practices such as pulp manufacturing, biological bleaching, and quality improvement of poultry and livestock feed. In the paper industry, the use of xylanase as a pulp bleaching aid can not only improve the whiteness and strength of paper, but also reduce the amount of chlorine, thereby reducing environmental pollution (Wong K K Y, CRC Crit, Rev et al. Biotechnol. 1992, 12: 413~435). In the food industry, it has become a reality to produce functional xylooligosaccharides by hydrolyzing xylan with xylanase (You Xin. Food Industry Science and Technology. 2001, 6: 1~4). In the feed industry, the use of xylanase can improve the nutritional value of feed, thereby increasing the utilization rate of feed. In the "Catalogue of Permitted Feed and Feed Additives" formulated by the Ministry of Agriculture of my country, xylanase is included in feed-grade enzyme preparations. However, the application of xylanase in the feed industry faces problems such as low yield, high cost, and difficulty in obtaining heat-resistant enzymes.

Alkaline xylanases are of particular interest since xylans are most soluble under alkaline conditions. At the same time, the pretreatment process of xylanase in application usually requires high temperature conditions, therefore, high temperature resistant xylanase with high thermal stability is particularly important. Moreover, the low fermentation yield of existing xylanases and complicated separation and purification processes have become the key limiting factors for the large-scale application of xylanases. Therefore, finding or producing high-temperature, alkali-resistant xylanase, and significantly improving the enzyme activity of xylanase and its secreted expression level in the host has become a very important direction in the current xylanase research.

At present, relevant domestic scientific research institutes have carried out extensive research on xylanase and its related contents. For example, Liu Weifeng, Mao Aijun, Zhu Lingxiang, Zhao Yun, Dong Zhiyang, etc. from the Institute of Microbiology, Chinese Academy of Sciences have studied the high-efficiency secretion and expression of the alkali-resistant xylanase gene in Bacillus pumilus (see Acta Microbiology, Vol.44.August.No.4.2004, 487-490), its enzyme activity is 968.108IU/mL, the optimum reaction temperature is 55°C, and the enzyme activity decreases by 90% when incubated at 60°C for 2 hours, and the xylanase is not resistant to high temperature.

China Agricultural University Wang Hai, Li Lite, Shi Bo and others used corncob enzymatic method to prepare xylo-oligosaccharides, (see Food Science: 2002, Vol, 23, No.5, 81-83) their xylan The enzyme activity is 3.6IU/ml, the fermentation enzyme activity is low, and the heat resistance or alkali resistance is not good enough.

Guptan N etc. abroad (Cloning, Expression, and Sequence Analysis of the Gene Encoding the Alkali-Stable, Thermostable Endoxylanase from Alkalophilic, Mesophilic Bacillus sp.Strain NG-27.Appl.Environ.Microbiol, 2000, 66(6): 2631-2635) A heat-resistant and alkali-resistant xylanase gene was cloned, but they only expressed the gene in Escherichia coli, and the enzyme activity measured in a 1000ml shake flask was only 7IU/ml.

Contents of the invention

The present invention constructs yeast genetically engineered bacteria capable of efficiently expressing heat-resistant and alkali-resistant xylanase by means of genetic engineering, and cultures, separates and purifies it to produce heat-resistant and alkali-resistant xylanase, thereby enabling efficient and economical transformation Preparation of xylooligosaccharides.

Microorganism used: the yeast genetic engineering bacterium (Pichia pastoris GS115/HB705) that the present invention relates to has the characteristic of highly expressing heat-resistant and alkali-resistant xylanase. Preserved by the Culture Collection Center, the preservation number is CCTCC No: M205033 (hereinafter referred to as GS115/HB705)

Mycological characteristics of GS115/HB705 strain:

a. Morphological characteristics: The asexual reproduction of Pichia pastoris is budding, sometimes with pseudohyphae. Sexual reproduction produces ascus containing 1-4 smooth round, hat-shaped or Saturn ascospores.

b. Physiological and biochemical characteristics: Pichia pastoris can use special substances as raw materials for growth, such as petroleum, methanol, ammoniacal nitrogen, organic acids, etc. The GS115/HB705 strain has the physiological and biochemical characteristics of Pichia pastoris, and at the same time has the characteristic of highly expressing the heat-resistant and alkali-resistant xylanase gene due to the insertion of the heat-resistant and alkali-resistant xylanase gene.

The present invention is achieved like this. Yeast genetically engineered bacteria GS115/HB705 (Pichia pastoris) constructed an environmental genome library, and screened out a heat-resistant and alkali-resistant xylanase gene. The results of Blast found that the gene was similar to the gene derived from Bacillus sp.N6-27 The xylanase gene AF015445 has 98% homology at the nucleotide level and 100% homology at the amino acid level. Primers were designed according to its sequence, the above-mentioned heat-resistant and alkali-resistant xylanase gene was amplified by PCR, and this gene was inserted into the integrated expression vector pHBM905 of Pichia pastoris, and then the obtained heat-resistant and alkali-resistant xylan The expression vector of the enzyme enzyme gene is introduced into Pichia pastoris, and then a yeast genetically engineered strain GS115/HB705 strain expressing heat-resistant and alkali-resistant xylanase enzyme with high efficiency is screened out. The optimum reaction pH of the enzyme we obtained is 8.5, the optimum reaction temperature is 70°C, and it still has more than 60% enzyme activity in the range of 30°C-85°C, especially in the range of 50°C-80°C, it still has 80% enzyme activity. % or more enzyme activity; its half-life at 70°C and pH 8.5 is 30 minutes; and its pH stability is very high, and it still has more than 80% enzyme activity in the range of pH 4.0-10.6. The purity of the enzyme produced by the genetically engineered bacterium reaches over 90%, and is easy to separate and purify.

The specific method is, according to the heat-resistant and alkali-resistant xylanase gene sequence screened, the plasmid DNA of the positive clone screened from the environmental genome library is used as a template by PCR method to amplify a 1.2Kb DNA fragment. It is inserted into the integrated expression vector of Pichia pastoris, and then introduced into the host of Pichia pastoris GS115 through chemical transformation, and then the yeast genetically engineered bacteria that highly express heat-resistant and alkali-resistant xylanase are screened out.

The highly expressed heat-resistant and alkali-resistant xylanase yeast genetically engineered strain GS115/HB705 was induced and cultured for 24-360 hours in a medium with methanol as a carbon source at a temperature of 24°C-28°C (Measure the enzyme activity at regular intervals, and stop the induction culture when the enzyme activity reaches 426IU/ml). The yeast genetically engineered bacteria that have completed the induction culture will be removed by high-speed centrifugation, and the supernatant, that is, the crude enzyme solution, will be collected and concentrated by filtration. A heat-resistant and alkali-resistant xylanase product is obtained.

The method for preparing xylooligosaccharides by hydrolyzing the above-mentioned crude enzyme solution of heat-resistant and alkali-resistant xylanase comprises the following steps:

1), the xylan extracted from the corncob is made into a corncob xylan solution with a concentration of 10%--20%;

2), add enzyme according to the ratio of every 50---100g corn cob xylan corresponding to 1ml crude enzyme solution;

3) The above mixed solution is hydrolyzed at 60°C---80°C for 4-12 hours;

4) Centrifuge the hydrolyzate to remove residues, take the supernatant, and then undergo microfiltration, ultrafiltration, and vacuum drying to obtain the finished xylooligosaccharide.

Centrifuge the hydrolyzate to remove the residue, take the supernatant, and then weigh it after microfiltration, ultrafiltration, and vacuum drying. The calculated yield is 20%---40%, and the thin layer chromatography or mass spectrum identification shows that --The xylooligosaccharide content of 7 sugars accounts for more than 80%.

The heat-resistant and alkali-resistant xylanase prepared by using the yeast genetically engineered bacteria GS115/HB705 of the present invention has high enzymatic activity, heat resistance up to 80°C, and alkali resistance up to pH 10.6. The production of xylooligosaccharide by using the heat-resistant and alkali-resistant xylanase has high purity and good benefit.

Detailed ways

By constructing an environmental genome library, the endo-β-xylanase gene was screened, and DNA sequencing analysis confirmed that the screened gene was 98 nucleotides from the xylanase gene AF015445 derived from Bacillus sp.NG-27 % homology, 100% homology at the amino acid level. The above genes were inserted into the Pichia pastoris expression vector by PCR method, and then introduced into the Pichia pastoris host GS115 by chemical transformation, and PCR identification was performed on several obtained transformants.

Example 1: Identification and confirmation of several GS115 transformants (such as 8-10) that have introduced the heat-resistant and alkali-resistant xylanase gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0---6.0; transfer Induce culture at 24°C-28°C in the induction medium with methanol as the only carbon source.

After 48 hours of induction culture, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

Select, induce, and cultivate the bacterial strain GS115/HB705 (GS115+ heat-resistant and alkali-resistant xylanase gene) that highly expresses the effect of heat-resistant and alkali-resistant xylanase, and repeat the above process to carry out larger-scale (100---1000ml) bottling amount) induction culture, during which sampling was carried out for SDS-PAGE analysis, when the enzyme production level reached more than 36IU/ml, the induction was stopped, the bacteria were centrifuged, and the supernatant containing heat-resistant and alkali-resistant xylanase was collected, which was Crude enzyme solution, the crude enzyme solution was tested for enzyme activity, and the enzyme activity was 36IU/ml.

Example 2: Identify and confirm that several GS115 transformants (such as 8-10) that have introduced the heat-resistant and alkali-resistant xylanase gene are cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0---6.0; transfer Induce culture at 24°C-28°C in the induction medium with methanol as the only carbon source.

The culture was induced for 192 hours, during the first 48 hours, samples were taken every 12 hours, after 48 hours, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

Select, induce, and cultivate the bacterial strain GS115/HB705 (GS115+ heat-resistant and alkali-resistant xylanase gene) that highly expresses the effect of heat-resistant and alkali-resistant xylanase, repeat the above process for larger scale (100----1000ml) Bottle volume) induction culture, during which sampling is carried out SDS-PAGE analysis, when the enzyme production level reaches more than 179IU/ml, stop induction, centrifuge the thalline, collect the supernatant that contains heat-resistant and alkali-resistant xylanase, namely It is a crude enzyme solution, and the crude enzyme solution is tested for enzyme activity, and the enzyme activity is 179IU/ml.

Example 3: Identification and confirmation of several GS115 transformants (such as 8-10) that have introduced the heat-resistant and alkali-resistant xylanase gene, cultured in shake flasks, so that the OD600 corresponding to the cell density reaches 2.0---6.0; transfer Induce culture at 24°C-28°C in the induction medium with methanol as the only carbon source.

During the induction culture for 360 hours, during the first 48 hours, samples were taken every 12 hours, after 48 hours, samples were taken every 24 hours, and the samples were analyzed by SDS-PAGE electrophoresis.

Select, induce, and cultivate the bacterial strain GS115/HB705 (GS115+ heat-resistant and alkali-resistant xylanase gene) that highly expresses the effect of heat-resistant and alkali-resistant xylanase, repeat the above process for larger scale (100----1000ml) Bottle volume) induction culture, during which sampling is carried out SDS-PAGE analysis, when the enzyme production level reaches more than 426IU/ml, stop induction, centrifuge the thalline, collect the supernatant that contains heat-resistant and alkali-resistant xylanase, namely It is a crude enzyme solution, and the crude enzyme solution is tested for enzyme activity, and the enzyme activity is 426IU/ml.

Hydrolysis time of crude enzyme solution: prepare 10%--20% corncob xylan solution from 100---1000g xylan extracted from corn cob, and then correspond to 50---100g corn cob xylan Add enzyme in proportion to 1ml of crude solution, and carry out hydrolysis reaction at 60°C-80°C for 4-12 hours.

Centrifuge the above-mentioned hydrolyzate to remove the residue, weigh the supernatant liquid successively through microfiltration, ultrafiltration, and vacuum drying. Thin-layer chromatography or mass spectrometry identification shows that the oligosaccharide content of 2---7 sugar is greater than 80%.

Claims (3)

1. A Pichia pastoris GS115/HB705, whose preservation number is CCTCC No: M205033. 2. The heat-resistant and alkali-resistant xylanase prepared by the yeast genetically engineered bacterium according to claim 1, characterized in that the yeast genetically engineered bacterium expressing heat-resistant and alkali-resistant xylanase according to claim 1 is used in In the medium with methanol as the carbon source, the temperature is 24°C-28°C, after 24-360 hours of induction culture, the enzyme activity is measured at regular intervals, and the induction culture is stopped when the enzyme activity reaches 36--426IU/ml The yeast genetically engineered bacteria that have completed the induction culture are removed by high-speed centrifugation, and the supernatant, that is, the crude enzyme solution, is filtered and concentrated to obtain a heat-resistant and alkali-resistant xylanase product. The most suitable xylanase The reaction pH is 8.5, the optimum reaction temperature is 70°C, it still has more than 60% of the enzyme activity in the range of 30°C-85°C, and it still has more than 80% of the enzyme activity in the range of 50°C-80°C; , The half-life at pH 8.5 is 30 minutes, and it still has more than 80% enzyme activity in the range of pH 4.0-10.6. 3. The method for preparing xylooligosaccharides by hydrolysis of heat-resistant and alkali-resistant xylanase according to claim 2, characterized in that: 1), the xylan extracted from the corn cob is prepared into a solution with a concentration of 10-20% xylan; 2), add the heat-resistant and alkali-resistant xylanase described in claim 2 according to the ratio of the heat-resistant and alkali-resistant xylanase crude enzyme liquid described in claim 2 corresponding to 1ml of every 50---100g xylan ; 3), the above mixed solution is hydrolyzed at 60°C-80°C for 4-12 hours; 4) Centrifuge the hydrolyzate to remove residues, take the supernatant, and then undergo microfiltration, ultrafiltration, and vacuum drying to obtain the finished xylooligosaccharide.