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High efficiency preparation method of high temperature alpha-amylase and mutant thereof

A technology for mutants and amylases, applied in the field of high-efficiency preparation of high-temperature α-amylases and their mutants

Active Publication Date: 2009-06-17
福建福大百特生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, the enzyme production level of BLA industrial production strains is maintained at about 6-10 mg / mL under industrial production conditions, which is still relatively large compared with the potential for protein secretion above 20 mg / mL (Veith et al.J MolMicrobiol Biotechnol, 2004) space

Method used

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  • High efficiency preparation method of high temperature alpha-amylase and mutant thereof
  • High efficiency preparation method of high temperature alpha-amylase and mutant thereof
  • High efficiency preparation method of high temperature alpha-amylase and mutant thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: the breeding of host cell

[0059] Construct the mutant plasmid pSK-Up-Gm of BLA coding gene according to the above-mentioned technical scheme R -Dn( figure 1 ), was transformed into Bacillus licheniformis B0204 strain by electroporation, and gentamicin resistance and no obvious starch hydrolysis transparent circle were used as selection markers to obtain a BLA mutant strain, which was named B0204 (ΔamyL). The genetic characteristics of the obtained mutant strains are deletion of amyL, yvdE and mal genes. Mutant B0204 (ΔamyL) was used as host cells for subsequent experiments.

Embodiment 2

[0060] Embodiment 2: BLA and its mutant coding gene

[0061] The BLA-encoded gene amyL (sequence SEQ ID NO: 1) (Niu Dandan et al. Acta Microbiology, 2006, 46(4): 576-580) was amplified from the genome of Bacillus licheniformis B0204 strain by PCR technology, and cloned into the expression vector pHY-WZX, pBL-WZX (Wang Zhengxiang Niu Dandan. Chinese invention patent, ZL 200510081648.7; Niu & Wang. J Ind Microbiol Biotechnol, 2007, 34: 357-362), obtain the recombinant expression plasmid pHY-amyL or pBL- amyL( figure 2 ).

[0062] Based on pBL-amyL, through the website http: / / genomes.urv.es / OPTIMIZER / , optimize the codon composition of the BLA coding gene, and through the whole gene synthesis technology (Niu Dandan et al. Journal of Applied and Environmental Biotechnology, 2007, 13 (4): 515-518), the new gene amyLc (sequence SEQ ID NO: 3) of codon-optimized coding BLA was obtained, and the coding product of the gene was named BLA-Lc, and the amino acid sequence and SEQ ID NO: ...

Embodiment 3

[0066] Example 3: Efficient secretory expression of BLA and its mutants

[0067] The recombinant expression plasmids pHY-amyL and pHY-amyLc obtained in Example 2 above were transformed into the host cell B0204 (ΔamyL) obtained in Example 1 by electroporation, and the transformants were obtained by screening on a selective plate and named respectively are B0204Δ-L and B0204Δ-Lc. Shake flask fermentation was carried out in a 250mL Erlenmeyer flask (filling volume: 50mL). The fermentation is carried out in a fermentation medium (yeast extract 0.5-1.5%, peptone 1.2-3.6%, glucose 8-20%; pH 7.0) at 42°C and 220r / min, and the fermentation time is 120h. The results are summarized in Table 2. The level of BLA synthesis and secretion of recombinant bacteria B0204Δ-Lc was almost 3 times that of B0204, which was about 73% higher than that of recombinant bacteria B0204Δ-L. After the recombinant bacteria were subcultured for 75 generations in the antibiotic-free medium, the plasmid maint...

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Abstract

A high temperature alpha-amylase and a high efficiency preparation method of the mutant thereof belongs to the fermentation engineering field. The invention provides a method for high efficiently secreting expressing a high temperature alpha-amylase and mutant thereof with alpha-amylase activity through a bacillus, especially a bacillus licheniformis mutant strain as a host cell. The synthesis and secretion level of the high temperature alpha-amylase or mutant thereof of the recombinant strain of the invention reaches 18-25mg / mL during fed-batch fermentation and is 3-4.5 times of the original strain high temperature alpha-amylase synthesis level. The high temperature alpha-amylase of the invention is useful for reducing the fermentation manufacture cost of the industrial enzyme, simplifying the fermentation manufacture process and reducing fermentation industrial environment pressure.

Description

technical field [0001] The invention is a method for efficiently preparing high-temperature alpha-amylase with starch hydrolysis activity and mutants thereof by using bacillus host cells. The high-temperature α-amylase with starch hydrolysis activity in the present invention is derived from the bacterial strain of Bacillus licheniformis, and the mutant of the high-temperature α-amylase is derived from the artificial mutant of α-amylase of the bacterial strain of Bacillus licheniformis. Further, the present invention uses a specific expression vector to efficiently secrete, express and prepare high-temperature α-amylase and its mutants in Bacillus, especially Bacillus licheniformis host cells. The invention belongs to the field of fermentation engineering. Background technique [0002] Starch is a class of polymer substances formed by the polymerization of glucose. Starch consists of two parts, amylose and amylopectin. Starch granules are generally round or irregular, with...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/75C12N9/28C12N1/21C12R1/10
Inventor 王正祥牛丹丹石贵阳
Owner 福建福大百特生物科技有限公司
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