CN101386824A - Engineering strain for producing mannanase - Google Patents
Engineering strain for producing mannanase Download PDFInfo
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- CN101386824A CN101386824A CNA2008101368439A CN200810136843A CN101386824A CN 101386824 A CN101386824 A CN 101386824A CN A2008101368439 A CNA2008101368439 A CN A2008101368439A CN 200810136843 A CN200810136843 A CN 200810136843A CN 101386824 A CN101386824 A CN 101386824A
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Abstract
The invention relates to an engineering strain for producing mannose, which relates to an engineering strain for producing enzyme. The invention provides an engineering strain for producing the mannose used for microbial fermentation. The engineering strain for producing the mannose, namely Bacillus licheniformis HDDMM02 belongs to Bacillus, and is preserved in the China Center for Type Culture Collection, with a preserving number of CCTCC No: M 208081. The Bacillus licheniformis HDDMM02 belongs to Gram-positive bacteria; an individual strain is a straight bar shape, the length of the individual strain is between 1.2 and 3.0 micrometers and the width of the individual strain is between 0.8 and 1.0 micrometer, and the individual strain is provided with a round end; and a colony of the Bacillus licheniformis HDDMM02 is circular, ivory-white and ridged, has orderly edge, has no glossy surface, has ruffles, is opaque, and is in the shape of adhesive water when lifted. By adopting the prior Bacillus licheniformis liquid culture medium and the prior fermentation condition for fermentation, the enzyme activity of the mannose in the fermentation broth reaches more than 300,000IU/mL when the microbial content concentration of the Bacillus licheniformis HDDMM02 in the fermentation broth is between 1x 10<7> and 1x 10<8> cfu/mL.
Description
Technical field
The present invention relates to a kind of product enzyme engineering bacterial strain.
Background technology
Mannase is a kind of hemicellulase, can the hydrolysis mannosans, vegetable polysaccharidess such as glucomannan, polygalactomannan and gala glucomannan, and have reduce the body's cholesterol level, alleviate just secrete, probiotics and reduce the physiological function of harmful bacterium in the lowering blood glucose, increase intestines; So be widely used in fields such as food, health care and medicine, feed, papermaking, washing, the broken glue of petroleum fracturing liquid and weaving, have application space widely.Adopting microbial fermentation is to obtain the ideal approach of mannase at present.
Summary of the invention
The purpose of this invention is to provide a kind of engineering strain that is used for the product mannase of microbial fermentation.The engineering strain Bacillus licheniformis HDDMM02 (Bacillus licheniformisHDDMM02) that produces mannase belongs to bacillus (Bacillus), be preserved in Chinese typical culture collection center, preservation date is on June 2nd, 2008, and preserving number is CCTCC No:M208081; Bacillus licheniformis HDDMM02 is a gram-positive microorganism, and the bacterial strain individuality is direct rod shape, and length is 1.2～3.0 μ m, and wide is 0.8～1.0 μ m, often with paired or catenation, and the tool nose circle; Bacillus licheniformis HDDMM02 bacterium colony is rounded, oyster white, and protuberance, neat in edge, tarnish has gauffer, and is opaque, provokes to be sticking watery.
Each cell of Bacillus licheniformis HDDMM02 of the present invention produces a gemma, and gemma is ellipse or ovum circle.Bacillus licheniformis HDDMM02 D-of the present invention wood-sugar fermentation is negative, sorbitol fermentation is positive, lactose fermentation is positive, the raffinose fermentation is positive, the trehalose fermentation is positive, glucose fermentation is positive, mannose ferment is positive, the L-arabinose fermentation is positive, Citrate trianion utilization test is positive, methyl red test is negative, the V-P test is positive, nitrate reduction test is positive, nitrite reduction test is positive, the starch hydrolysis experiment is positive, the propanedioic acid test is positive, gelatin liquification test is positive, 55 ℃ of tests of growth temperature are negative, the NaCl salt tolerance test of mass concentration 2% is positive, the NaCl salt tolerance test of mass concentration 3% is positive, the NaCl salt tolerance test of mass concentration 7% is positive, the NaCl salt tolerance test of mass concentration 10% is positive.
Adopt existing Bacillus licheniformis liquid nutrient medium and fermentation condition to ferment to Bacillus licheniformis HDDMM02 of the present invention.Adopt the liquid nutrient medium and the fermentation condition fermentation Bacillus licheniformis HDDMM02 of the present invention that use in " research of Bacillus licheniformis TS-01 liquid culture ", in the fermented liquid Bacillus licheniformis HDDMM02 bacterium to count concentration be 1 * 10
7～1 * 10
8During cfu/mL in the fermented liquid enzyme activity of mannase reach more than the 300000IU/mL.
The engineering strain HDDMM02 (Bacillus licheniformisHDDMM02) that the present invention produces mannase belongs to bacillus (Bacillus) for Bacillus licheniformis (Bacillus licheniformis), be preserved in Chinese typical culture collection center, preservation date is on June 2nd, 2008, and preserving number is CCTCC No:M208081.
Description of drawings
Fig. 1 is embodiment one Bacillus licheniformis HDDMM02 strain morphology figure (100 times of enlarged views), and Fig. 2 is the constructed phylogenetic tree of 16SrDNA gene order of embodiment one Bacillus licheniformis HDDMM02 and close bacterial strain thereof.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the engineering strain Bacillus licheniformis HDDMM02 (Bacillus licheniformis HDDMM02) that present embodiment is produced mannase belongs to bacillus (Bacillus), be preserved in Chinese typical culture collection center, preservation date is on June 2nd, 2008, and preserving number is CCTCC No:M208081; Bacillus licheniformis HDDMM02 is a gram-positive microorganism, and the bacterial strain individuality is direct rod shape, and length is 1.2～3.0 μ m, and wide is 0.8～1.0 μ m, often with paired or catenation, and the tool nose circle; Bacillus licheniformis HDDMM02 bacterium colony is rounded, oyster white, and protuberance, neat in edge, tarnish has gauffer, and is opaque, provokes to be sticking watery.
Each cell of present embodiment Bacillus licheniformis HDDMM02 produces a gemma, and gemma is ellipse or ovum circle.Present embodiment Bacillus licheniformis HDDMM02 strain morphology as shown in Figure 1.
Present embodiment Bacillus licheniformis HDDMM02D-wood-sugar fermentation is negative, sorbitol fermentation is positive, lactose fermentation is positive, the raffinose fermentation is positive, the trehalose fermentation is positive, glucose fermentation is positive, mannose ferment is positive, the L-arabinose fermentation is positive, Citrate trianion utilization test is positive, methyl red test is negative, the V-P test is positive, nitrate reduction test is positive, nitrite reduction test is positive, the starch hydrolysis experiment is positive, the propanedioic acid test is positive, gelatin liquification test is positive, 55 ℃ of tests of growth temperature are negative, the NaCl salt tolerance test of mass concentration 2% is positive, the NaCl salt tolerance test of mass concentration 3% is positive, the NaCl salt tolerance test of mass concentration 7% is positive, the NaCl salt tolerance test of mass concentration 10% is positive.
Present embodiment Bacillus licheniformis HDDMM02 adopts 3, and 5-dinitrosalicylic acid colorimetry (DNS method) separation screening from the hemp retting liquid obtains.
With primer A1 (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and A2 (5 '-ACGGCTACCTTGTTACGACTT-3 ') present embodiment Bacillus licheniformis HDDMM02 is carried out 16S rDNA sequence amplification.Amplification reaction system is as follows: Bacillus licheniformis HDDMM02DNA template 10 μ L, concentration are base title (dNTP) 4 μ L, the 10 * damping fluid (Mg of 2.5mmol/L
2+Free) 5 μ L, concentration are the MgCl of 25mmol/L
24 μ L, concentration are that primer 5 μ L, the enzyme work of 10pmol/L complements to 50 μ L for Taq enzyme (archaeal dna polymerase) the 0.2 μ L of 5U/ μ L and with deionized water.Amplification reaction condition is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ of extension 1min, 35 circulations, last 72 ℃ are extended 7min.
The 16S rDNA sequence Genbank comparison result (having enumerated the highest bacterial classification of similarity in the table 1) as shown in table 1 of present embodiment Bacillus licheniformis HDDMM02.
Table 1
| The most similar bacterial classification (accession number) The strains which have the highest identity from NCBI (Accession) in the ncbi database | Similarity Similarity |
| Bacillus?vallismortis(AB021198) | 99％ |
| Brevibacterium?halotolerans(AJ620368) | 99％ |
| Bacillus?pumilus(AM062682) | 99％ |
| Brevibacterium?halotolerans(AM747812) | 99％ |
| Bacillus?mojavensis(AY436360) | 99％ |
| Bacillus?licheniformis(AY971527) | 99％ |
| Bacillus?licheniformis(DQ167473) | 99％ |
| Bacillus?axarquiensis(DQ993670) | 99％ |
| Bacillus?mojavensis(DQ993678) | 99％ |
| Bacillus?licheniformis(EF656455) | 99％ |
| Bacillus?subtilis(EU047884) | 99％ |
By the constructed phylogenetic tree of the 16S rDNA gene order of present embodiment Bacillus licheniformis HDDMM02 and close bacterial strain thereof as shown in Figure 2.
Present embodiment utilizes BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST) a pair of sequencing result to carry out homology analysis, obtains relevant kind 16S rDNA sequence information from database; (alignment) arranged in the contraposition of the sequence of nearly edge bacterial classification among aim sequence and the Genbank being carried out default parameter by DNAMAN (Version5.1) software again; Use the ortho position addition method (neighbor-joining) in this software to carry out Phylogenetic Analysis then.Above method has all been carried out 1000 times bootstrap (bootstraping), has confirmed the verity of evolutionary tree that present embodiment is built.
Embodiment two: present embodiment adopts the liquid nutrient medium of use in " research of Bacillus licheniformis TS-01 liquid culture " (research Gu Chuntao, Peng Aiming, Sa Renna, the Tong Jianming of Bacillus licheniformis TS-01 liquid culture, " feed wide-angle " 07 phase in 2003) and the Bacillus licheniformis HDDMM02 in the fermentation condition fermentation embodiment one.
It is 1 * 10 that the middle Bacillus licheniformis HDDMM02 bacterium of present embodiment fermented liquid (liquid nutrient medium) is counted concentration
7～1 * 10
8During cfu/mL after testing in the fermented liquid enzyme activity of mannase be 320000IU/mL.
Claims (3)
1, a kind of engineering strain that produces mannase, the engineering strain Bacillus licheniformis HDDMM02 (Bacillus licheniformis HDDMM02) that it is characterized in that producing mannase belongs to bacillus (Bacillus), be preserved in Chinese typical culture collection center, preservation date is on June 2nd, 2008, and preserving number is CCTCC No:M 208081; Bacillus licheniformis HDDMM02 is a gram-positive microorganism, and the bacterial strain individuality is direct rod shape, and length is 1.2～3.0 μ m, and wide is 0.8～1.0 μ m, often with paired or catenation, and the tool nose circle; Bacillus licheniformis HDDMM02 bacterium colony is rounded, oyster white, and protuberance, neat in edge, tarnish has gauffer, and is opaque, provokes to be sticking watery.
2, a kind of engineering strain that produces mannase according to claim 1 is characterized in that each cell of Bacillus licheniformis HDDMM02 produces a gemma, and gemma is ellipse or ovum circle.
3, a kind of engineering strain that produces mannase according to claim 1 is characterized in that Bacillus licheniformis HDDMM02 D-wood-sugar fermentation is negative, sorbitol fermentation is positive, lactose fermentation is positive, the raffinose fermentation is positive, the trehalose fermentation is positive, glucose fermentation is positive, mannose ferment is positive, the L-arabinose fermentation is positive, Citrate trianion utilization test is positive, methyl red test is negative, the V-P test is positive, nitrate reduction test is positive, nitrite reduction test is positive, the starch hydrolysis experiment is positive, the propanedioic acid test is positive, gelatin liquification test is positive, 55 ℃ of tests of growth temperature are negative, the NaCl salt tolerance test of mass concentration 2% is positive, the NaCl salt tolerance test of mass concentration 3% is positive, the NaCl salt tolerance test of mass concentration 7% is positive, the NaCl salt tolerance test of mass concentration 10% is positive.
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|---|---|---|---|
| CN2008101368439A CN101386824B (en) | 2008-07-30 | 2008-07-30 | Engineering strain for producing mannanase |
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|---|---|---|---|
| CN2008101368439A CN101386824B (en) | 2008-07-30 | 2008-07-30 | Engineering strain for producing mannanase |
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| CN101386824B CN101386824B (en) | 2010-09-08 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102011200A (en) * | 2010-12-29 | 2011-04-13 | 黑龙江大学 | Hemp degumming method |
| CN102168049A (en) * | 2011-01-21 | 2011-08-31 | 天津工业生物技术研究所 | Bacterial strain for producing gel breaking enzyme and application thereof |
| CN103184206A (en) * | 2011-08-16 | 2013-07-03 | 青岛蔚蓝生物集团有限公司 | Mannase and mutants thereof |
| CN115232773A (en) * | 2022-08-05 | 2022-10-25 | 刘洪涛 | Bacillus licheniformis and application thereof in degradation of bletilla striata polysaccharide |
-
2008
- 2008-07-30 CN CN2008101368439A patent/CN101386824B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102011200A (en) * | 2010-12-29 | 2011-04-13 | 黑龙江大学 | Hemp degumming method |
| CN102168049A (en) * | 2011-01-21 | 2011-08-31 | 天津工业生物技术研究所 | Bacterial strain for producing gel breaking enzyme and application thereof |
| CN102168049B (en) * | 2011-01-21 | 2012-11-21 | 天津工业生物技术研究所 | Bacterial strain for producing gel breaking enzyme and application thereof |
| CN103184206A (en) * | 2011-08-16 | 2013-07-03 | 青岛蔚蓝生物集团有限公司 | Mannase and mutants thereof |
| CN103184206B (en) * | 2011-08-16 | 2015-04-15 | 青岛蔚蓝生物集团有限公司 | Mannase and mutants thereof |
| CN115232773A (en) * | 2022-08-05 | 2022-10-25 | 刘洪涛 | Bacillus licheniformis and application thereof in degradation of bletilla striata polysaccharide |
| CN115232773B (en) * | 2022-08-05 | 2023-12-19 | 武汉市四圣元生物科技有限责任公司 | Bacillus licheniformis and application thereof in degradation of bletilla striata polysaccharide |
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| Publication number | Publication date |
|---|---|
| CN101386824B (en) | 2010-09-08 |
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