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Cold-adapted marine yeast BoHai Sea-9145 low-temperature alkaline lipase gene, amino acid sequence and recombinant lipase

A lipase gene, marine yeast technology, applied in genetic engineering, plant genetic improvement, fermentation, etc., can solve the problems of complex metabolites, low occupancy, cumbersome extraction and purification steps, etc.

Active Publication Date: 2012-02-29
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the domestically developed lipase market share is low, and 80% of the lipase market in the world is still occupied by Danish Novozymes (NOVO), and the price is relatively high ($600-1500 / kg).
There are two main reasons for the analysis: one is that the enzymatic properties of lipase developed in China cannot meet the new application requirements
For example, most of the lipases currently used are mesophilic enzymes (the optimum temperature is basically around 50°C), but in the fields of food, washing, pharmaceuticals, lipid processing, environmental restoration, etc., the participation of low-temperature lipases is required. Second, the metabolites of wild-type lipase-producing microbial strains are complex, and the cumbersome extraction and purification steps increase production costs and reduce economic benefits

Method used

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  • Cold-adapted marine yeast BoHai Sea-9145 low-temperature alkaline lipase gene, amino acid sequence and recombinant lipase
  • Cold-adapted marine yeast BoHai Sea-9145 low-temperature alkaline lipase gene, amino acid sequence and recombinant lipase
  • Cold-adapted marine yeast BoHai Sea-9145 low-temperature alkaline lipase gene, amino acid sequence and recombinant lipase

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Experimental program
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Effect test

Embodiment 1

[0146] Obtaining of embodiment 1 lipase gene conservative sequence

[0147] The inventor screened a cold-adapted marine yeast Bohai Sea-9145 that produces low-temperature alkaline lipase, which belongs to a subspecies of Yarrowia lipolytica (patent authorization number ZL200410096668.7); Cloning of the lipase gene.

[0148] 11 Acquisition of Conserved Sequence of Marine Low-temperature Lipase Gene

[0149] The degenerate primers F1 and R1 were designed according to the conserved sequences of lipase genes of different species and the codon preference of Y. lipolytica. The sequences are as follows

[0150] F1 5'-GTSTTYMGAGGHACYTT-3'

[0151] R1 5'-CC SARRGAGTGDCCRGT-3'

[0152] The total DNA of the screened wild cold-adaptive marine yeast Bohai Sea-9145 strain was used as a template for PCR amplification under the following conditions:

[0153] Reaction conditions: pre-denaturation at 94°C for 5 minutes; denaturation temperature at 94°C for 1 minute; annealing temperature at...

Embodiment 2

[0164] The preparation of embodiment 2 recombinant lipase

[0165] 2.1 Cloning of lipase LipY gene

[0166] According to the marine low-temperature alkaline lipase gene sequence, two expression primers F2 and R2 were designed,

[0167] F2 5'-GAC GAATTC

[0168] GCAGGCGTGTCTCAAGG-3'

[0169] R2 5'-AAC GCGGCCGC GTTCTCAACTTGTGGGG-3'

[0170] The underlined marks are EcoRI and NotI restriction sites respectively, and a base encoding 6×histidine (indicated in italics) is added to the 5' end of the primer F2.

[0171] The genomic DNA of the cold-adapted marine yeast Bohai Sea-9145 strain was used as a template, and F2 and R2 were used as primers for PCR amplification. The conditions are as follows:

[0172] Reaction conditions: pre-denaturation at 94°C for 5 minutes; denaturation temperature at 94°C for 1 minute; annealing temperature at 54°C for 1 minute; extension temperature at 72°C for 1 minute; after 30 cycles, extension at 72°C for 10 minutes.

[0173] The PCR produ...

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Abstract

The invention relates a gene for coding low-temperature alkaline lipase (LipY). The gene is obtained by cloning cold-adapted marine yeast (BoHai Sea-9145), which can secrete low-temperature alkaline lipase and is screened from sea mud of Bohai, by a method of combining degenerate primer polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The gene contains a 1206bp open reading frame (ORF), codes 401 amino acids and contains a signal peptide with 28 amino acids at the amino acid end. Each coded amino acid contains a conserved sequence (G-X-S-X-G) of the lipase and a potential N-glycosylation site. The lipase gene is cloned on an eukaryotic expression vector (pIc9K) and performs heterologous expression in Pichia pastoris (Gs115). High-activity recombinant lipase is obtained from supernate 96 hours after fermentation, wherein methanol serves as an inducer. An electrophoretic pure lipase sample is obtained by purifying the recombinant lipase by a nickel ion affinity column. The recombinant lipase has a series of advantages of high low-temperature catalytic activity, stable performance under the alkaline condition and the like, and has wide application prospect in the low-temperature catalytic fields of daily chemicals, textile, food processing and the like.

Description

technical field [0001] The invention relates to the technical field of marine microorganism cloning, in particular to the cloning, amino acid sequence and enzymatic characteristics of the recombinant lipase of a low-temperature alkaline lipase gene of cold-adapted marine yeast (bacterial strain) Bohai Sea-9145. Background technique [0002] Lipase (Lipase EC 3.1.1.3.) is a class of enzymes that can catalyze the hydrolysis of triacylglycerides, diacylglycerides and monoacylglycerides. The catalyzed reaction of lipase has two notable features. One is that no coenzymes are required, the reaction conditions are mild, and there are few by-products; the other is that the substrate in the lipase-catalyzed reaction is insoluble in water and can play a role in a heterogeneous system (ie, an oil-water interface) or an organic phase. [0003] Lipase has a wide range of sources, and animals, plants, and microorganisms can all produce lipase. Microorganisms produce the most types of li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/20
Inventor 孙谧盛军王跃军刘均忠郝建华
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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