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Hyperthermophilic lipase lipk and its related biomaterials and applications

A biomaterial, lipase technology, applied in applications, biochemical equipment and methods, enzymes, etc., can solve problems such as lack of biological activity and difficulty in finding hyperthermophilic lipases

Active Publication Date: 2021-09-14
INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical catalysts usually used in practical applications do not have biological activity, which is a major advantage of enzyme preparations in industrial reactions, and lipases that exist stably and continue to exert catalytic activity under harsh industrial conditions are rare In particular, hyperthermophilic lipase with an optimum temperature exceeding 80°C is almost hard to find

Method used

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  • Hyperthermophilic lipase lipk and its related biomaterials and applications
  • Hyperthermophilic lipase lipk and its related biomaterials and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, preparation lipase

[0058] 1 Preparation of recombinant bacteria

[0059] 1.1 Preparation of recombinant bacteria E.coli BL21(DE3) / pET30a(+)-RTLipK-his-Y containing codon-optimized genes

[0060] According to the lipase gene cDNA obtained by Trichoderma lentiforme ACCC 30425 whole genome sequencing technology, the signal peptide was manually deleted to obtain the TLipK gene, which was named TLipK-W gene. The TLipK-W gene is to delete the 1st-159th nucleotide of SEQ ID No.3, and delete the 1762-1785th nucleotide of SEQ ID No.3, and keep other nucleosides of SEQ ID No.3 DNA molecules obtained without acid change. The TLipK-W gene is the original sequence of Trichoderma lentiforme ACCC 30425 without codon optimization. The amino acid sequence encoded by the TLipK-W gene is the protein at positions 54-587 of SEQ ID No. 2 (its name is TLipK). TLipK is a wild-type protein.

[0061] Select appropriate enzyme cutting sites (EcoRI and XhoI), delete the origin...

Embodiment 2

[0078] Embodiment 2, RTLipK-his is alkaline hyperthermophilic lipase

[0079] E.coli BL21(DE3) / pET30a(+)-RTLipK-his-Y was inoculated into 30 mL LB vial liquid medium (containing 50 μg / mL kanamycin sulfate) at an inoculum size of 0.5%, at 37 Cultivate and activate in a shaking shaker at 220 rpm for 12-16 hours. Then take an appropriate amount of activated bacterial solution according to the inoculum amount of 1%, and inoculate it into a 300mL large bottle of LB culture solution (containing 50μg / mL kanamycin sulfate), and continuously cultivate it in a shaking shaker at 37°C and 220rpm 2.5-3 hours (Use the ultraviolet spectrophotometer to determine the OD600 value of the culture solution to 0.8, and use the LB liquid medium containing 50 μg / mL kanamycin sulfate as the blank control), add IPTG (pass through a 0.22 μm filter membrane to filter and sterilize) ) to an IPTG content of 0.6 mM, induced culture in a shaking shaker at 30° C. and 220 rpm for 6 hours. Transfer the above-...

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Abstract

The invention discloses hyperthermophilic lipase LipK and related biological materials and applications. The hyperthermophilic lipase LipK is any protein in A1)-A3): A1) the amino acid sequence is the protein of SEQ ID No.2; A2) the amino acid sequence is the protein at position 53-587 of SEQ ID No.2 ; A3) the amino acid sequence is the protein at position 54‑587 of SEQ ID No.2. The optimum temperature of the enzymatic reaction of the hyperthermophilic lipase is 80°C, the optimum pH of the enzyme reaction is 9.5, and the lipase activity at 37°C and pH value of 9‑10 is at 37°C and pH value It is more than 92% of the lipase activity under the condition of 9.5, and the lipase activity under the condition of 80-90°C and pH value of 9.5 is 90% of the lipase activity under the condition of 80°C and pH value of 9.5 above.

Description

technical field [0001] The invention relates to hyperthermophilic lipase LipK and related biological materials and applications in the field of biotechnology. Background technique [0002] Lipase (EC 3.1.1.3) is considered as one of the most important commercial enzymes and has attracted great attention in the rapidly growing field of biotechnology. Lipases can catalyze the hydrolysis of triacylglycerols at the oil-water interface, releasing diacylglycerides, long-chain fatty acids (>10 carbons) and glycerol. During the hydrolysis process, the lipase combines with the acyl group of the substrate to form a lipase-acyl complex, and then transfers the acyl group to the hydroxyl group of the water molecule to achieve hydrolysis. Under non-aqueous conditions, lipase can transfer the acyl group of a carboxylic acid to a nucleophile. [0003] Lipases derived from microorganisms are widely used in various fields, including industry, food, feed and medical fields. Compared with...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/82C12N15/70
CPCC12N9/20C12N15/70C12N15/82C12Y301/01003
Inventor 顾金刚王宇洲马锐李世贵龚明波向杰陈敬师
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI