Phospholipases, nucleic acids encoding them and methods for making and using them

A phospholipase, nucleic acid technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, fat oil/fat refining, etc., can solve problems such as yield loss and quality decline

Inactive Publication Date: 2011-09-07
VERENIUM CORP (US)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The neutralization process is very effective at removing free fatty acids and phospholipids, but the process also results in significant yield loss and quality loss

Method used

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  • Phospholipases, nucleic acids encoding them and methods for making and using them
  • Phospholipases, nucleic acids encoding them and methods for making and using them
  • Phospholipases, nucleic acids encoding them and methods for making and using them

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0517] Example 1: BLAST program for sequence identity exploration

[0518] This example illustrates a typical sequence identity program used to determine whether a nucleic acid is within the scope of the invention. The NCBI BLAST 2.2.2 program was applied, with to blastp selected by default. In addition to the default filtering setting, all default values ​​apply (ie, all parameters are set to default except filtering is set to OFF); where the "FF" setting is applied, it disables filtering. Due to the short length of the sequences, applying default filtering often leads to Karlin-Altschul violations. Default values ​​used in this example:

[0519]

[0520] Other default settings are: low complexity filtering off, for protein word length 3, BLOSUM62 matrix, gap presence penalty -11 and gap extension penalty -1. The "-W" option is set to the default value of 0. This means that, if not set, the word length defaults to 3 for proteins and 11 for nucleotides. Settings are ...

Embodiment 2

[0525] Example 2: Simulation of PLC-mediated degumming

[0526] This example illustrates the simulation of phospholipase C (PLC) mediated degumming.

[0527] Due to the poor solubility of phosphatidylcholine (PC) in water, it was initially dissolved in ethanol (100 mg / ml). For initial assays, stock solutions of PC were prepared by dissolving in 50 mM morpholinopropanesulfonic acid or 60 mM citric acid / NaOH at pH 6. PC stock solution (10 μl, 1 μg / μl) was added to 500 μl refined soybean oil (with 2% water) in an Eppendorf tube. To obtain an emulsion, the contents of the tube were mixed by vortex mixer for 3 minutes (see Figure 5 A). The oil and water phases were separated by centrifugation at 13,000 rpm for 1 minute ( Figure 5 B). The reaction tube was pre-incubated at the desired temperature (37°C, 50°C, or 60°C), and 3 μl of PLC (0.9 U / μl) from Bacillus cereus was added to the aqueous phase ( Figure 5 C). Disappearance of PC was analyzed by TLC using chloroform / met...

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PUM

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Abstract

The invention provides a separated, synthetic or recombinant nucleic acid, the polypeptide coded by the nucleic acid and the use thereof. The nucleic acid includes the nucleic acid sequence described by the invention for coding the polypeptide having the phosphatidase activity. The phospholipase activity includes, e.g., phospholipase A, B, C and D activity, patatin activity, lipid acyl hydrolase (LAH) activity. In one aspect, the nucleic acid codes the phospholipase lack of the natural precursor (signal) sequence. In the other aspect, the nucleic acid also includes the heterogenous nucleic acid sequence which codes the precursor (signal) sequence or the catalytic domain. The precursor (signal) sequence or the catalytic domain includes the phosphatidase precursor (signal) sequence or the catalytic domain or the non-phosphatidase precursor (signal) sequence or the catalytic domain of the yeast precursor (signal) sequence.

Description

[0001] This application is a divisional application. The filing date of the original application is April 21, 2003, the application number is 03813594.9 (PCT / US03 / 12556), and the title of the invention is "phospholipase, nucleic acid encoding phospholipase and the preparation and application of Methods". field of invention [0002] The present invention generally relates to phospholipases, polynucleotides encoding phospholipases, methods of making and using these polynucleotides and polypeptides. In particular, the present invention provides novel polypeptides having phospholipase activity, nucleic acids encoding them, and antibodies that bind them. Industrial processes and products involving the use of these phospholipases are also provided. Background technique [0003] Phospholipases are enzymes that hydrolyze the ester bonds of phospholipids. Corresponding to their importance in phospholipid metabolism, these enzymes are widespread in eukaryotes and prokaryotes. Phosp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12Q1/68C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N9/16C12N11/00C07K16/40C12N15/10C07K7/08C07K14/00C07K19/00C11D3/386C11D7/42C11B3/00C11B3/06A01H5/00A01K67/027C07K7/00C12N15/09C12P7/02C12P21/08C12Q1/02C12Q1/34G01N33/53G01N37/00
CPCC12N9/18Y02E50/13C07K2319/00A01K2217/05C12N9/16
Inventor S·格拉马蒂科瓦G·黑尔伍德D·拉姆N·巴顿
Owner VERENIUM CORP (US)
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