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Catalytically inactive proteins and method for recovery of enzymes from plant-derived materials

An inactive, xylanase-active technology, applied in the field of expression of mutant xylanase in microorganisms and yeast, can solve the problems of poor enzymatic activity, poor recoverability, sharp recovery, etc.

Inactive Publication Date: 2009-08-05
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most commercial xylanases designed for feed applications were not selected due to the poor resiliency of their enzymatic activity in formulated feeds
This problem is especially acute for some enzymes whose recovery is only 10-20%

Method used

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  • Catalytically inactive proteins and method for recovery of enzymes from plant-derived materials
  • Catalytically inactive proteins and method for recovery of enzymes from plant-derived materials
  • Catalytically inactive proteins and method for recovery of enzymes from plant-derived materials

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0178] Example 1: Site-directed mutagenesis to change the catalytic nucleophile of xylanase from glutamic acid to alanine, producing a catalytically inactive protein

[0179] Xylanase Xy1A1A was identified by activity-based screening of libraries from environmental samples. The gene encoding wild-type Xy1A1A xylanase (SEQ ID NO. 3) was cloned into the bacterial expression vector pTrcHis. The vector is named pTrcHis_Xy1A1A, which is derived from figure 1 and represented by SEQ ID NO.114. To create this construct, the possible signal sequence of Xy1A1A was removed from the full-length gene sequence resulting in a truncated xylanase gene. Further, after inserting the truncated xylanase gene into pTrcHis, the open reading frame containing the xylanase gene contained five additional 5' terminal codon. Sequence alignment of the translated protein of the full-length Xy1A1A coding sequence and other glycosyl hydrolases family 11 xylanases in the literature shows that the 79th posi...

Embodiment 2

[0194] Example 2: Preparation of catalytically inactive xylanase proteins in bacterial expression hosts

[0195] The pTrcHis_Xy1A1A_E79A vector was transformed into BL21 Star (pLysS) cells using standard techniques [Sambrook et al.] and spread to 100 μg / mL ampicillin (LB amp100 ) on Luria medium agar plates. Pick a single colony and inoculate into 3.5 mL containing 50 μg / mL ampicillin and 25 μg / mL chloramphenicol (TB amp50-chlor25 ) in Terrific medium, grown overnight at 37°C with continuous shaking. After culturing overnight, a portion of the culture was removed and stored as a frozen glycerol stock solution at -80°C.

[0196] From this glycerol stock, inoculate 20 mL of TB with a sterile loop amp50-chlor25 into a 250mL culture flask. Cultures were grown overnight at 37°C with shaking at 200-250 rpm. The next day, dilute 5 mL of the overnight culture to 1.5 L of TB amp50-chlor25 middle. The culture was grown at 37°C with shaking until the OD600 reached 0.6-1.0. Then, ...

Embodiment 3

[0197] Example 3: Preparation of expression constructs for the production of Xy1A1A_E79A in the yeast host Pichia pastoris

[0198] Construction of pCR4Blunt_Xy1A1A_E79A

[0199] Synthetic oligonucleotides (primers 3 and 4) and Pfu DNA polymerase (Stratagene, LaJol1a, CA) were used to PCR amplify the BD6002E79A gene from pTrcHis2-BD6002E79, and the parameters of the thermocycler used were set as follows:

[0200] Primer 3

[0201] 5'-TTTCCCTCTCGAGAAAAGAGCTTCGACAGACTACTGGCAAAATTGG (SEQ ID NO. 123)

[0202] Primer 4

[0203] 5'-TTTTCCTTTTGCGGCCGCCTATTACCAGACCGTTACGTTAGAGTAC (SEQ ID NO. 124)

[0204]

[0205] Primer 3 was designed to anneal at the codon corresponding to amino acid 5 on the xylanase-containing pTrcHis open reading frame. In addition, primer 3 added an XhoI restriction site and a Kex2 protease cleavage signal (Leu-Glu-Lys-Arg) in front of the mature xylanase coding sequence. Primer 4 contained a double stop codon after the xylanase gene. The BD6002E79A PCR...

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Abstract

An inactive xylanase molecule for the recovery of xylanase activity in plant-derived material containing active xylanase enzyme(s) and xylanase inhibitors. The inactive xylanase molecule of binds to xylanase inhibitors in the plant-derived material, thereby allowing accurate measurement of xylanase enzyme activity of the enzyme contained in the plant-derived material. The invention further includes amino acid molecules depicted by SEQ ID NOS. 4 through 112, wherein the catalytically active sites of each of the amino acids have been modified resulting in inactive xylanase molecules. A method of production of the inactive xylanase molecules includes expression of the inactive xylanase molecule in microbial or eukaryal (e.g., yeast including Pichia pastoris) host cell an expression cassette comprising a promoter operably linked to a nucleic acid molecule encoding the inactive xylanase molecule and using the expressed molecule in an assay to recover the xylanase enzyme activity in plant-derived material, for example, in plant-derived material such as animal feed.

Description

field of invention [0001] The present invention relates to mutant xylanase coding sequences useful for producing catalytically inactive proteins. The present invention also relates to the expression of these mutant xylanases in microorganisms and yeast. The present invention also relates to the use of a catalytically inactive protein to improve the reversibility of xylanase activity from plant source materials such as formulated animal feed. Background of the invention [0002] Xylan is a linear polysaccharide formed from β-1,4 linked D-xylopyranose. Xylans often contain alpha-1,2, alpha-1,3, or alpha-1,2 and alpha-1,3 linked L-arabinofuranoside side chains. These substituted xylans are commonly referred to as arabinoxylans. Xylan and arabinoxylan are among the main non-starch polysaccharides (NSP) in plants. These NSPs form viscous solutions that can be troublesome in baking, brewing and animal feed applications. For example, when preparing dough in baking applications...

Claims

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Application Information

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IPC IPC(8): C12N9/42
CPCC12N9/24C12N9/2482C12Y302/01008
Inventor M·W·鲍尔J·德方特斯
Owner SYNGENTA PARTICIPATIONS AG
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