Pectin degrading alcohol from i(Bacillus licheniformis)

A technology of Bacillus licheniformis and Bacillus, which is applied in the direction of bacteria, lyase, hydrolase, etc., and can solve problems such as optimization and inclusion

Inactive Publication Date: 2001-02-07
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These microorganisms also frequently produce cellulases and / or hemicellulases, complex multicomponent enzyme preparations derived from these microorganisms may be difficult to optimize for various applications, and they may even contain enzymes with deleterious effects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0308] Cloning, expression, purification and identification of pectate lyase (Ⅱ) from Bacillus licheniformis

[0309] Genomic DNA preparation

[0310] Bacillus licheniformis strain ATCC14580 was propagated in liquid medium 3 specified in ATCC (American Type Culture Collection, USA). After culturing at 37°C and 300rpm for 18 hours, the cells were collected, and bacterial genomic DNA was rapidly extracted with guanidine isothiocyanate by Pitcher et al. (Pitcher, D.G. Saunders, N.A. Owen, R.J. Genomic DNA was isolated using the method described in Microbiology Letters 8: 151-156) et al.

[0311] Use the following two oligonucleotide PCR primer sets to amplify the DNA coding sequence of pectate lyase II (see above, the amino acid sequence is shown in SEQ ID NO: 8) of the present invention: Pecl. B. lich. upper. SacⅡ5'-CTA ACT GCA GCC GCG GCA GCT TCT GCC TTA AAC TCG GGC -3'Pecl. B. lich. lower. NotⅠ5'-GCG TTG AGA CGC GCG GCC GCT GAA TGC CCC GGA CGT TTC ACC -3'

[0312] Re...

Embodiment 2

[0367] Cloning, expression, purification and identification of pectate lyase (Ⅰ) from Bacillus licheniformis

[0368] Genomic DNA preparation

[0369] Bacillus licheniformis strain ATCC14580 was propagated in liquid medium 3 specified by ATCC (American Type Culture Collection, USA). After incubating at 37°C and 300rpm for 18 hours, the cells were collected and rapidly extracted with guanidine isothiocyanate by the method described by Pitcher et al. Bacterial Genomic DNA, Applied Microbiology Letters 8: 151-156) Isolation of genomic DNA.

[0370] Sequences defining the invention

[0371] The sequence is defined by the following two primers, which can be used in subsequent PCR reactions to amplify the complete open reading frame of the pectate lyase of the present invention: Pecl3. orf. PstⅠ5'-CAC ATC TGCAGC ATG AAG AGA TTA GCA GGT ACG GTT ATT TTG TC-3'Pecl3. Licheniformis. lower. NotⅠ5'- CTC ATC ATG CGG CCG CAG GGG CCT TTA TTT GCA ATC AGT G -3'

[0372] Restriction site...

Embodiment 3

[0392] Cloning, expression, purification and identification of pectin lyase (Ⅲ) from Bacillus licheniformis

[0393] Cloning of the gene encoding pectin lyase from Bacillus licheniformis

[0394] The pectin lyase coding DNA sequence (SEQ ID NO: 1) of the present invention was amplified by PCR using the following two oligonucleotide PCR primer sets: Pect. upper. PstⅠ5'-CAT AAA TCT GCA GCC GCG GCA GCA AAC GAA GAT TAT CCG GAA C -3'Pect. lower. NotⅠ5'-GAA AGG AAA AGC GGC CGC CAA ATA TTG AAA AGT GAG CGC AAT GTCG-3'

[0395] Restriction sites PstI and NotI are underlined.

[0396] A PCR reaction was carried out as described in Example 1 using the chromosomal DNA isolated from Bacillus licheniformis as described above as a template. The appearance of a DNA fragment of about 1.5kb in size indicated that the gene segment was correctly amplified.

[0397] Subcloning of PCR fragments

[0398] Subcloning was performed as described in Example 1, but the purified PCR fragment was dig...

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PUM

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Abstract

Pectin degrading enzymes derived from or endogenous to Bacillus licheniformis or other Bacillus species which are at least 99 % homologous to Bacillus Licheniformis based on aligned 16S rDNA sequences have optimum activity at pH higher than 8. The pectin degrading enzymes belong to the enzyme classes pectate lyases (EC 4.2.2.2), pectin lyases (EC 4.2.2.10) and polygalacturonases (EC 3.2.1.15) and are useful in industrial processes under alkaline conditions such as in textile processing and as an active ingredient, e.g. in laundry detergents and hard surface cleaning products.

Description

[0001] The present invention relates to a preparation of pectin-degrading enzymes; preferably to microbial pectin-degrading enzymes, in particular to microbial enzymes which exhibit pectin-degrading activity as their main enzyme activity in the neutral and alkaline pH range, especially to sources Cloned pectin-degrading enzymes from Bacillus licheniformis; the invention also relates to methods of producing such enzymes and methods of using these enzymes in the textile, detergent and cellulosic fiber processing industries. Background of the invention [0002] Pectin polymers are important components of plant cell walls. Pectin is a heteropolysaccharide whose backbone consists of alternating homogalacturonans (smooth regions) and rhamnogalacturonans (hairy regions). The smooth region is a linear polymer of 1,4-linked α-D-galacturonic acid. Galacturonic acid residues can be methylated to varying degrees at the carboxyl group, usually in a non-random manner to fully methylate the...

Claims

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Application Information

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IPC IPC(8): A23K1/165A23L2/84C11D3/386C12G1/00C12N1/15C12N1/19C12N1/21C12N5/10C12N9/24C12N9/88C12N15/09C12R1/10
CPCC12N9/88
Inventor L·N·安德森M·舒莱恩N·E·K·兰吉M·E·布约恩瓦德K·施诺尔
Owner NOVO NORDISK AS
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