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Pullulanase variants and methods for preparing such variants with predetermined properties

a technology of pulverizing enzymes and variants, applied in the field of pulverizing enzyme variants and methods for preparing such variants with predetermined properties, can solve the problems of reducing the saccharification yield and being highly undesirable, and achieve the effects of improving ph optimum, improving properties, and modifying physicochemical properties

Inactive Publication Date: 2004-03-11
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to a method for modifying the amino acid sequence of a pullulanase to create variants with improved properties. These variants can have altered pH optimum, increased thermostability, improved specific activity, or altered substrate specificity. The method involves analyzing the structure of the pullulanase to identify relevant amino acid residues or structural regions, and then constructing a variant of the pullulanase by modifying the identified residues or regions. The resulting variant is tested for the desired property. The invention also includes variants of pullulanase with improved thermostability, pH dependent activity, or increased specific activity. The DNA encoding these variants and methods of preparing them are also provided. The invention is useful for industrial applications such as the production of sweeteners from starch.

Problems solved by technology

If active amylase from the liquefaction step is present during saccharification (i.e., no denaturing), this level can be as high as 1-2% or even higher, which is highly undesirable as it lowers the saccharification yield significantly.

Method used

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  • Pullulanase variants and methods for preparing such variants with predetermined properties
  • Pullulanase variants and methods for preparing such variants with predetermined properties
  • Pullulanase variants and methods for preparing such variants with predetermined properties

Examples

Experimental program
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Effect test

example 1

[0491] Construction of Bacillus deramificans D620A Variant

[0492] Gene specific primer 132011 and mutagenic primer 132012 are used to amplify by PCR an approximately 410 bp DNA fragment from the pCA36 plasmid.

[0493] The 410 bp fragment is purified from an agarose gel and used as a Mega-primer together with primer 136054 in a second PCR carried out on the same template.

[0494] The resulting approximately 1110 bp fragment is digested with restriction enzymes BsiW I and Mlu I and the resulting approximately 330 bp DNA fragment is purified and ligated with the pCA36 plasmid digested with the same enzymes. Competent Bacillus subtilis SHA273 (amylase and protease low) cells are transformed with the ligation and chlorampenicol resistant transformants are checked by colony PCR.

[0495] The mutagenesis primer 132012 introduced the D620A substitution (written in bold in the primer sequence) and introduced simultaneously a Bgl I restriction site (underlined in the primer seq.), which facilitates e...

example 2

[0497] Construction of Bacillus deramificans E649A Variant

[0498] This variant constructed as described in Example 1, except that mutagenic primer 132013 is used. The mutagenesis primer 132013 introduced the E649A substitution (written in bold in the primer sequence) and a NarI restriction site (underlined in the primer sequence), which facilitates easy pinpointing of mutants.

[0499] Primer 132013:

[0500] 5' gcactttacggggcgccatggacggg 3' (SEQ ID NO: 10)

example 3

[0501] Thermostability test of variant of the invention The below mentioned variants were constructed in Bacillus deramificans pullullanase using a megaprimer approach similar to the one described above. The following primers were used in order to introduce the various amino acid changes indicated below (all primer are written directional (5'-3'):

3 deletion (1-111) Nr.167 TVB400: CATTCTGCAGCGGCCGCAAACGCTT-ATTTAGATGCTTCAAACC (SEQ ID NO:11) deletion (1-113) Nr.168-tvb401: CATTCTGCAGCGGCCGCAGATGATCTTGGGAATACCTATAC (SEQ ID NO:12) D562P Nr.170-tvb496: CTTTGCCACGCAGATCTCTCCCTTCGATA-AAATTG (SEQ ID NO:13) G292P NR.171-tvb497: CATTCAAACGGATCCCTATCAGGCAAAG (SEQ ID NO:14) G794P Nr.172-tvb498: GTTATAATGCACCCGATGCGGTCAATG (SEQ ID NO:15) D148P Nr. 173-tvb499: CAGCAAATAAGCCCATTCCAGTGACATCTGTG (SEQ ID NO:16) N119P Nr. 174-tvb500: CTTATTTAGATGCATCACCCCAGGTGC (SEQ ID NO:17) N400S Nr.175-tvb567: CAACTGCGATCGCACCAAGTGGAACGAG (SEQ ID NO:18) N400L Nr.176-tvb568: GCGATCGCACCACTTCGAACGAGAGGC (SEQ ID NO:19)...

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Abstract

The present invention relates to a method for producing a variant of a parent pullulanase, the variant having at least one altered property as compared to the parent pullulanase. The invention also relates to pullulanase variants and to the use of pullulanase variants of the invention for use in particular starch conversion processes.

Description

[0001] The present invention relates to variants of pullulanases and to methods for constructing such variants with altered properties, including stability (e.g., thermostability), pH dependent activity, substrate specificity, such as increased isoamylase activity, or specific activity; specific activity at a given pH and / or altered substrate specificity, such as an altered pattern of substrate cleavage or an altered pattern of substrate inhibition.[0002] Starches such as corn, potato, wheat, manioc and rice starch are used as the starting material in commercial large-scale production of sugars, such as high fructose syrup, high maltose syrup, maltodextrins, amylose, G4-G6 oligosaccharides and other carbohydrate products such as fat replacers.[0003] Degradation of Starch Starch usually consists of about 80% amylopectin and 20% amylose. Amylopectin is a branched polysaccharide in which linear chains alpha-1,4 D-glucose residues are joined by alpha-1,6 glucosidic linkages. Amylopectin...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/44
CPCC12N9/2457C12Y302/01068C12Y302/01041C12N9/246
Inventor SVENDSEN, ALLANANDERSEN, CARSTENBORCHERT, TORBEN VEDEL
Owner NOVOZYMES AS
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