In vitro methods of chemical conversion using non-stereospecific glutathione lyases

Patent metadata

Field	Value
Publication number	US-11447754-B2
Application number	US-202017038971-A
Country	US
Kind code	B2
Filing date	Sep 30, 2020
Priority date	Aug 14, 2017
Publication date	Sep 20, 2022
Grant date	Sep 20, 2022

Abstract

Enzymes for depolymerizing lignin. The enzymes include dehydrogenases, β-etherases, and glutathione lyases. The dehydrogenases can comprise one or more or LigD, LigO, LigN, and LigL. The β-etherases can comprise one or more of LigE, LigF, LigP, and BaeA. The glutathione lyases can comprise any one or more of LigG and a number of non-stereospecific, optionally recombinant glutathione lyases derived from Sphingobium sp. SYK-6, Novosphingobium aromaticivorans, Escherichia coli, Streptococcus sanguinis, Phanerochaete chrysosporium, and other microorganisms. The enzymes can be combined in compositions and/or used in methods of processing lignin or other aromatic compounds in vitro.

First claim

We claim:

1. A method of chemical conversion, comprising contacting a first compound in vitro with a non-stereospecific glutathione lyase to yield a second compound, wherein:
the non-stereospecific glutathione lyase comprises:
an amino acid sequence at least 80% identical to any of:

SEQ ID NO: 18 (NaGST Nu );

residues 21-313 of SEQ ID NO: 20 (recombinant

NaGST Nu );

SEQ ID NO: 22 (SYK6GST Nu );

residues 21-324 of SEQ ID NO: 24 (recombinant

SYK6GST Nu );

SEQ ID NO: 26 (ecYghU);

residues 21-313 of SEQ ID NO: 28 (recombinant

ecYghU);

SEQ ID NO: 30 (ecYfcG);

SEQ ID NO: 32 (ssYghU); 

and

SEQ ID NO: 36 (PcUre2pB 1);

and
at least four of:
threonine or a conservative variant of threonine at a position corresponding to position 51 of SEQ ID NO:18 (NaGST Nu );
asparagine or a conservative variant of asparagine at a position corresponding to position 53 of SEQ ID NO:18 (NaGST Nu );
glutamine or a conservative variant of glutamine at a position corresponding to position 86 of SEQ ID NO:18 (NaGST Nu );
lysine, a conservative variant of lysine, arginine, or a conservative variant of arginine at a position corresponding to position 99 of SEQ ID NO:18 (NaGST Nu );
isoleucine or a conservative variant of isoleucine at a position corresponding to position 100 of SEQ ID NO:18 (NaGST Nu );
glutamate or a conservative variant of glutamate at a position corresponding to position 116 of SEQ ID NO:18 (NaGST Nu );
serine, threonine, a conservative variant of serine, or a conservative variant of threonine at a position corresponding to position 117 of SEQ ID NO:18 (NaGST Nu ); and
arginine or a conservative variant of arginine at a position corresponding to position 177 of SEQ ID NO:18 (NaGST Nu );

the first compound has a structure of Formula I or a salt thereof:

wherein:
R 1 , R 2 , and R 3 are each independently —H, —OH, —O-alkyl, —O-lignin, or -lignin;
R 4 is —H, —OH, —SH, —COOH, —SO 3 H, or —O-lignin; and
SG is glutathione bound in an S or R configuration; and

the second compound has a structure of Formula II or a salt thereof:

wherein:
R 1 , R 2 , and R 3 are each independently —H, —OH, —O-alkyl, —O-lignin, or -lignin; and
R 4 is —H, —OH, —SH, —COOH, —SO 3 H, or —O-lignin.

2. The method of claim 1 , wherein the non-stereospecific glutathione lyase comprises an amino acid sequence at least about 85% identical to any of:

SEQ ID NO: 18 (NaGST Nu );

residues 21-313 of SEQ ID NO: 20 (recombinant

NaGST Nu );

SEQ ID NO: 22 (SYK6GST Nu );

residues 21-324 of SEQ ID NO: 24 (recombinant

SYK6GST Nu );

SEQ ID NO: 26 (ecYghU);

residues 21-313 of SEQ ID NO: 28 (recombinant

ecYghU);

SEQ ID NO: 30 (ecYfcG);

SEQ ID NO: 32 (ssYghU);

and

SEQ ID NO: 36 (PcUre2pB1).

3. The method of claim 1 , wherein the non-stereospecific glutathione lyase comprises an amino acid sequence at least about 90% identical to any of:

SEQ ID NO: 18 (NaGST Nu );

residues 21-313 of SEQ ID NO: 20 (recombinant

NaGST Nu );

SEQ ID NO: 22 (SYK6GST Nu );

residues

Assignees

• Wisconsin Alumni Res Found

Inventors

• Donohue Timothy James
• Gall Daniel Leo
• Kontur Wayne S
• Kim Hoon
• Ralph John
• Noguera Daniel R
• Fox Brian
• Bingman Craig

Classifications

• C12P7/22

  aromatic · CPC title

• Y02P20/582

  Recycling of unreacted starting or intermediate materials · CPC title

• C12Y205/01018

  Glutathione transferase (2.5.1.18) · CPC title

• C12Y114/16005

  Alkylglycerol monooxygenase (1.14.16.5) · CPC title

• C12N9/88

  Lyases (4.) · CPC title