Glucoamylase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-10494685-B2
Application number	US-201615767551-A
Country	US
Kind code	B2
Filing date	Oct 12, 2016
Priority date	Oct 14, 2015
Publication date	Dec 3, 2019
Grant date	Dec 3, 2019

Abstract

The present invention relates to glucoamylase variants having an increase in specific activity and/or an increase in melting temperature measured by TSA of at least 0.5 degrees Celsius, and further wherein the variants have at least at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the amino acid sequence of SEQ ID NO: 3. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

First claim

The invention claimed is:

1. A glucoamylase variant, comprising a substitution at one or more positions selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, I13, K15, V18, L19, N25, S27, K28, S30, V36, V37, T43, D45, S57, V59, F60, I71, S73, T74, L77, D82, D83, V85, T86, E88, L91, S95, P97, T103, D114, S134, L137, T139, N142, L145, S146, N147, N149, Y152, V153, T154, S155, N156, L157, W158, P159, I160, Q162, V169, S170, S175, T176, Y177, D184, S186, R199, A202, A203, T206, Q210, T211, S212, Q213, V214, S215, Y217, T218, T219, Q220, A221, D222, N223, L224, F227, Y231, P234, S235, Y238, T240, T243, G244, G245, G246, R247, S248, A252, T254, L255, Y262, S265, G267, A270, A271, K279, S282, L284, V294, Y295, S296, I297, N298, S299, G300, A302, S303, N304, T309, E314, S316, Q318, G319, T326, V330, N339, E342, S343, E348, S351, T352, Q359, S362, G363, V364, T365, A366, S371, S372, T378, S381, I383, N385, F386, A392, N394, K396, Y408, K410, D412, S414, S417, V419, A426, S427, E433, A434, N436, N437, T438, Q439, G442, A446, L448, V450, N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N495, S501, A502, T506, I509, T510, N512, S516, A518, I519, N527, N528, A530, E534, D536, P537, N538, N539, I541, A545, S546, G547, S548, N552, and T554, wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a glucoamylase variant having an increase in melting temperature measured by Thermal Shift Assay (TSA) of at least 0.5 degrees Celsius, and further wherein the variants have at least at least 95%, but less than 100%, sequence identity to the amino acid sequence of SEQ ID NO: 3.

2. The glucoamylase variants according to claim 1 , comprising a substitution at one or more positions selected from the group consisting of: Q1K, Q1R, S2E, S2K, S2L, S2P, S2R, V3L, V3G, V3R, D4R, D4S, D4G, D4A, D4W, S5L, S5V, S5G, S5C, S5R, S8Q, S8H, S8A, S8Y, S9C, S9Q, S9M, S9W, S9D, S9G, I13V, I13R, I13S, I13L, I13E, K15G, K15R, V18M, V18Q, L19G, L19F, N25S, N25A, 527A, S27L, S27G, S27V, S27C, K28C, K28R, 530Q, 530A, 530K, 530T, 530L, V36K, V36G, V36W, V36A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, D45L, D45P, S57P, S57L, S57G, S57F, S57R, S57T, 557A, V59T, V59S, V59E, F60S, I71M, I71S, I71T, I71V, S73H, S73R, S73N, S73V, S73G, T74V, L77S, L77P, L77R, D82N, D82R, D82V, D82G, D83L, D83C, D83W, V85Q, V85G, V85P, T86R, T86V, E88Q, E88R, E88G, L91S, L91P, L91G, S95A, S95T, S95V, P97T, P97I, P97R, T103Y, T103A, T103G, D114G, D114N, D114M, D114R, D114C, S134P, S134A, S134V, S134W, S134D, S134H, S134L, S134G, L137W, L137S, L137A, L137V, L137G, L137D, L137R, L137P, T139D, T139P, T139V, N142Y, N142H, N142C, L145C, L145D, L145G, L145V, L145S, S146W, S146L, S146R, S146G, S146P, N147Q, N147V, N147L, N147K, L147D, L147Y, L147H, L147S, N149H, N149T, N149R, N149K, N149S, Y152S, Y152A, Y152R, Y152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C, Y152W, V153E, V153S, V153G, V153W, V153Y, T154N, T154R, T154K, T154P, T154V, S155C, S155P, S155R, S155G, S155A, N156I, N156A, N156V, N156R, N156T, N156K, L157P, L157R, L157A, L157G, L157W, W158T, W158A, W158M, W158V, W158R, W158P, P159S, P159G, P159L, P159V, P159A, P159R, P159Q, P159E, I160A, I160G, I160N, I160T, I160R, I160V, Q162L, Q162V, Q162H, Q162P, Q162R, V169A, V169L, V169W, V169S, V169D, V169R, V169E, S170A, S170P, S170R, S170M, S175L, S175C, S175W, T176R, T176L, T176N, T176A, T176S, T176I, Y177H, D184P, D184W, D184S, D184Y, D184G, S186A, S186R, S186W, R199F, R199E, R199L, R199C, R199K, A202R, A202W, A202E, A202S, A202V, A203M, A203W, A203P, A203L, T206C, T206P, T206G, T206A, T206R, Q210C, Q210G, Q210S, Q210R, Q210L, Q210P, Q210V, T211R, T211A, T211H, T211K, T211Q, T211G, T211W, T211E, T211I, T211V, T211P, T211L, T211D, S212D, S212E, S212L, S212P S212T, Q213W, Q213V, Q213D, Q213A, Q213T, Q213R, Q213G, Q213S, V214G, V214R, V214W, V214A, V214I, S215R, S215G, S215L, S215Y, S215P, S215E, S215W, Y217G, Y217C, Y217A, Y217S, Y217T, Y217F, T218H, T218C, T218A, T218M, T218Q, T218G, T219R, T219D, T219S, T219G, T219C, Q220R, Q220V, Q220D, Q220S, Q220L, A221V, A221T, A221L, A221P, A221R, A221E, D222V, D222W, D222T, D222G, D222L, D222R, D222N, D222F, D222M, N223A, N223S, N223R, N223F, N223P, N223G, N223L, L224G, L224D, L224K, L224V, L224R, F227G, F227W, Y231S, Y231T, Y231R, Y231L, Y231A, Y231V, Y231N, P234D, P234L, P234S, P234V, S235C, S235R, S235N, S235G, S235W, Y238R, Y238A, Y238Q, Y238C, Y238E, T240C, T240I, T240L, T240S, T243V, T243S, T243L, T243R, G244R, G244C, G244P, G244D, G244W, G245R, G245S, G245V, G245W, G245M, G246L, G246E, G246S, G246R, G246K, G246W, G246D, R247E, S248Y, S248P, S248V, S248L, S248F, S248A, S248E, S248W, S248K, S248T, A252E, A252T, A252Y, A252V, A252L, T254D, T254W, T254V, T254G, T254A, L255R, L255Q, L255P, L255G, Y262C, Y262Q, Y262S, Y262G, Y262V, Y262A, Y262W, S265C, S265P, S265G, S265L, G267W, G267C, A270L, A270M, A271W, A271Y, A271L, K279R, K279W, K279E, K279P, K279G, K279F, S282W, S282T, S282K, S282R, L284N, L284Q, L284T, L284S, L284R, L284G, L284V, V294G, V294W, V294E, V294S, Y295V, Y295R, S296F, S296L, S296W, S296K, I297S, I297P, I297K, I297F, I297R, I297W, N298W, N298G, N298C, N298V, N298L, N298A, S299P, S299C, S299M, S299L, S299T, G300S, G300A, G300P, G300L, G300W, A302G, A302L, A302C, A302R, A302V, S303P, S303V, S303C, S303A, S303R, N304T, N304R, N304Q, N304L, N304V, T309G, T309I, T309R, T309M, E314Y, E314T, E314V, E314G, E314S, E314L, E314A, S316T, S316L, S316G, S316F, S316R, S316P, S316V, S316Q, Q318L, Q318R, G319R, G319Q, G319P, G319A, T326V, T326G, T326W, T326N, T326A, V330S, V330L, V330P, V330R, V330A, V330G, N339P, N339A, N339T, E342M, E342W, E342N, E342L, E342R, S343R, S343C, E348W, E348F, E348P, E348V, E348G, E348M, S351P, S351C, S351G, S351R, S351L, S351W, T352P, T352L, T352G, T352Q, T352Y, Q359K, Q359P, Q359R, Q359S, Q359A, S362P, S262R, S262G, S262M, G363R, G363T, G363P, V364A, V364C, V364E, V364S, V364G, V364L, T365S, T365G, T365W, T365L, T365H, A366D, A366T, A366P, A366R, A366H, S371A, S371G, S372A, S372E, S372C, S372L, S372R, T378G, T378L, T378D, T378H, T378A, T378P, S381K, I383A, I383G, I383C, I383L, I383T, I383M, N385R, N385W, N385S, N385G, N385D, F386S, F386W, F386Q, F386V, F386I, F386G, F386C, F386A, F386T, F386L, A392V, A392L, A392E, A392G, N394D, N394R, N394Y, N394W, N394E, K396I, K396W, K396P, K396Y, K396F, Y408V, Y408E, Y408P, Y408S, Y408K, Y408L, K410S, K410R, D412M, D412S, D412N, D412W, D412L, D412R, S414C, S414R, S414G, S414V, S414W, S414H, S417Y, V419S, V419G, V419C, V419A, V419K, V419R, V419T, A426M, A426N, A426K, A426R, S427G, S427A, S427P, S427N, S427D, S427L, E433C, A434Q, A434G, N436S, N436P, N436D, N437K, N437R, N437T, N437P, T438E, T438G, Q439W, Q439S, Q439G, Q439C, Q439R, Q439Y, G442V, G442D, G442C, G442A, G442L, G442W, G442E, G442M, G442R, A446G, A446D, A446R, A446E, A446I, L448G, L448P, L448E, V450P, V450S, V450C, V450E, V450L, V450N, N470H, N470D, N470K, N470V, N470L, E472I, V474W, V474C, V474A, V474L, V474G, W475P, W475A, W475R, N478L, N478I, N478P, N478R, N478W, N478S, N478G, N478K, N478A, S484G, S484Y, S484P, S484A, S484N, V485A, V485W, V485K, V485G, V485R, D486I, D486K, D486Y, D486S, D486A, D486W, D486L, A487S, A487V, A487L, A487G, A487C, A487K, S492L, S492R, S492T, S492W, S492P, S492C, A493V, A493R, A493D, A493W, D494N, D494R, D494G, D494L, D494E, D494Q, N495L, N495W, N495G, N495R, N495C, S501R, S501L, S501M, S501K, S501W, A502C, A502Q, A502W, A502G, A502V, T506A, T506P, T506V, I509E, I509D, I509S, I509F, I509W, I509R, T510F, T510E, T510R, T510P, T510V, T510A, T510L, N512Q, N512K, N512H, N512R, N512V, S516R, S516W, S516P, S516K, S516Y, S516C, A518D, A518G, A518Y, A518V, A518R, A518L, A518T, I519W, I519L, I519R, I519F, I519K, N527T, N527K, N527P, N527L, N528D, N528G, N528K, N528V, N528E, N528L, A530R, A530C, A530G, A530V, A530S, A530T, E534W, E534Q, E534C, E534V, E534G, E534R, E534F, E534K, D536

Assignees

• Novozymes As

Inventors

• Gupta Shikha
• Svendsen Allan
• Srivastava Vivek
• Malten Marco
• Poulsen Thomas Agersten
• Ramaiya Preethi
• Nelson Beth
• Rao Vasudeva P
• Balumuri Padmavathi

Classifications

• C12N9/2428 Primary

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12Y302/01003 Primary

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12P19/20

  produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title