Glucoamylase variants

Abstract

Aspects of the present disclosure include glucoamylase (GA) variants having at least one improved property over a parent GA, compositions containing the GA variants, nucleic acids encoding the GA variants, and methods for producing and using the same. In some aspects, the GA variant is a variant of a parent GA from Humicola grisea.

1 - 398 . (canceled) 
     
     
         399 . A variant of a parent glucoamylase (GA) enzyme, where the variant differs from the parent enzyme in that it has one or a combination of amino acid substitutions that are selected from the group consisting of:
 (a) 468 selected from the group consisting of: A, E, D, G, H, I, K, L, M, N, P, Q, R, T and W;   (b) 466 selected from the group consisting of: D, K, M, R, Y, C, P, T, W, A, E, F, H, L, N, S, I, V and Q; and   (c) 472 selected from the group consisting of: R, D, I, M, Q, S and Y,   
       where the position of each amino acid substitution corresponds to SEQ ID NO:3, and where the GA variant has starch hydrolysis activity; has at least 80% sequence identity to SEQ ID NO:4; and has at least one improved property over the parent GA enzyme selected from: (a) expression, (b) hydrolytic activity on DP2 substrate, (c) hydrolytic activity on DP7 substrate at pH 6.8, (d) hydrolytic activity on DP7 substrate at pH 5.5, (e) hydrolytic activity on panose substrate, (f) hydrolytic activity on pullulan substrate, (g) hydrolytic activity on granular corn starch (CS), (h) thermostability, (i) glucose inhibition, and (j) reversion activity. 
     
     
         400 . The GA variant of  claim 399 , comprising at least one further amino acid substitution at a position selected from the group consisting of: T35, K41, P60, L97, N100, T107, A175, K183, E229, T245, G263, P293, E294, N299, L300, R308, K334, S342, Y364, I367, V369, Q373, G374, S382, V393, S394, T397, K400, S402, T406, N407, V409, N410, A411, A414, I462, L467, P469, S471, V476, A477, K478, S479, Q480, S483, T484, P503, K505, Q506, A522, T544, N556, G587, A589, T599, N600, T604, S607, P609, A31, I37, N46, K57, D85, L105, K171, K179, S184, P213, A221, T239, Q250, L252, N260, S261, K262, N264, E274, A295, A325, K328, A366, Y368, N371, K372, I376, S380, V381, P384, R387, V390, S391, T395, G396, S399, S401, S403, F405, I408, A448, D462, S475, S486, A491, S511, S515, A524, V537, S538, A540, W541, G542, V551, P552, K561, A562, T564, L565, N573, A585, V590, Q591, V597, G601, I603, D608, I613, T614, S620, S632, A32, V33, N38, T39, T74, H99, T104, N111, K118, Q121, I164, Q168, I174, G177, S180, T181, V190, K191, A198, L227, L233, Q236, D238, E240, A243, A248, V251, Q255, Y275, N290, F291, D292, G296, D298, T301, F302, A311, A315, S319, G329, A331, Q332, V345, A360, D365, W370, T379, F386, L389, S392, Y398, K413, A416, D417, S429, A434, K443, A460, R463, R464, A465, R473, L481, T493, S499, S501, V531, R536, V548, N550, A553, L554, G555, D558, T559, S560, S572, D574, S578, K584, S588, I595, K596, K602, W605, E606, R611, S612, S619, V629, D34, P42, I43, K47, A50, N55, A58, A62, A64, I68, S72, R73, D75, P77, T83, L90, S96, G98, Y101, T103, I109, Q110, V113, S115, V122, S123, S126, T128, F129, A146, T148, E150, A165, L166, Y169, A170, A182, V185, P188, L194, T197, F206, F218, S222, H224, A226, Y232, L237, P249, Q256, A257, F258, W259, Y265, V267, S268, G272, G273, S277, D280, A281, I284, A286, S287, Q303, S306, E307, H313, Y316, D318, N322, V336, A337, N348, A355, N356, A359, L363, S375, T377, V378, L383, F385, D388, T404, E421, V422, A424, K425, Y426, A432, N440, S446, T451, F457, P470, R487, I488, V495, A496, A497, F502, S504, A513, P516, P520, T521, S527, V529, T532, N534, E535, T539, E543, I545, V547, G549, K571, L576, I579, I583, T586, Y592, Y594, G622, Q627, T628, N630 and D631, where the position of each amino acid substitution corresponds to SEQ ID NO:3; optionally wherein the at least one further amino acid substitution is selected from Table 8. 
     
     
         401 . The variant of  claim 400 , wherein the GA variant comprises an amino acid substitution at position:
 (a) T35 selected from the group consisting of: A, P, C, F, K, L, Q, R, W, Y and H; and/or   (b) K41 selected from the group consisting of: C, S, T, W, D, H, L, N, P, R and E; and/or   (c) P60 selected from the group consisting of: V, G, H, M, R, I, W, F, T and Q; and/or   (d) L97 selected from the group consisting of: A, G, Q, E, T, V, F, C, H, I and N; and/or   (e) N100 selected from the group consisting of: P, E, V, C, I, M, Q, T, F, G, K, S, W, Y, A, L and R; and/or   (f) T107 selected from the group consisting of: A, W, Y, H, G, I, S, V, K, C, D, E, M and N; and/or   (g) A175 selected from the group consisting of: F, E, H, K, M, N, Q, R, S, T, V and C; and/or   (h) K183 selected from the group consisting of: H, E, I, Q, R, V, Y, D, F, T and W; and/or   (i) E229 selected from the group consisting of: A, D, G, M, T, W, I, L, Y, C, F, K and V; and/or   (j) T245 selected from the group consisting of: L, C, I, R, H, M, Q, S, V, Y, F and W; and/or   (k) G263 selected from the group consisting of: A, C, E, H, L, Q, R, S, T and Y; and/or   (l) P293 selected from the group consisting of: T, A, C, E, F, G, H, L, R, S, Y and Q; and/or   (m) E294 selected from the group consisting of: D, G, I, K, L, M, N, S, V, W, A and C; and/or   (n) N299 selected from the group consisting of: A, C, E, G, L, Q, T, V, P, R and S; and/or   (o) L300 selected from the group consisting of: I, K, N, A, C, D, E, F, G, H, P, Q, R, S, T, V, W and Y; and/or   (p) R308 selected from the group consisting of: I, F, L, A, D, E, G, H, K, M, Q, T, W and V; and/or   (q) K334 selected from the group consisting of: C, G, I, L, T, W, D, V, A, E, F, R and Y; and/or   (r) S342 selected from the group consisting of: G, L, T, W, Y, C, E, F, N, Q, K, M, P, R and V; and/or   (s) Y364 selected from the group consisting of: E, R, W, G, Q, H, I, L, M, N, S and V; and/or   (t) 1367 selected from the group consisting of: G, E, F, K, L, M, S, V, W and Y; and/or   (u) V369 selected from the group consisting of: F, Q, C, I, K, L, N, S, T, Y, A, E, G and M; and/or   (v) Q373 selected from the group consisting of: D, E, F, G, L, N, R, S, T, C, H, K and V; and/or   (w) G374 selected from the group consisting of: D, E, F, K, R, T, V, W, C, L, M, P, S, A and Y; and/or   (x) S382 selected from the group consisting of: D, H, C, P, Q, E, G, N, T and V; and/or   (y) V393 selected from the group consisting of: A, W, E, G, T, Y, F, H, I, L and N; and/or   (z) S394 selected from the group consisting of: F, G, H, I, N, A, C, D, L, M, P and Y; and/or   (aa) T397 selected from the group consisting of: A, G, F, L, Q, V, W, Y, K and R; and/or   (ab) K400 selected from the group consisting of: A, E, G, L, R, S, V, W, Y and P; and/or   (ac) S402 selected from the group consisting of: D, I, N, P, T, W, R, F, K and M; and/or   (ad) T406 selected from the group consisting of: C, A, D, G, H, I, L, N, P, R, S, V, W and E; and/or   (ae) N407 selected from the group consisting of: C, G, I, K, L, Q, R, A, P, S and W; and/or   (af) V409 selected from the group consisting of: C, G, H, K, D, E, L, M, R, W and Y; and/or   (ag) N410 selected from the group consisting of: I, G, Q, Y, C, F, H, K, L, P, T, V, W, A and D; and/or   (ah) A411 selected from the group consisting of: P, G, I, L, N, Q, R, S, W and C; and/or   (ai) A414 selected from the group consisting of: C, F, G, I, P, Q, S, D, E, R, T and W; and/or   (aj) 1461 selected from the group consisting of: C, F, G, K, M, S, T, V, Y, N, Q and R; and/or   (ak) L467 selected from the group consisting of: C, G, Q, T, W, D, H, N, Y, A, F, S and V; and/or   (al) P469 selected from the group consisting of: R, C, H, T, V, G, N, S, E, I, Q and W; and/or   (am) S471 selected from the group consisting of: D, C, M, E, G, P, Q, L, A, H, I, N and V; and/or   (an) V476 selected from the group consisting of: A, D, E, G, H, N, S, L, M, P, Q, Y, R, T, I and W; and/or   (ao) A477 selected from the group consisting of: D, E, I, K, Q, R, M, W, S and V; and/or   (ap) e K478 selected from the group consisting of: E, I, P, R, S, T, Y, F, Q and V; and/or   (aq) S479 selected from the group consisting of: A, G, L, M, N, Q, R, T, Y, F, I and K; and/or   (ar) Q480 selected from the group consisting of: A, C, D, T, V, H, I, L, M and F; and/or   (as) S483 selected from the group consisting of: F, P, W, Y, A, I, K, N, R, T, H and L; and/or   (at) T484 selected from the group consisting of: I, M, V, K, P, Q, Y, F, L and S; and/or   (au) P503 selected from the group consisting of: A, D, F, G, L, M, N, Q, R, S, T, V and Y; and/or   (av) K505 selected from the group consisting of: D, E, G, N, T, M, S, V, H and P; and/or   (aw) Q506 selected from the group consisting of: A, F, L, N, P, S, E, M, T, V, Y and C; and/or   (ax) A522 selected from the group consisting of: E, P, N, T, V, Y, L, R, K, C and D; and/or   (ay) T544 selected from the group consisting of: P, A, I, L, M, N, S, Y, G, K, V and R; and/or   (az) N556 selected from the group consisting of: P, A, G, H, I, M, S, T, W, D, E, L, V and Y; and/or   (ba) G587 selected from the group consisting of: D, F, I, N, Q, T, Y, C, P, L and R; and/or   (bb) A589 selected from the group consisting of: C, D, K, L, M, R, E, Q, T and V; and/or   (bc) T599 selected from the group consisting of: M, C, E, R, D, K, L, V, Y, A, F, P, Q and S; and/or   (bd) N600 selected from the group consisting of: A, E, M, Q, R, W, Y, P, V and K; and/or   (be) T604 selected from the group consisting of: K, M, S, Y, A, D, H, I, Q, R, F, G and L; and/or   (bf) S607 selected from the group consisting of: W, L, Y, G, I, Q, V, A, H, M and N; and/or   (bg) P609 selected from the group consisting of: K, L, N, Q, R, T, V, Y, A, G and W; and/or   (bh) A31 selected from the group consisting of: I, T, Y, F, L and Q; and/or   (bi) I37 selected from the group consisting of: G, M, A, S, C and T; and/or   (bk) N46 selected from the group consisting of: D, E, P, V, C and S; and/or   (bl) K57 selected from the group consisting of: E, S, A, C, I, V, T, Q and R; and/or   (bm) D85 selected from the group consisting of: N, Y, L, S, C, G, M, E and V; and/or   (bn) L105 selected from the group consisting of: M, R, T, I, F and Y; and/or   (bo) K171 selected from the group consisting of: C, G, H, R, V and W; and/or   (bp) K179 selected from the group consisting of: D, P, H, L, Q, R and W; and/or   (bq) S184 selected from the group consisting of: A, I, M, L, C, F, V and R; and/or   (br) P213 selected from the group consisting of: A, M, N, H, Q and R; and/or   (bs) A221 selected from the group consisting of: M, R, S, I, L and T; and/or   (bt) T239 selected from the group consisting of: A, C, F, M, V, I and R; and/or   (bu) Q250 selected from the group consisting of: D, F, H, M, Y and E; and/or   (by) L252 selected from the group consisting of: H, A, I, N, S, M and V; and/or   (bw) N260 selected from the group consisting of: C, H, R, T, F, S and W; and/or   (bx) S261 selected from the group consisting of: E, F, K, T, W and Y; and/or   (by) K262 selected from the group consisting of: H, M, P, R, V, W and C; and/or   (bz) N264 selected from the group consisting of: A, F, P, Q, V and E; and/or   (ca) E274 selected from the group consisting of: V, D, I, L, Q and F; and/or   (cb) A295 selected from the group consisting of: F, G, V, W, Y, D and N; and/or   (cc) A325 selected from the group consisting of: E, C, F, L, P and Q; and/or   (cd) K328 selected from the group consisting of: C, L, Q, W, E, F and R; and/or   (ce) A366 selected from the group consisting of: Q, C, M, V, G, S and T; and/or   (cf) Y368 selected from the group consisting of: E, H, K, T, W and M; and/or   (cg) N371 selected from the group consisting of: C, L, M, D, Q, R, Y and A; and/or   (ch) K372 selected from the group consisting of: C, D, F, S, L, N and Q; and/or   (ci) I376 selected from the group consisting of: C, T, V, Y, A, F, L and M; and/or   (cj) S380 selected from the group consisting of: A, F, K, L, Q, R, W and Y; and/or   (ck) V381 selected from the group consisting of: F, G, K, M, N, S, A, C and Q; and/or   (cl) P384 selected from the group consisting of: D, F, S, Y, E, T and V; and/or   (cm) R387 selected from the group consisting of: C, I, Q, Y, G, L and K; and/or   (cn) V390 selected from the group consisting of: H, I, K, Y, F, L and R; and/or   (co) S391 selected from the group consisting of: N, H, L, F, I and R; and/or   (cp) T395 selected from the group consisting of: K, Q, R, V, Y, F and L; and/or   (cq) G396 selected from the group consisting of: E, L, Q, R, K and V; and/or   (cr) S399 selected from the group consisting of: Q, T, R, W, K and C; and/or   (cs) S401 selected from the group consisting of: H, T, A, Y, P and Q; and/or   (ct) S403 selected from the group consisting of: I, P, W, Y, A and V; and/or   (cu) F405 selected from the group consisting of: A, H, W, Y, N and S; and/or   (cv) I408 selected from the group consisting of: Q, A, T, V, S and Y; and/or   (cw) A448 selected from the group consisting of: F, P, T, N, I and Q; and/or   (cx) D462 selected from the group consisting of: H, A, E, F, I, L, S, M and T; and/or   (cy) S475 selected from the group consisting of: K, N, Q, E, F, I, R and L; and/or   (cz) S486 selected from the group consisting of: V, T, Y, D, G, K, L, I and M; and/or   (da) A491 selected from the group consisting of: H, I, L, M, Y, K, R and V; and/or   (db) S511 selected from the group consisting of: I, W, H, K, L and N; and/or   (dc) S515 selected from the group consisting of: D, M, N, T, A, I, K and V; and/or   (dd) A524 selected from the group consisting of: F, L, N, Q, R, T, W, S and Y; and/or   (de) V537 selected from the group consisting of: M, T, A, C, K and R; and/or   (df) S538 selected from the group consisting of: G, A, C, K, Q, R, T and F; and/or   (dg) A540 selected from the group consisting of: Y, E, F, L, T, V, W and M; and/or   (dh) W541 selected from the group consisting of: A, I, L, R, V and Y; and/or   (di) G542 selected from the group consisting of: D, A, M, N, K and R; and/or   (dj) V551 selected from the group consisting of: S, N, F, K, L, Q and Y; and/or   (dk) P552 selected from the group consisting of: A, G, I, K, M, N, Y and R; and/or   (dl) K561 selected from the group consisting of: D, E, G, I, L, N, P, Q and R; and/or   (dm) A562 selected from the group consisting of: I, K, L, Y, C, G, V and S; and/or   (dn) T564 selected from the group consisting of: N, L, M, S, K and R; and/or   (do) L565 selected from the group consisting of: E, H, M, Q, S, T and V; and/or   (dp) N573 selected from the group consisting of: G, E, F, H, I, Y and A; and/or   (dq) A585 selected from the group consisting of: D, F, S, T, W, Y and N; and/or   (dr) V590 selected from the group consisting of: W, A, G, P, S, I and L; and/or   (ds) Q591 selected from the group consisting of: L, Y, C, R, P, S and T; and/or   (dt) V597 selected from the group consisting of: R, L, M, Q, S, W, F and K; and/or   (du) G601 selected from the group consisting of: E, I, R, A, H, N, P, Q and M; and/or   (dv) I603 selected from the group consisting of: Q, T, Y, A, K, F, L, N and P; and/or   (dw) D608 selected from the group consisting of: L, P, R, T, E, F and I; and/or   (dx) I613 selected from the group consisting of: A, F, L, S, T and V; and/or   (dy) T614 selected from the group consisting of: L, M, Q, V, Y and S; and/or   (dz) S620 selected from the group consisting of: D, M, Q, E, H, P and R; and/or   (ea) S632 selected from the group consisting of: L, C, D, E, G and T; and/or   (eb) A32 selected from the group consisting of: S, T, E and Q; and/or   (ec) V33 selected from the group consisting of: L, P and I; and/or   (ed) N38 selected from the group consisting of: G, F, H, K and I; and/or   (ee) T39 selected from the group consisting of: E, K, L and Q; and/or   (ef) T74 selected from the group consisting of: D, I, K and V; and/or   (eg) H99 selected from the group consisting of: G, A, M, V and Y; and/or   (eh) T104 selected from the group consisting of: V, D and E; and/or   (ei) N111 selected from the group consisting of: F, M, D, Q and H; and/or   (ej) K118 selected from the group consisting of: M, L, F and Y; and/or   (ek) Q121 selected from the group consisting of: V, L and T; and/or   (el) I164 selected from the group consisting of: K, V and A; and/or   (em) Q168 selected from the group consisting of: H, A, L and Y; and/or   (en) I174 selected from the group consisting of: V, F and Y; and/or   (eo) G177 selected from the group consisting of: D, H, K and S; and/or   (ep) S180 selected from the group consisting of: A, C and F; and/or   (eq) T181 selected from the group consisting of: A, S and W; and/or   (er) V190 selected from the group consisting of: A, I, M and L; and/or   (es) K191 selected from the group consisting of: V, W and R; and/or   (et) A198 selected from the group consisting of: Y, C, G and S; and/or   (eu) L227 selected from the group consisting of: A, C and I; and/or   (ev) L233 selected from the group consisting of: T, V and M; and/or   (ew) Q236 selected from the group consisting of: W, F, D, N and Y; and/or   (ex) D238 selected from the group consisting of: L, Q and R; and/or   (ey) E240 selected from the group consisting of: Q, W and P; and/or   (ez) A243 selected from the group consisting of: G, H, R and P; and/or   (fa) A248 selected from the group consisting of: G, C and S; and/or   (fb) V251 selected from the group consisting of: L, Q and R; and/or   (fc) Q255 selected from the group consisting of: E, G, L and S; and/or   (fd) Y275 selected from the group consisting of: E, G, L and S; and/or   (fe) N290 selected from the group consisting of: E, I, Q and V; and/or   (ff) F291 selected from the group consisting of: Y, L and M; and/or   (fg) D292 selected from the group consisting of: F, C and S; and/or   (fh) G296 selected from the group consisting of: L, P, Q and V; and/or   (fi) D298 selected from the group consisting of: A, E, I and N; and/or   (fi) T301 selected from the group consisting of: A, I, C and L; and/or   (fk) F302 selected from the group consisting of: G, M and W; and/or   (fl) A311 selected from the group consisting of: R, C, G, M and V; and/or   (fm) A315 selected from the group consisting of: G, C, E, M and Q; and/or   (fn) S319 selected from the group consisting of: N, M and Q; and/or   (fo) G329 selected from the group consisting of: T, C and D; and/or   (fp) A331 selected from the group consisting of: V, K, L, R and M; and/or   (fq) Q332 selected from the group consisting of: V, W, E, L and P; and/or   (fr) V345 selected from the group consisting of: I, Q and K; and/or   (fs) A360 selected from the group consisting of: C, G and S; and/or   (ft) D365 selected from the group consisting of: A, E, Q and R; and/or   (fu) W370 selected from the group consisting of: S, T, V, F and M; and/or   (fv) T379 selected from the group consisting of: E, H, N, Q and R; and/or   (fw) F386 selected from the group consisting of: H, L, T, V and W; and/or   (fx) L389 selected from the group consisting of: A, Q, S and T; and/or   (fy) S392 selected from the group consisting of: Y, I and P; and/or   (fz) Y398 selected from the group consisting of: N, Q, F, L and H; and/or   (ga) K413 selected from the group consisting of: T, E, F, L and R; and/or   (gb) A416 selected from the group consisting of: C, G and S; and/or   (gc) D417 selected from the group consisting of: N, T, E and Q; and/or   (gd) S429 selected from the group consisting of: D, G, M, T and A; and/or   (ge) A434 selected from the group consisting of: F, S, W and Y; and/or   (gf) K443 selected from the group consisting of: I, M, L and H; and/or   (gg) A460 selected from the group consisting of: C, G, M and T; and/or   (gh) R463 selected from the group consisting of: A, K and M; and/or   (gi) R464 selected from the group consisting of: F, V, H and K; and/or   (gj) A465 selected from the group consisting of: I, W, M, R and Y; and/or   (gk) R473 selected from the group consisting of: T, C and M; and/or   (gl) L481 selected from the group consisting of: F, H, R and C; and/or   (gm) T493 selected from the group consisting of: C, S, V and Q; and/or   (gn) S499 selected from the group consisting of: A, C, H, N and M; and/or   (go) S501 selected from the group consisting of: V, A, I and Q; and/or   (gp) V531 selected from the group consisting of: S, T, A and C; and/or   (gq) R536 selected from the group consisting of: C, Q, L, Y and H; and/or   (gr) V548 selected from the group consisting of: F, M and T; and/or   (gs) N550 selected from the group consisting of: C, G, M, S and T; and/or   (gt) A553 selected from the group consisting of: E, K, P, Q and Y; and/or   (gu) L554 selected from the group consisting of: M, V and T; and/or   (gv) G555 selected from the group consisting of: K, A and H; and/or   (gw) D558 selected from the group consisting of: F, L, Q and R; and/or   (gx) T559 selected from the group consisting of: E, G, I and L; and/or   (gy) S560 selected from the group consisting of: F, A, K, M and V; and/or   (gz) S572 selected from the group consisting of: P, E, L, V and K; and/or   (ha) D574 selected from the group consisting of: I, M, N and Y; and/or   (hb) S578 selected from the group consisting of: F, A and C; and/or   (hc) K584 selected from the group consisting of: F, T and V; and/or   (hd) S588 selected from the group consisting of: H, L, P and Y; and/or   (he) I595 selected from the group consisting of: G, M, T and L; and/or   (hf) K596 selected from the group consisting of: D, E and N; and/or   (hg) K602 selected from the group consisting of: D, I, R and F; and/or   (hi) W605 selected from the group consisting of: T, Y and F; and/or   (hj) E606 selected from the group consisting of: G, K and Q; and/or   (hk) R611 selected from the group consisting of: S, N and H; and/or   (hi) S612 selected from the group consisting of: C, E, W, F and H; and/or   (hm) S619 selected from the group consisting of: L, E and Q; and/or   (hn) V629 selected from the group consisting of: C, S, I, M and Q; and/or   (ho) D34 of M; and/or   (hp) P42 selected from the group consisting of: C and Q; and/or   (hq) I43 of V; and/or   (hr) K47 of R; and/or   (hs) A50 selected from the group consisting of: D and N; and/or   (ht) N55 of D; and/or   (hu) A58 of C; and/or   (hv) A62 of H; and/or   (hw) A64 of P; and/or   (hx) I68 of V; and/or   (hy) S72 selected from the group consisting of: C and Q; and/or   (hz) R73 of M; and/or   (ia) D75 selected from the group consisting of: H and R; and/or   (ib) P77 of D; and/or   (ic) T83 of V; and/or   (id) L90 selected from the group consisting of: I and P; and/or   (ie) S96 selected from the group consisting of: A and Y; and/or   (if) G98 selected from the group consisting of: Y and F; and/or   (ig) Y101 of F; and/or   (ih) T103 selected from the group consisting of: Q and V; and/or   (ii) I109 selected from the group consisting of: T and A; and/or   (ij) Q110 selected from the group consisting of: E and C; and/or   (ik) V113 selected from the group consisting of: E and L; and/or   (il) S115 of A; and/or   (im) V122 of L; and/or   (in) S123 selected from the group consisting of: A and P; and/or   (io) S126 of A; and/or   (ip) T128 of V; and/or   (iq) F129 of L; and/or   (ir) A146 of P; and/or   (is) T148 selected from the group consisting of: L and M; and/or   (it) E150 selected from the group consisting of: P and R; and/or   (iu) A165 of S; and/or   (iv) L166 selected from the group consisting of: M and C; and/or   (iw) Y169 selected from the group consisting of: E and F; and/or   (ix) A170 selected from the group consisting of: C and I; and/or   (iy) A182 of V; and/or   (iz) V185 selected from the group consisting of: N and Q; and/or   (ja) P188 of K; and/or   (jb) L194 of I; and/or   (jc) T197 selected from the group consisting of: A and G; and/or   (jd) F206 of Y; and/or   (je) F218 of W; and/or   (jf) S222 selected from the group consisting of: M and Q; and/or   (jg) H224 of A; and/or   (jh) A226 of S; and/or   (ji) Y232 of F; and/or   (jj) L237 of I; and/or   (jk) P249 of D; and/or   (jl) Q256 of F; and/or   (jm) A257 selected from the group consisting of: G and F; and/or   (jn) F258 of N; and/or   (jo) W259 of Y; and/or   (jp) Y265 of W; and/or   (jq) V267 selected from the group consisting of: K and R; and/or   (jr) S268 of C; and/or   (js) G272 of C; and/or   (jt) G273 of H; and/or   (ju) S277 of T; and/or   (jv) D280 of V; and/or   (jw) A281 of L; and/or   (jx) I284 selected from the group consisting of: L and V; and/or   (jx) A286 selected from the group consisting of: L and T; and/or   (jy) S287 selected from the group consisting of: C and Q; and/or   (jz) Q303 of E; and/or   (ka) S306 of C; and/or   (kb) E307 selected from the group consisting of: D and P; and/or   (kc) H313 of Y; and/or   (kd) Y316 of W; and/or   (ke) D318 of M; and/or   (kf) N322 selected from the group consisting of: H and C; and/or   (kg) V336 selected from the group consisting of: C and I; and/or   (kh) A337 of S; and/or   (ki) N348 of G; and/or   (kj) A355 selected from the group consisting of: S and C; and/or   (kk) N356 of S; and/or   (kl) A359 selected from the group consisting of: G and S; and/or   (km) L363 selected from the group consisting of: A and V; and/or   (kn) S375 selected from the group consisting of: A and L; and/or   (ko) T377 selected from the group consisting of: S and A; and/or   (kp) V378 selected from the group consisting of: L and T; and/or   (kq) L383 selected from the group consisting of: C and N; and/or   (kr) F385 of L; and/or   (ks) D388 selected from the group consisting of: A and C; and/or   (kt) T404 of Q; and/or   (ku) E421 selected from the group consisting of: C and Q; and/or   (kv) V422 of F; and/or   (kw) A424 selected from the group consisting of: C and Q; and/or   (kx) K425 selected from the group consisting of: M and R; and/or   (ky) Y426 of F; and/or   (kz) A432 selected from the group consisting of: D and S; and/or   (la) N440 of D; and/or   (lb) S446 of G; and/or   (lc) T451 of S; and/or   (ld) F457 of A; and/or   (le) R487 of S; and/or   (lf) I488 of V; and/or   (lg) V495 of I; and/or   (lh) A496 of P; and/or   (li) A497 selected from the group consisting of: Q and S; and/or   (lj) F502 of I; and/or   (lk) S504 of T; and/or   (ll) A513 selected from the group consisting of: D and G; and/or   (lm) P516 selected from the group consisting of: E and A; and/or   (ln) P520 of V; and/or   (lo) T521 selected from the group consisting of: W and S; and/or   (lp) S527 of F; and/or   (lq) V529 of W; and/or   (lr) T532 selected from the group consisting of: W and Y; and/or   (ls) N534 selected from the group consisting of: D and E; and/or   (lt) E535 of Q; and/or   (lu) T539 of S; and/or   (lv) E543 selected from the group consisting of: C and I; and/or   (lw) 1545 of V; and/or   (lx) V547 selected from the group consisting of: A and C; and/or   (ly) G549 of A; and/or   (lz) K571 selected from the group consisting of: R and T; and/or   (ma) L576 of K; and/or   (mb) I579 of M; and/or   (mc) I583 of M; and/or   (md) T586 selected from the group consisting of: L and R; and/or   (me) Y592 of H; and/or   (mf) 44Y594 selected from the group consisting of: M and H; and/or   (mg) G622 selected from the group consisting of: P and W; and/or   (mh) Q627 selected from the group consisting of: E and A; and/or   (mi) T628 of L; and/or   (mj) N630 of C; and/or   (mk) D631 selected from the group consisting of: Q and S; and/or   (ml) P470 selected from the group consisting of: K, S, A, C, E, F, G, I, L, M, N, Q, R, V and W,   
       where the position of each amino acid substitution corresponds to SEQ ID NO:3. 
     
     
         402 . The variant of  claim 399 , wherein the variant enzyme has at least 90% identity to SEQ ID NO: 4. 
     
     
         403 . A bacterial or fungal host cell comprising the variant enzyme of  claim 399 . 
     
     
         404 . The host cell of  claim 403 , wherein the fungal cell is:
 (a) a filamentous fungal cell selected from the group consisting of:  Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum, Penicillium, Humicola, Humicola insolens, Humicola grisea, Chrysosporium, Chrysosporium lucknowense, Myceliophthora thermophila, Gliocladium, Aspergillus, Fusarium, Neurospora, Hypocrea, Emericella, Aspergillus niger, Aspergillus awamori, Aspergillus aculeatus , and  Aspergillus nidulans ; or   (b) a yeast cell selected from the group consisting of:  Saccharomyces cervisiae, Saccharomycopsis fibuligera, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluveromyces lactis, Candida utilis, Candida albicans, Pichia stipitis, Pichia pastoris, Yarrowia hpolytica, Hansenula polymorpha, Phaffia rhodozyma, Arxula adeninivorans, Debaryomyces hansenii , and  Debaryomyces polymorphus.      
     
     
         405 . The host cell of  claim 403 , wherein the bacterial cell is  Zymomonas mobilis.    
     
     
         406 . The host cell of  claim 404 , wherein the host cell expresses the variant enzyme encoded by an isolated polynucleotide, vector, or expression vector. 
     
     
         407 . A method of producing a variant GA enzyme comprising culturing the host cell according to  claim 403 , in a suitable culture medium under suitable conditions to produce the variant. 
     
     
         408 . The method of  claim 407 , further comprising isolating the produced variant enzyme. 
     
     
         409 . A method for hydrolyzing a starch substrate, comprising contacting the starch substrate with the variant enzyme according to  claim 399 . 
     
     
         410 . The method of  claim 409 , wherein the method is selected from the group consisting of: a starch conversion process, an alcohol fermentation process, a direct starch to glucose process, and a simultaneous saccharification and fermentation (SSF) process. 
     
     
         411 . The method of  claim 409 , wherein the starch substrate is derived from a source selected from the group consisting of grains, grasses, tubers and roots and more specifically wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, potato, sweet potato, and tapioca.