Method for increasing the production of ethanol from corn fiber in a starch hydrolysis process

Abstract

Described are compositions and methods relating the use of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to increase the production of ethanol from a corn fiber product in a starch hydrolysis process.

Description

METHOD FOR INCREASING THE PRODUCTION OF ETHANOL FROM CORN FIBER IN A STARCH HYDROLYSIS PROCESS CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 62 / 434,883, filed December 15, 2016. TECHNICAL FIELD

[002] The present compositions and methods relate to the use of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to increase the production of ethanol from a corn fiber material derived from a predominantly corn kernel feedstock. BACKGROUND

[003] Many commercial fermentation process are designed to maximize the conversion of plant-derived complex carbohydrates into simple fermentable sugars. These sugars may be simultaneously or sequentially utilized by microorganisms to produce commercially-valuable biochemicals or biofuels. One well-known such process is the conversion of starch into glucose followed by yeast fermentation to produce ethanol. A commonly used feedstock is corn kernels, which may include some corn stover, resulting in a feedstock that is predominantly starch but contains a significant amount of corn fiber.

[004] Corn fiber is sometimes separated from corn carbohydrates by mechanical fractionation, resulting in essentially two (or more) different feedstocks having different chemical and enzymatic processing requirements. In addition, corn-ethanol processing generates by-products that include non-fermentable plant materials. There is considerable interest in treating corn fiber and corn processing by-products that contain corn fiber to extract more fermentable sugars, thereby improving the overall yield of commercially valuable biochemicals or biofuels. SUMMARY

[005] The present compositions and methods relate to the use of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to increase the production of ethanol from a corn fiber product in a starch hydrolysis process. Aspects and embodiments of the compositions and methods are described in the following, independently- numbered paragraphs. 1. In one aspect, a method for increasing the production of fermentable sugars in an ethanol fermentation process is provided, comprising: chemically-pretreating corn fiber material derived from a predominantly corn kernel feedstock; and contacting the pretreated corn fiber with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to release fermentable sugars from the corn fiber; wherein the contacting with \alpha-glucosidase releases fermentable sugars from the corn fiber material derived from a predominantly corn kernel feedstock that otherwise would not be available for fermentation. 2. In some embodiments of the method of paragraph 1, the corn fiber product is the stillage resulting from distillation of a fermentation product, or a derivative, thereof. 3. In some embodiments of the method of paragraph 1, the com fiber product is produced by physically separating corn fiber from starch in a substrate for ethanol fermentation. 4. In some embodiments of the method of paragraph 1, the corn fiber product is exposed to a temperature of at least 80°C prior to the contacting with α-glucosidase. 5. In some embodiments of the method of paragraph 1, the corn fiber product is subjected to an alcohol distillation step prior to the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase. 6. In some embodiments of the method of paragraph 1, the corn fiber product contains between about 1-12% corn fiber (wt / wt). 7. In some embodiments of the method of any of the preceding paragraphs, the pretreatment is acid pretreatment. 8. In some embodiments of the method of any of the preceding paragraphs, the pretreatment is alkaline pretreatment. 9. In some embodiments of the method of any of the preceding paragraphs, the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidase is a GH31 enzyme. 10. In some embodiments of the method of any of the preceding paragraphs, the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidase has at least 70% amino acid sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. 11. In some embodiments of the method of any of the preceding paragraphs, the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase is performed in the presence of one or more additional enzymes. 12. In another aspect, a method for increasing the production of fermentable sugars from chemically-pretreated stillage is provided, comprising: fermenting a predominantly corn kernel feedstock with an ethanolagen to produce ethanol; evaporating at least a portion of the ethanol and recovering stillage; chemically-pretreating the stillage; contacting the stillage with α-glucosidase to release fermentable sugars; fermenting the released fermentable sugars with an ethanolagen to produce additional ethanol; and recovering the additional ethanol; wherein the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase releases fermentable sugars from the corn fiber material derived from the predominantly corn kernel feedstock that otherwise would not be available for fermentation. 13. In some embodiments of the method of paragraph 12, the pretreatment is acid pretreatment. 14. In some embodiments of the method of paragraph 12, the pretreatment is alkaline pretreatment. 15. In some embodiments of the method of any of paragraphs 12-14, the corn fiber product contains between about 1-12% corn fiber (wt / wt). 16. In some embodiments of the method of any of paragraphs 12-15, the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase is a GH31 enzyme. 17. In some embodiments of the method of any of paragraphs 12-16, the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase has at least 70% amino acid sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. 18. In some embodiments of the method of any of paragraphs 12-17, the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase is performed in the presence of one or more additional enzymes. 19. In another aspect, a polypeptide having <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase activity is provided, selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having preferably at least 94% identity to the polypeptide of SEQ ID NO: 3; (b) a polypeptide encoded by a polynucleotide that hybridizes under preferably at least low stringency conditions, more preferably at least medium stringency conditions, even more preferably at least medium-high stringency conditions, most preferably at least high stringency, and even most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 7, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 7, or (iii) a full-length complementary strand of (i) or (ii); (c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 94% identity to the polypeptide coding sequence of SEQ ID NO: 3; (d) a variant comprising a substitution, deletion, and / or insertion of one or more (e.g., several) amino acids of the polypeptide of SEQ ID NO: 3; (e) a mature polypeptide produced by the processing of the polypeptide of SEQ ID NO: 6 by a signal peptidase or post translational modification during secretion from an expression host; and (f) a fragment of a polypeptide of (a), (b), (c), (d), or (e) that has <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase activity. 20. In another aspect, a polynucleotide comprising a nucleotide sequence that encodes the polypeptide of paragraph 19 is provided.

[006] These and other aspects and embodiments of present compositions and methods will be apparent from the description, including the accompanying Figures. BRIEF DESCRIPTION OF THE DRAWINGS

[007] Figure 1 is a plasmid map of pTTT-PvaGlu1.

[008] Figure 2 is a graph showing the amount of glucose (mg / mL) released from a whole stillage substrate using various amounts of different <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidases (<com:Math><semantics>μg / g<annotation encoding="application / x-tex">\mu g / g< / annotation>< / semantics>< / com:Math> dry solids) at pH 5 and 50°C for 24 hours. DETAILED DESCRIPTION 1. Introduction

[009] The present compositions and methods relate to an improvement on current methods that utilize enzymes to release fermentable sugars from plant-derived carbohydrates. Specifically, the compositions and methods involve the addition of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase, which has a complex substrate specificity, to corn fiber derived from a predominantly corn kernel feedstock that has been pretreated in preparation for enzymatic digestion. The present compositions and methods should not be confused with the addition of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to a cellulosic feedstock in a cellulose-based biofuel process.

[0010] Prior to describing the present compositions and methods in detail, the following terms are defined for clarity. Terms not defined should be accorded their ordinary meanings as used in the relevant art.

[0011] As used herein, an "α-glucosidase" (EC 3.2.1.20) is an enzyme capable of the hydrolysis of terminal, non-reducing, <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> (1-4)-linked -D-glucose residues from a substrate, with the resulting release of D-glucose. <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidases are not to be confused with <com:Math><semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>< / com:Math>-glucosidases (E.C. 3.2.1.21) or other carbohydrate-processing enzymes, including but not limited to <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- amylases (EC 3.2.1.1), β-amylases (EC 3.2.1.2), glucoamylases (EC 3.2.1.3), trehalases (EC 3.2.1.28), pullulanses (EC 3.2.1.41), and other enzymes having predominantly different substrate specificities.

[0012] As used herein, "background <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase activity," and similar terms, refer to enzyme activity that is not attributable to the addition of exogenous <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to plant fiber.

[0013] As used herein, "physical treatment," or similar expressions, refer to mechanical and / or thermal treatments, which may or may not result in chemical modifications, that modifying a fiber substrate to increase its suitability for digestion by enzymes, particularly <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase.

[0014] As used herein, "thermal pretreatment" refers to exposure to a temperature suitable to evaporate alcohol, particularly ethanol, as in the case of recovering fuel alcohol from a fermentation process, or to otherwise modify a fermentation substrate by heat, so as to modify the substrate to increase enzymatic digestion. Thermal pretreatment typically involves a temperature of at least 80°C.

[0015] As used herein, "chemical treatment" refers to non-enzymatic modification of a substrate, such as by acid or alkaline treatment (i.e., pH adjustment), exposure to oxidizing or reducing agents, and the like.

[0016] As used herein, a "predominantly corn kernel feedstock" is a feedstock containing at least 80%, at least 85%, at least 90%, at least 95%, at least 97% and even at least 98% corn kernels. The remaining material may include corn stover, other plant components, insects and the like.

[0017] As used herein, a "corn fiber product" is material produced as a result of processing a predominantly corn kernel-based substrate in a typical fuel ethanol processing plant, where the steps of starch hydrolysis and fermentation are performed to produce ethanol. An exemplary corn fiber product is whole stillage.

[0018] As used herein, "wet distillers' grains (WDG)" primarily contain unfermented grain residues, specifically protein, fiber, fat and up to 70% moisture.

[0019] As used herein, "dried distillers' grains with solubles (DDGS) is WDG that has been dried with the concentrated thin stillage (liquid remaining after boiling of the ethanol) to about 10-12 percent moisture. The fiber content can be up to about 5-50%.

[0020] As used herein, the singular articles "a," "an," and "the" encompass the plural referents unless the context clearly dictates otherwise. The following abbreviations / acronyms have the following meanings unless otherwise specified: EC enzyme commission CAZy carbohydrate active enzyme W / V weight / volume W / W weight / weight V / V volume / volume wt% weight percent <com:Math><semantics>∘C<annotation encoding="application / x-tex">^{\circ}C< / annotation>< / semantics>< / com:Math> degrees Centigrade g or gm gram μg microgram mg milligram kg kilogram μL and μl microliter mL and ml milliliter mm millimeter <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>m micrometer mol mole mmol millimole <com:Math><semantics>𝑴<annotation encoding="application / x-tex">\mathbf{M}< / annotation>< / semantics>< / com:Math> molar mM millimolar <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>M micromolar nm nanometer U unit ppm parts per million hr and h hour EtOH ethanol III. a-glucosidases

[0021] All embodiments of the present compositions and methods relate to the use of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidase to improving ethanol production from corn fiber derived from a predominantly corn kernel feedstock.

[0022] <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidases (EC 3.2.1.20) hydrolyze terminal, non-reducing <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-1,4- linked glucose residues in various substrates, releasing glucose. They degrade disaccharides and oligosaccharides quickly while polysaccharides are attacked more slowly. α-glucosidases display broad substrate specificity for <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosides, which include panose, isomaltose, isopanose, maltotriose, turanose, and maltose. Aside from being able to hydrolyze synthetic <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucoside and oligosaccharide substrates, <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidases also can hydrolyze <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucans such as soluble starch and glycogen.

[0023] <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> -glucosidases can be divided into three types based on substrate specificity. Type I <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidases hydrolyze heterogeneous substrates, such as aryl glucosides and sucrose, more efficiently than they hydrolyze maltose. Type II α-glucosidases prefer maltose and isomaltose as substrates, and have low activities toward aryl glucosides. Type III <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosdiases have the same substrate specificity as type II but can additionally hydrolyze polysaccharides such as amylose and starch.

[0024] <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase activity can be found in a wide variety of organisms. <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidases from bacteria, yeast, and insects typically belong to the GH13 class, and have four conserved sequence regions. α-glucosidases from plants, animals, molds, and two species of bacteria are members of GH31 class. Additional α-glucosidase of members of GH97 class.

[0025] In some embodiments of the present compositions and methods, the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase are of the GH31 class, as described in, e.g., Okuyama, M. et al. (2016) Cell. Mol. Life Sci. 73:2727-51. Exemplary GH31 enzymes are E1, E2, E3 and E4, having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, as shown, below:

[0026] E1 GH31 α-glucosidase (SEQ ID NO: 1): STTAPSQPQFTIPASADVGAQLIANIDDPQAADAQSVCPGYKASKVQHNSRGFTASLQLAGRP CNVYGTDVESLTLSVEYQDSDRLNIQILPTHVDSTNASWYFLSENLVPRPKASLNASVSQSDL FVSWSNEPSFNFKVIRKATGDALFSTEGTVLVYENQFIEFVTALPEEYNLYGLGEHITQFRLQ RNANLTIYPSDDGTPIDQNLYGQHPFYLDTRYYKGDRQNGSYIPVKSSEADASQDYISLSHGV FLRNSHGLEILLRSQKLIWRTLGGGIDLTFYSGPAPADVTRQYLTSTVGLPAMQQYNTLGFHQ CRWGYNNWSDLADVVANFEKFEIPLEYIWTDIDYMHGYRNFDNDQHRFSYSEGDEFLSKLHES GRYYVPIVDAALYIPNPENASDAYATYDRGAADDVFLKNPDGSLYIGAVWPGYTVFPDWHHPK AVDFWANELVIWSKKVAFDGVWYDMSEVSSFCVGSCGTGNLTLNPAHPSFLLPGEPGDIIYDY PEAFNITNATEAASASAGASSQAAATATTTSTSVSYLRTTPTPGVRNVEHPPYVINHDQEGHD LSVHAVSPNATHVDGVEEYDVHGLYGHQGLNATYQGLLEVWSHKRRPFIIGRSTFAGSGKWAG HWGGDNYSKWWSMYYSISQALSFSLFGIPMFGADTCGFNGNSDEELCNRWMQLSAFFPFYRNH NELSTIPQEPYRWASVIEATKSAMRIRYAILPYFYTLFDLAHTTGSTVMRALSWEFPNDPTLA AVETQFMVGPAIMVVPVLEPLVNTVKGVFPGVGHGEVWYDWYTQAAVDAKPGVNTTISAPLGH IPVYVRGGNILPMQEPALTTREARQTPWALLAALGSNGTASGQLYLDDGESIYPNATLHVDFT ASRSSLRSSAQGRWKERNPLANVTVLGVNKEPSAVTLNGQAVFPGSVTYNSTSQVLFVGGLQN LTKGGAWAENWVLEW

[0027] E2 GH31 <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase (SEQ ID NO: 2): AAIVRRNGASPSCPGYKASNVKTVDGEIVSADLNLAGPACNVYGTDLDDLKLQVEYQSEQRLH VKIYDAAEQVYQVPTAVLPRPSSANIPPAKSDLKFSMTNDPFSFTIKRRSNGEILFDTSGHPL IFESQYLGLRTKLPDSPNIYGLGEHTGSFRLPTKNYTRTLWSRDAYGTPKDTNLYGNHPVYFD YRGSNGTHGVFLLNSNGMDVDIDVDSDGQYLQYNTLGGVLDFYFLSGPDPKAVATQYAETVGK PVMMPYWGFGFHNCRYGYQDIYEVAEIIANYSAANIPLETQWTDIDYMDLRKVFTLDPYRYPL KLVQEVVSYLHKHNQHYIMMVDPAVAYQNYSAFNNGVAADAFLKFSNGSIYQGVVWPGPTAFP DWFAPQTQEFWNSEFSTFFDPAHGVDIDALWIDMNEASNFCDFPCSNPAAYAAANGDPPTPPP VRLSPPRPIPGFGPDFQPTCVATVSFDCDAQTYFGENILILGNSTTLGAGDVHMAPVMSANNY PIWQLTVQMPPNGTFSYQYVRKESDGSYIYEQTNRTVTTGDCTSGTLKVSDTITTSSGPHKRS ELRPLVRSPFPAEDLTRRQSGSMLGLPNRNLLNPPYTIHNAAGNLSEKTINTDLIHAGGYAEY DTHNLYGTMMSATSREAMLNRRPAVRPLVITRSTFAGAGRQVGHWLGDNFADWDHYRWTIAEL QEFAALFQIPMVGSDICGYDGNTTDNLCSRWVFLGAFSPFFRDHSDNQSPPHELYRTPQIAAA ARAAIDIRYRLLDYAYTVLWTQTQTGAPMLNPMFFEYPADSNTADLQYQFFWGDSIMVAPVTD NDSTTVNVYFPKDQFYDFYTGAPVSGEGNTVTLTDVGFDTIPLYFKGGSIVPMRVRSANTTAE LRQQDFVVVIAPDSHGDATGQLYLDDGESINQPHTSEIQFSYRGGHFSMTGKFDYDPGNVVIS QITLLGADGAGKGGSYNSTTKVATYKVNAKLTGKFEASLH

[0028] E3 GH31 α-glucosidase (SEQ ID NO: 3), also referred to, herein, as PvaGlu1: AAISPAATSSTASWGPVFTVPASADEGAQLIANINDPQSVNAQTVCPGYVASNVQNNEFGFTA TLNLAGKACNVYGTDVDSLNLTVQYQASDRLNINIGPAHVDASNESWYILSDDLVYKPTVDGT ASISQSDLLVSWSNEPSFNFKVIRKANKDVLFNTEGTVLVYENQFIEFVSALPENYNLYGLGE RIHGLRLGNNFTATTYAADAADPIDANIYGTHPFYLDTRYYEVDSKQGTYTLLTTNETDQSKE YTSFSHGVFLRNAHGQEVLLRPEGITWRTLGGSIDLYFYSGPTQADVTRSYQTSTVGLPTMQQ YYTFGYHQCRWGYQNWSVMADVVSSFAKFQIPLETIWSDIDYMNAYRDFENDPIRFSYSEGAE FLGQLHENGQHWVPIVDSAIYIPNSENASDAYDVYTRGEADGVWMTNPDGSLYIGAVWPGYTV FPDWHNPKAHEFWSNEIATWHQKVAFDGIWIDMSEVSSFCVGSCGTGNLTLNPVHPSFLLPGE PGAVIYDYPESFNVTNSTEAASASASVSQAAATASASASTTTSYLRTTPTPGVRDVNHPPYV INNVQPGHDLAVHAVSPNATHIDGVSEYDVHNLWGYQILNATYHGLLKVWEDKRPFIIGRSTF AGSGKWAGHWGGDNTSLWAYMFFSIPQALSFSLFGIPMFGVDTCGFNGNSDEELCNRWMQLSA FFPFYRNHNVLSAISQEPYVWASVIDASKAAMKIRYALLPYIYTLFYLAHTTGSTVMRAVSWE FPNDPSLAAIDTQFLLGPSLMVVPVLEPQVDYVKGVFPGVGNGEVWYDWYTQSVFDAKPGVNT TISAPLGHIPVFVRGGSILPMQEPALTTRDARKTPWALLTALGGNGTASGQLYIDDGESITPN ATLNVDFVASNSNLVASPRGSWVEKNPLANVTVLGVPTAPSSVTFNGAAVPRASVAYNSTSKT LFVGGLQDFTKTGAWADKWVLKW

[0029] E4 GH31 <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase (SEQ ID NO: 4): AATQTSSSAYVQTTLESSVDVGANLIANIDDPEAINAQSACPGYRASNVQNTSRGWTATLKLA GKACNVYGTDVESLNFTLEYLSSTRVNIQITPSHVDSSNASWYHLSEDVVPRPKADKNASAKD SHFEVSWSNEPSFGFKVARKATGDVLFNTIGSKLVYENQFIEFVTALPDDYNLYGLGEHIQQL RLLKNSTFTLYAADTGDPVDLNTYGSHAFYLDTRYYEVNDKGSHTLVSSDQATTSKNYVSYSH GVFLRNAHGQEILLGTGKLTWRTIGGSIDLTLYAGPTQTEVTKDYQLSTIGLPAMQQYFTFGY HQCRWGYTNWSEVEDVVANFQKFEIPLENIWNDIDYMHGYRDFDNDQNRYSYEEGAVFLEKLH KAGIHYIPIVDSALYIPDPNNASDAYDTYTRGAELDVFLKNPDGSTYIGAVWPGYTVFADWHH PKAGDFWANELVTWHERVAFDGIWIDMNEVSSFCVGSCGSGKLSQNPVHPPFSLPGEPGNIIY DYPEGFNATNSTEAASASAASASQASAAAATGQSAATTTTPYLRTTPTPGVRDVNHPPYVINH AQTGHDLAVHAVSPNATHSDGVQEYDVHSLYGHSIIRATYEGLLKVFPEKRPFIIGRSTFAGT GKWAGHWGGDNNSKWSYMFWSIPQALQFSLFGVPMFGVDTCGFNGNTDEELCNRWMQLSAFFP FYRNHNVLSAISQEPYRWASVAEASKAAMKIRYAILPYMYTLFQQAHTTGSTVMRALAWEFPN DPSLAAVDTOFLLGPSIMVVPVLAPOATSVKGVFPGIKOGEVWYDWYTOTAVDAOPHVNTTIA APLGHIPVFVRGDSVLPMQEPALTTRDARNTPWTILAALGDKGTASGELYLDDGESLEPNATL TVTFKATKSSLSAEPRGNWQEKNALGNVNVLGVAHKPNGVTLNGKAVPAASVHYNSTSQVLSV TDLQKMTSKGAFANNWVLKW

[0030] Additional <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase include but are not limited to those having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or even at least 99% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and / or SEQ ID NO: 4.

[0031] In some embodiments, \alpha-glucosidase is added to corn fiber in an amount of less than 100, less than 70, less than 50, less than 40, less than 30, less than 25, or even less than 20 µg enzyme per g fiber. In some embodiments, the increase in glucose release from corn fiber is at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or even at least 70%, compared to the amount of glucose released under equivalent conditions in the absence of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase. In some embodiments, the increase in ethanol obtained from corn fiber is at least 0.1%, at least 0.2%, at least 0.5%, at least 1.0%, or even at least 2.0% compared to the amount of produced under equivalent conditions in the absence of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase. The absence of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase encompasses conditions where a minor amount of background <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase may be present, i.e., less than 5, less than 3, less than 2, or even less than 1 µg enzyme per g fiber. IV. Corn fiber materials from predominantly corn kernel feedstocks

[0032] The present compositions and methods involve the addition of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to corn fiber materials derived from a predominantly corn kernel feedstock that has been pretreated in preparation for enzymatic digestion.

[0033] In some embodiments, the compositions and methods relate to producing additional ethanol from a stillage by-product of an ethanol production facility that utilizes predominantly corn kernel feedstock. Whole stillage, or simply stillage, refers to the solid mixture that remains after distilling ethanol and other low molecular components from a fermentation mixture (or beer). Typically, whole stillage falls to the bottom of distillation columns and is eventually transferred to one or more holding tanks to make room for a fresh batch of beer. Whole stillage may then be processed, e.g., via centrifugation, to separate it into mostly-liquid thin stillage and wet distillers' grain (or wet cake). Wet cake may be sold as animal feed directly or dried to produce distillers' dry grain (DDG). Syrup obtained from the drying the thin stillage may be added to the DDG to produce DDG with solubles (DDGS), which is a preferred form of animal feed.

[0034] The present compositions and methods are directed at using whole stillage for the production of additional fermentable sugars. Derivatives of whole stillage, such as thin stillage, wet cake, DDG, and DDGS may also be used, although the additional processing steps are deemed unnecessary.

[0035] An exemplary process for the use of whole stillage for the production of fermentable sugars is described in US Patent No. 8, 633,033 (Assigned to Quad County Corn Processors, Holstein, Iowa, USA). However, the importance and commercial value of using <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to release fermentable carbohydrates from pretreated corn fiber in a starch-hydrolysis process are previously undescribed.

[0036] In other embodiments, the present compositions and methods relate to producing additional ethanol from corn fiber materials that are mechanically separated from carbohydrate material in a predominantly corn kernel feedstock at the beginning of a corn starch production process. The corn fibers are typically separated mechanically, primarily to remove unwanted fiber from carbohydrate portion of the feedstock, which fibers can lead to fouling and clogging equipment and increase the demand for material handling. Following chemical pretreatment of the fibers to render them amenable to enzymatic digestion, the fibers are subsequently treated with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase.

[0037] In some embodiments, the amount of fiber present in the corn fiber materials derived from a predominantly corn kernel feedstock is less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, or even less than about 10% of the total dry weight of the substrate. Exemplary amounts are about 1-30%, about 1-25%, about 1- 20%, about 1-15%, and about 1-12%. <com:Math><semantics>𝑽•<annotation encoding="application / x-tex">\mathbf{V}_{\bullet}< / annotation>< / semantics>< / com:Math> Chemical, thermal, or mechanical pretreatment

[0038] The present compositions and methods require some form of chemical, thermal, or mechanical pretreatment, or combination of, the corn fibers to prepare them for enzymatic digestion. Exemplary treatments are with dilute acid, optionally with heat and pressure, or more concentrated acid, typically at lower temperatures and pressures. Alternative treatments are with dilute ammonia. The optimum pH for α-glucosidase treatment largely depend on the optimum pH for the enzyme and is not believed to be critical.

[0039] In another embodiment, heating of the fiber substrate is performed wherein high pressure steam is injected into the substrate mixture to increase its temperature to about 215- 300°F (i.e., about 102-150°C), preferably in a container or vessel able to maintain a pressure above the vapor pressure of water in order to avoid boiling the sample. Heating by steam injection is beneficial because it aids in disruption of the fiber structure to improve subsequent enzymatic hydrolysis.

[0040] In another embodiment, mechanical pretreatment is used to produce uniform particle size of less than about 1,600 microns (µm). In some embodiments, at least 95% of the pretreated biomass particles are mechanically treated to have a particle size of from about 100 microns to about 800 microns, or a particle size from about 100 microns to about 500 microns. Such pretreated biomass particles can be generated by, for example, using a hammer mill or a colloid mill. EXAMPLES EXAMPLE 1. Cloning of Paecilomyces variotii a-glucosidase (PvaGlu1)

[0041] Paecilomyces variotii NRRL1115 strain was selected as a potential source for various enzymes, useful for industrial applications. The entire genome of the P. variotii NRRL1115 strain was sequenced and the nucleotide sequence of a putative <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase, designated "PvaGlu1" was identified by sequence identity. The gene encoding PvaGlu1 is set forth as SEQ ID NO:5: ATGGGCGCTTCACCCACTACATGCTCGCTTCCGCTTGGCTGCCTCTGACCCTGGGCGCCCC ATCTCTCCCGCTGCTACCAGCAGCACCGCTTCGTGGGGCCCCGTTTTCACCGTCCCCGCCTCC GCCGACGAGGGCGCTCAGCTCATTGCCAACATTAACGACCCCCAGTCTGTTAACGCCCAGACC <Formule mathématique disponible dans le document PDF, Math available in the PDF document>\operatorname{GTTTGCCCCGGCTACGTTGCTTCTAACGTCCAGAACAACGAGTTCGGCTTCACCGCCACCCTG <com:Math><semantics>𝙰𝙰𝙲𝙲𝚃𝙲𝙶𝙲𝙲𝙶𝙶𝙲𝙰𝙰𝙶𝙶𝙲𝚃𝚃𝙶𝙲𝙰𝙰𝙲𝙶𝚃𝚃𝚃𝙰𝙲𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝙰𝙲𝙶𝚃𝙲𝙶𝙰𝚃𝙰𝙶𝙲𝙲𝚃𝙶𝙰𝙰𝙲𝙲𝚃𝙶𝙰𝙲𝙲𝙶𝚃𝙲<annotation encoding="application / x-tex">\texttt{AACCTCGCCGGCAAGGCTTGCAACGTTTACGGCACCGACGTCGATAGCCTGAACCTGACCGTC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝖢𝖠𝖦𝖳𝖠𝖢𝖢𝖠𝖦𝖦𝖢𝖦𝖠𝖦𝖳𝖦𝖠𝖢𝖢𝖦𝖠𝖢𝖳𝖦𝖠𝖠𝖢𝖠𝖳𝖳𝖠𝖠𝖢𝖠𝖳𝖳𝖦𝖦𝖢𝖢𝖢𝖢𝖦𝖢𝖢𝖢𝖠𝖢𝖦𝖳𝖳𝖦𝖠𝖢𝖦𝖢𝖢𝖳𝖢𝖢𝖠𝖠𝖢<annotation encoding="application / x-tex">\textsf{CAGTACCAGGCGAGTGACCGACTGAACATTAACATTGGCCCCGCCCACGTTGACGCCTCCAAC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝙰𝙶𝚃𝙲𝚃𝚃𝙶𝙶𝚃𝙰𝙲𝙰𝚃𝚃𝙲𝚃𝙶𝙰𝙶𝙲𝙶𝙰𝙲𝙶𝙰𝙲𝙲𝚃𝙶𝙶𝚃𝚃𝚃𝙰𝙲𝙰𝙰𝙶𝙲𝙲𝚃𝙰𝙲𝙲𝙶𝚃𝙶𝙶𝙰𝙲𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝙲𝚃𝚃𝙲𝚃<annotation encoding="application / x-tex">\texttt{GAGTCTTGGTACATTCTGAGCGACGACCTGGTTTACAAGCCTACCGTGGACGGCACCGCTTCT}< / annotation>< / semantics>< / com:Math> ATCTCTCAGTCTGACCTGCTGGTTAGCTGGTCTAACGAGCCTTCTTTCAACTTCAAGGTTATT <com:Math><semantics>𝙲𝙶𝚃𝙰𝙰𝙶𝙶𝙲𝚃𝙰𝙰𝙲𝙰𝙰𝙶𝙶𝙰𝙲𝙶𝚃𝙲𝙲𝚃𝙶𝚃𝚃𝙲𝙰𝙰𝙲𝙰𝙲𝙲𝙶𝙰𝙶𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝚃𝙲𝙲𝚃𝙲𝙶𝚃𝚃𝚃𝙰𝙲𝙶𝙰𝙶𝙰𝙰𝙲𝙲𝙰𝙶<annotation encoding="application / x-tex">\texttt{CGTAAGGCTAACAAGGACGTCCTGTTCAACACCGAGGGCACCGTCCTCGTTTACGAGAACCAG}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>mTTCATCGAGTTCGTCAGCGCTCTCCCTGAGAACTACAACCTGTACGGCCTGGGCGAGCGCATT<annotation encoding="application / x-tex">{m TTCATCGAGTTCGTCAGCGCTCTCCCTGAGAACTACAACCTGTACGGCCTGGGCGAGCGCATT}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝙰𝙲𝙶𝙶𝙲𝙲𝚃𝙶𝙲𝙶𝙰𝙲𝚃𝙲𝙶𝙶𝙲𝙰𝙰𝙲𝙰𝙰𝙲𝚃𝚃𝙲𝙰𝙲𝙲𝙶𝙲𝙲𝙰𝙲𝙲𝙰𝙲𝙲𝚃𝙰𝙲𝙶𝙲𝙲𝙶𝙲𝙲𝙶𝙰𝙲𝙶𝙲𝙲𝙶𝙰𝙲𝙲𝙲𝙲𝙲<annotation encoding="application / x-tex">\texttt{CACGGCCTGCGACTCGGCAACAACTTCACCGCCACCACCTACGCCGCCGACGCCGACCCCC}< / annotation>< / semantics>< / com:Math> ATCGACGCTAACATATATGGCACCCACCCTTTCTACCTGGACACCCGATACTACGAGGTTGAC <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{ t TCCAAGCAGGGCACCTACACCCTGCTGACCACCAACGAGACCGACC <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{ t TCTTTCTCTCACGGCGTTTTCCTCCGAAACGCTCACGGCCAGGAAGTTCTGCTGCGCCCTGAG <com:Math><semantics>𝙶𝙶𝙲𝙰𝚃𝙲𝙰𝙲𝙲𝚃𝙶𝙶𝙲𝙶𝙰𝙰𝙲𝙲𝙲𝚃𝙶𝙶𝙶𝙲𝙶𝙶𝙲𝙰𝙶𝙲𝙰𝚃𝚃𝙶𝙰𝙲𝙲𝚃𝙶𝚃𝙰𝙲𝚃𝚃𝙲𝚃𝙰𝙲𝚃𝙲𝙲𝙶𝙶𝙲𝙲𝙲𝚃𝙰𝙲𝙲𝙲𝙰𝙶<annotation encoding="application / x-tex">\texttt{GGCATCACCTGGCGAACCCTGGGCGGCAGCATTGACCTGTACTTCTACTCCGGCCCTACCCAG}< / annotation>< / semantics>< / com:Math> <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{\tt GCTGACGTCACCCGATCTTACCAGACCAGCACCGTTGGCCTGCCT ACCTTCGGCTACCACCAGTGCCGATGGGGCTACCAGAACTGGTCTGTCATGGCTGACGTTGTT <com:Math><semantics>𝖠𝖦𝖢𝖳𝖢𝖳𝖳𝖳𝖢𝖦𝖢𝖢𝖠𝖠𝖦𝖳𝖳𝖢𝖢𝖠𝖦𝖠𝖳𝖳𝖢𝖢𝖳𝖢𝖳𝖦𝖦𝖠𝖦𝖠𝖢𝖢𝖠𝖳𝖳𝖳𝖦𝖦𝖠𝖦𝖢𝖦𝖠𝖢𝖠𝖳𝖢𝖦𝖠𝖢𝖳𝖠𝖢𝖠𝖳𝖦𝖠𝖠𝖢<annotation encoding="application / x-tex">\textsf{AGCTCTTTCGCCAAGTTCCAGATTCCTCTGGAGACCATTTGGAGCGACATCGACTACATGAAC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝙲𝚃𝚃𝙰𝙲𝙲𝙶𝙰𝙶𝙰𝙲𝚃𝚃𝙲𝙶𝙰𝙶𝙰𝙰𝙲𝙶𝙰𝙲𝙲𝙲𝚃𝙰𝚃𝙲𝙲𝙶𝙰𝚃𝚃𝙲𝚃𝙲𝚃𝚃𝙰𝙲𝚃𝙲𝚃𝙶𝙰𝙶𝙶𝙶𝙲𝙶𝙲𝚃𝙶𝙰𝙶𝚃𝚃𝙲𝙲𝚃𝙲<annotation encoding="application / x-tex">\texttt{GCTTACCGAGACTTCGAGAACGACCCTATCCGATTCTCTTACTCTGAGGGCGCTGAGTTCCTC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝙶𝙲𝙲𝙰𝙶𝙲𝚃𝙶𝙲𝙰𝙲𝙶𝙰𝙶𝙰𝙰𝙲𝙶𝙶𝙲𝙲𝙰𝙶𝙲𝙰𝙲𝚃𝙶𝙶𝙶𝚃𝚃𝙲𝙲𝚃𝙰𝚃𝚃𝙶𝚃𝚃𝙶𝙰𝙲𝚃𝙲𝙲𝙶𝙲𝙲𝙰𝚃𝙲𝚃𝙰𝙲𝙰𝚃𝙲𝙲𝙲𝚃<annotation encoding="application / x-tex">\texttt{GGCCAGCTGCACGAGAACGGCCAGCACTGGGTTCCTATTGTTGACTCCGCCATCTACATCCCT}< / annotation>< / semantics>< / com:Math> AACAGCGAGAACGCCAGCGACGCTTACGACGTTTACACCCGAGGCGAGGCCGACGGCGTTTGG <com:Math><semantics>mATGACCAACCCGACGGCAGCCTGTACATTGGCGCCGTTTGGCCCGGCTACACCGTTTTCCCCC<annotation encoding="application / x-tex">{m ATGACCAACCCGACGGCAGCCTGTACATTGGCGCCGTTTGGCCCGGCTACACCGTTTTCCCCC}< / annotation>< / semantics>< / com:Math> GACTGGCACAACCCTAAGGCTCACGAGTTCTGGTCTAACGAGATTGCTACCTGGCACCAGAAG <Formule mathématique disponible dans le document PDF, Math available in the PDF document>$\tt GTCGCTTTCGACGGCATTTGGATTGACATGTCTGAGGTCAGCTCTTTCTGCGTTGGCTCTTGC$ <com:Math><semantics>𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝙶𝙲𝙰𝙰𝙲𝙲𝚃𝙶𝙰𝙲𝙲𝙲𝚃𝙲𝙰𝙰𝙲𝙲𝙲𝙲𝙶𝚃𝙲𝙲𝙰𝙲𝙲𝙲𝚃𝚃𝙲𝚃𝚃𝚃𝙲𝙲𝚃𝙶𝙲𝚃𝙲𝙲𝙲𝙲𝙶𝙶𝙲𝙶𝙰𝙶𝙲𝙲𝙲𝙶𝙶𝙲<annotation encoding="application / x-tex">\texttt{GGCACCGGCAACCTGACCCTCAACCCCGTCCACCCTTCTTTCCTGCTCCCCGGCGAGCCCGGC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝙲𝚃𝙶𝚃𝚃𝙰𝚃𝙰𝚃𝙰𝚃𝙶𝙰𝙲𝚃𝙰𝙲𝙲𝙲𝚃𝙶𝙰𝙶𝚃𝙲𝚃𝚃𝚃𝙲𝙰𝙰𝙲𝙶𝚃𝙲𝙰𝙲𝙲𝙰𝙰𝙲𝚃𝙲𝚃𝙰𝙲𝙲𝙶𝙰𝙶𝙶𝙲𝚃𝙶𝙲𝚃𝚃𝙲𝚃𝙶𝙲𝚃<annotation encoding="application / x-tex">\texttt{GCTGTTATATATGACTACCCTGAGTCTTTCAACGTCACCAACTCTACCGAGGCTGCTTCTGCT}< / annotation>< / semantics>< / com:Math> TCAGCCGCATCGGTCTCCCAGGCTGCTGCTACCGCTTCTGCCTCTGCCTCAACCACCACCTCT <com:Math><semantics>tTACCTGCGAACCACCCCTACCCCCGGCGTCCGAGACGTTAACCACCCTCCTTACGTTATTAAC<annotation encoding="application / x-tex">{ t TACCTGCGAACCACCCCTACCCCCGGCGTCCGAGACGTTAACCACCCTCCTTACGTTATTAAC}< / annotation>< / semantics>< / com:Math> AACGTCCAGCCTGGCCACGACCTCGCCGTCCACGCCGTTAGCCCCAACGCTACCCACATTGAC GGCGTTAGCGAGTACGACGTCCACAACCTGTGGGGCTACCAGATTCTGAACGCTACCTAC <com:Math><semantics>𝙶𝙶𝙲𝙲𝚃𝙲𝙲𝚃𝙲𝙰𝙰𝙶𝙶𝚃𝚃𝚃𝙶𝙶𝙶𝙰𝙶𝙶𝙰𝙲𝙰𝙰𝙶𝙲𝙶𝙲𝙲𝙲𝚃𝚃𝚃𝙲𝙰𝚃𝚃𝙰𝚃𝚃𝙶𝙶𝙲𝙲𝙶𝙰𝙰𝙶𝙲𝙰𝙲𝙲𝚃𝚃𝙲𝙶𝙲𝚃𝙶𝙶𝙲<annotation encoding="application / x-tex">\texttt{GGCCTCCTCAAGGTTTGGGAGGACAAGCGCCCTTTCATTATTGGCCGAAGCACCTTCGCTGGC}< / annotation>< / semantics>< / com:Math> AGCGGCAAGTGGGCTGGCCACTGGGGCGGCGACAACACCTCTCTGTGGGGCTTACATGTTCTTC <com:Math><semantics>𝙰𝙶𝙲𝙰𝚃𝚃𝙲𝙲𝚃𝙲𝙰𝙶𝙶𝙲𝚃𝙲𝚃𝙶𝚃𝙲𝚃𝚃𝚃𝙲𝙰𝙶𝙲𝙲𝚃𝙶𝚃𝚃𝙲𝙶𝙶𝙲𝙰𝚃𝙲𝙲𝙲𝙲𝙰𝚃𝙶𝚃𝚃𝙲𝙶𝙶𝙲𝙶𝚃𝚃𝙶𝙰𝙲𝙰𝙲𝙲𝚃𝙶𝙲<annotation encoding="application / x-tex">\texttt{AGCATTCCTCAGGCTCTGTCTTTCAGCCTGTTCGGCATCCCCATGTTCGGCGTTGACACCTGC}< / annotation>< / semantics>< / com:Math> <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{\tt GGCTTCAACGGCAACTCCGACGAAGAACTGTGCAACCGATGGATG <com:Math><semantics>𝙲𝙲𝚃𝚃𝚃𝙲𝚃𝙰𝙲𝙲𝙶𝙰𝙰𝙰𝙲𝙲𝙰𝙲𝙰𝙰𝙲𝙶𝚃𝙲𝙲𝚃𝙲𝙰𝙶𝙲𝙶𝙲𝚃𝙰𝚃𝚃𝙰𝙶𝙲𝙲𝙰𝙰𝙶𝙰𝙰𝙲𝙲𝚃𝚃𝙰𝙲𝙶𝚃𝚃𝚃𝙶𝙶𝙶𝙲𝙲𝚃𝙲𝙲<annotation encoding="application / x-tex">\texttt{CCTTTCTACCGAAACCACAACGTCCTCAGCGCTATTAGCCAAGAACCTTACGTTTGGGCCTCC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝚃𝙲𝙰𝚃𝚃𝙶𝙰𝙲𝙶𝙲𝚃𝚃𝙲𝚃𝙰𝙰𝙶𝙶𝙲𝙲𝙶𝙲𝙲𝙰𝚃𝙶𝙰𝙰𝙶𝙰𝚃𝚃𝙲𝙶𝙰𝚃𝙰𝙲𝙶𝙲𝚃𝙲𝚃𝙶𝙲𝚃𝙶𝙲𝙲𝚃𝚃𝙰𝙲𝙰𝚃𝙰𝚃𝙰𝙲𝙰𝙲𝙲<annotation encoding="application / x-tex">\texttt{GTCATTGACGCTTCTAAGGCCGCCATGAAGATTCGATACGCTCTGCTGCCTTACATATACACC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝚃𝙶𝚃𝚃𝙲𝚃𝙰𝙲𝙲𝚃𝙲𝙶𝙲𝚃𝙲𝙰𝙲𝙰𝙲𝙲𝙰𝙲𝙲𝙶𝙶𝙲𝙰𝙶𝙲𝙰𝙲𝙲𝙶𝚃𝙲𝙰𝚃𝙶𝙲𝙶𝙰𝙶𝙲𝚃𝙶𝚃𝙲𝚃𝙲𝚃𝚃𝙶𝙶𝙶𝙰𝙶𝚃𝚃𝙲𝙲𝙲𝚃<annotation encoding="application / x-tex">\texttt{CTGTTCTACCTCGCTCACACCACCGGCAGCACCGTCATGCGAGCTGTCTCTTGGGAGTTCCCT}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙰𝙰𝙲𝙶𝙰𝙲𝙲𝙲𝚃𝙰𝙶𝙲𝙲𝚃𝙲𝙶𝙲𝙲𝙶𝙲𝙲𝙰𝚃𝚃𝙶𝙰𝙲𝙰𝙲𝙲𝙲𝙰𝙶𝚃𝚃𝙲𝙲𝚃𝙲𝙲𝚃𝙶𝙶𝙶𝙲𝙲𝙲𝚃𝙰𝙶𝙲𝙲𝚃𝙲𝙰𝚃𝙶𝙶𝚃𝚃𝙶𝚃𝙲<annotation encoding="application / x-tex">\texttt{AACGACCCTAGCCTCGCCGCCATTGACACCCAGTTCCTCCTGGGCCCTAGCCTCATGGTTGTC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝙲𝙲𝙶𝚃𝙲𝙲𝚃𝙶𝙶𝙰𝙶𝙲𝙲𝚃𝙲𝙰𝙶𝙶𝚃𝚃𝙶𝙰𝙲𝚃𝙰𝙲𝙶𝚃𝚃𝙰𝙰𝙶𝙶𝙶𝙲𝙶𝚃𝚃𝚃𝚃𝙲𝙲𝙲𝙲𝙶𝙶𝙲𝙶𝚃𝚃𝙶𝙶𝙲𝙰𝙰𝙲𝙶𝙶𝙲𝙶𝙰𝙶<annotation encoding="application / x-tex">\texttt{CCCGTCCTGGAGCCTCAGGTTGACTACGTTAAGGGCGTTTTCCCCGGCGTTGGCAACGGCGAG}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝚃𝚃𝚃𝙶𝙶𝚃𝙰𝙲𝙶𝙰𝙲𝚃𝙶𝙶𝚃𝙰𝙲𝙰𝙲𝙲𝙲𝙰𝙶𝚃𝙲𝚃𝙶𝚃𝚃𝚃𝚃𝙲𝙶𝙰𝙲𝙶𝙲𝙲𝙰𝙰𝙶𝙲𝙲𝙲𝙶𝙶𝙲𝙶𝚃𝚃𝙰𝙰𝙲𝙰𝙲𝙲𝙰𝙲𝙲𝙰𝚃𝙲<annotation encoding="application / x-tex">\texttt{GTTTGGTACGACTGGTACACCCAGTCTGTTTTCGACGCCAAGCCCGGCGTTAACACCACCATC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙰𝙶𝙲𝙶𝙲𝚃𝙲𝙲𝚃𝙲𝚃𝙶𝙶𝙲𝙲𝙰𝙲𝙰𝚃𝙲𝙲𝙲𝙲𝙶𝚃𝚃𝚃𝚃𝙲𝙶𝚃𝙲𝙲𝙶𝙰𝙶𝙶𝙲𝙶𝙶𝙲𝙰𝙶𝙲𝙰𝚃𝚃𝙲𝚃𝙶𝙲𝙲𝚃𝙰𝚃𝙶𝙲𝙰𝙰𝙶𝙰𝙶<annotation encoding="application / x-tex">\texttt{AGCGCTCCTCTGGCCACATCCCCGTTTTCGTCCGAGGCGGCAGCATTCTGCCTATGCAAGAG}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝙲𝙲𝙶𝙲𝚃𝙲𝚃𝙶𝙰𝙲𝙲𝙰𝙲𝙲𝙲𝙶𝙲𝙶𝙰𝙲𝙶𝙲𝚃𝙲𝙶𝙰𝙰𝙰𝙶𝙰𝙲𝙲𝙲𝙲𝚃𝚃𝙶𝙶𝙶𝙲𝚃𝙲𝚃𝙲𝙲𝚃𝙶𝙰𝙲𝙲𝙶𝙲𝚃𝙲𝚃𝙶𝙶𝙶𝙲𝙶𝙶𝙲<annotation encoding="application / x-tex">\texttt{CCCGCTCTGACCACCCGCGACGCTCGAAAGACCCCTTGGGCTCTCCTGACCGCTCTGGGCGGC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙰𝙰𝙲𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝙲𝚃𝚃𝙲𝚃𝙶𝙶𝙲𝙲𝙰𝙶𝙲𝚃𝙶𝚃𝙰𝙲𝙰𝚃𝙲𝙶𝙰𝙲𝙶𝙰𝙲𝙶𝙶𝙲𝙶𝙰𝙶𝚃𝙲𝚃𝙰𝚃𝚃𝙰𝙲𝙲𝙲𝙲𝚃𝙰𝙰𝙲𝙶𝙲𝙲𝙰𝙲𝙲<annotation encoding="application / x-tex">\texttt{AACGGCACCGCTTCTGGCCAGCTGTACATCGACGACGGCGAGTCTATTACCCCTAACGCCACC}< / annotation>< / semantics>< / com:Math> CTGAACGTGGACTTCGTTGCTTCCAACTCTAACCTCGTTGCGTCACCTCGGGGCTCTTGGGTT <com:Math><semantics>𝖦𝖠𝖦𝖠𝖠𝖦𝖠𝖠𝖢𝖢𝖢𝖳𝖢𝖳𝖢𝖦𝖢𝖳𝖠𝖠𝖢𝖦𝖳𝖳𝖠𝖢𝖢𝖦𝖳𝖢𝖢𝖳𝖢𝖦𝖦𝖢𝖦𝖳𝖢𝖢𝖢𝖳𝖠𝖢𝖢𝖦𝖢𝖳𝖢𝖢𝖳𝖠𝖦𝖢𝖳𝖢𝖢𝖦𝖳𝖳𝖠𝖢𝖢<annotation encoding="application / x-tex">\textsf{GAGAAGAACCCTCTCGCTAACGTTACCGTCCTCGGCGTCCCTACCGCTCCTAGCTCCGTTACC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>tTTCAACGGCGCCGCTGTTCCTCGCGCTTCCGTCGCTTACAACTCTACCTCCAAGACCCTGTTC<annotation encoding="application / x-tex">{ t TTCAACGGCGCCGCTGTTCCTCGCGCTTCCGTCGCTTACAACTCTACCTCCAAGACCCTGTTC}< / annotation>< / semantics>< / com:Math> <Formule mathématique disponible dans le document PDF, Math available in the PDF document>\operatorname{GTTGGCGGCCTGCAAGACTTCACCAAGACCGGCGCTTGGGCTGACAAGTGGGTTCTCAAGTGG

[0042] The amino acid sequence of the PvaGlu1 precursor protein is set forth as SEQ ID NO: 6. The predicted native signal peptide is shown in italics and underlined: MGGFTHYMLASAWLPLTLGAAISPAATSSTASWGPVFTVPASADEGAQLIANINDPQSVNAQT VCPGYVASNVQNNEFGFTATLNLAGKACNVYGTDVDSLNLTVQYQASDRLNINIGPAHVDASN ESWYILSDDLVYKPTVDGTASISQSDLLVSWSNEPSFNFKVIRKANKDVLFNTEGTVLVYENQ FIEFVSALPENYNLYGLGERIHGLRLGNNFTATTYAADAADPIDANIYGTHPFYLDTRYYEVD SKQGTYTLLTTNETDQSKEYTSFSHGVFLRNAHGQEVLLRPEGITWRTLGGSIDLYFYSGPTQ ADVTRSYQTSTVGLPTMQQYYTFGYHQCRWGYQNWSVMADVVSSFAKFQIPLETIWSDIDYMN AYRDFENDPIRFSYSEGAEFLGQLHENGQHWVPIVDSAIYIPNSENASDAYDVYTRGEADGVW MTNPDGSLYIGAVWPGYTVFPDWHNPKAHEFWSNEIATWHQKVAFDGIWIDMSEVSSFCVGSC GTGNLTLNPVHPSFLLPGEPGAVIYDYPESFNVTNSTEAASASASVSQAAATASASASTTTS YLRTTPTPGVRDVNHPPYVINNVQPGHDLAVHAVSPNATHIDGVSEYDVHNLWGYQILNATYH GLLKVWEDKRPFIIGRSTFAGSGKWAGHWGGDNTSLWAYMFFSIPQALSFSLFGIPMFGVDTC GFNGNSDEELCNRWMQLSAFFPFYRNHNVLSAISQEPYVWASVIDASKAAMKIRYALLPYIYT LFYLAHTTGSTVMRAVSWEFPNDPSLAAIDTQFLLGPSLMVVPVLEPQVDYVKGVFPGVGNGE VWYDWYTQSVFDAKPGVNTTISAPLGHIPVFVRGGSILPMQEPALTTRDARKTPWALLTALGG NGTASGQLYIDDGESITPNATLNVDFVASNSNLVASPRGSWVEKNPLANVTVLGVPTAPSSVT FNGAAVPRASVAYNSTSKTLFVGGLQDFTKTGAWADKWVLKW

[0043] The amino acid sequence of the mature form of PvaGlu1 (also referred to herein as E3 GH31 <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase) is set forth as SEQ ID NO:3, which is shown, above. Example 2. Expression and purification of PvaGlu1

[0044] The nucleotide sequence of a synthetic gene encoding PvaGlu1 optimized for expression in Trichoderma reesei is set forth as SEQ ID NO. 7: ATGGGCGCTTCACCCACTACATGCTCGCTTCCGCTTGGCCTCTGACCCTGGGCGCCCC ATCTCTCCCGCTGCTACCAGCAGCACCGCTTCGTGGGGCCCCGTTTTCACCGTCCCCGCCTCC GCCGACGAGGGCGCTCAGCTCATTGCCAACATTAACGACCCCCAGTCTGTTAACGCCCAGACC GTTTGCCCCGGCTACGTTGCTTCTAACGTCCAGAACAACGAGTTCGGCTTCACCGCCACCCTG <com:Math><semantics>𝙰𝙰𝙲𝙲𝚃𝙲𝙶𝙲𝙲𝙶𝙶𝙲𝙰𝙰𝙶𝙶𝙲𝚃𝚃𝙶𝙲𝙰𝙰𝙲𝙶𝚃𝚃𝚃𝙰𝙲𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝙰𝙲𝙶𝚃𝙲𝙶𝙰𝚃𝙰𝙶𝙲𝙲𝚃𝙶𝙰𝙰𝙲𝙲𝚃𝙶𝙰𝙲𝙲𝙶𝚃𝙲<annotation encoding="application / x-tex">\texttt{AACCTCGCCGGCAAGGCTTGCAACGTTTACGGCACCGACGTCGATAGCCTGAACCTGACCGTC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝙰𝙶𝚃𝙰𝙲𝙲𝙰𝙶𝙶𝙲𝙶𝙰𝙶𝚃𝙶𝙰𝙲𝙲𝙶𝙰𝙲𝚃𝙶𝙰𝙰𝙲𝙰𝚃𝚃𝙰𝙰𝙲𝙰𝚃𝚃𝙶𝙶𝙲𝙲𝙲𝙲𝙶𝙲𝙲𝙲𝙰𝙲𝙶𝚃𝚃𝙶𝙰𝙲𝙶𝙲𝙲𝚃𝙲𝙲𝙰𝙰𝙲<annotation encoding="application / x-tex">\texttt{CAGTACCAGGCGAGTGACCGACTGAACATTAACATTGGCCCCGCCCACGTTGACGCCTCCAAC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝙰𝙶𝚃𝙲𝚃𝚃𝙶𝙶𝚃𝙰𝙲𝙰𝚃𝚃𝙲𝚃𝙶𝙰𝙶𝙲𝙶𝙰𝙲𝙶𝙰𝙲𝙲𝚃𝙶𝙶𝚃𝚃𝚃𝙰𝙲𝙰𝙰𝙶𝙲𝙲𝚃𝙰𝙲𝙲𝙶𝚃𝙶𝙶𝙰𝙲𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝙲𝚃𝚃𝙲𝚃<annotation encoding="application / x-tex">\texttt{GAGTCTTGGTACATTCTGAGCGACGACCTGGTTTACAAGCCTACCGTGGACGGCACCGCTTCT}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>tATCTCTCAGTCTGACCTGCTGGTTAGCTGGTCTAACGAGCCTTCTTTCAACTTCAAGGTTATT<annotation encoding="application / x-tex">{ t ATCTCTCAGTCTGACCTGCTGGTTAGCTGGTCTAACGAGCCTTCTTTCAACTTCAAGGTTATT}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝙶𝚃𝙰𝙰𝙶𝙶𝙲𝚃𝙰𝙰𝙲𝙰𝙰𝙶𝙶𝙰𝙲𝙶𝚃𝙲𝙲𝚃𝙶𝚃𝚃𝙲𝙰𝙰𝙲𝙰𝙲𝙲𝙶𝙰𝙶𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝚃𝙲𝙲𝚃𝙲𝙶𝚃𝚃𝚃𝙰𝙲𝙶𝙰𝙶𝙰𝙰𝙲𝙲𝙰𝙶<annotation encoding="application / x-tex">\texttt{CGTAAGGCTAACAAGGACGTCCTGTTCAACACCGAGGGCACCGTCCTCGTTTACGAGAACCAG}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>mTTCATCGAGTTCGTCAGCGCTCTCCCTGAGAACTACAACCTGTACGGCCTGGGCGAGCGCATT<annotation encoding="application / x-tex">{m TTCATCGAGTTCGTCAGCGCTCTCCCTGAGAACTACAACCTGTACGGCCTGGGCGAGCGCATT}< / annotation>< / semantics>< / com:Math> <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{\tt CACGGCCTGCGACTCGGCAACAACTTCACCGCCACCACCTACGCCGCCGACGCCGACCCCCCCC ATCGACGCTAACATATATGGCACCCACCCTTTCTACCTGGACACCCGATACTACGAGGTTGAC <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{ t TCCAAGCAGGGCACCTACACCCTGCTGACCACCAACGAGACCGACC <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{ t TCTTTCTCTCACGGCGTTTTCCTCCGAAACGCTCACGGCCAGGAAGTTCTGCTGCGCCCTGAG <com:Math><semantics>𝙶𝙶𝙲𝙰𝚃𝙲𝙰𝙲𝙲𝚃𝙶𝙶𝙲𝙶𝙰𝙰𝙲𝙲𝙲𝚃𝙶𝙶𝙶𝙲𝙶𝙶𝙲𝙰𝙶𝙲𝙰𝚃𝚃𝙶𝙰𝙲𝙲𝚃𝙶𝚃𝙰𝙲𝚃𝚃𝙲𝚃𝙰𝙲𝚃𝙲𝙲𝙶𝙶𝙲𝙲𝙲𝚃𝙰𝙲𝙲𝙲𝙰𝙶<annotation encoding="application / x-tex">\texttt{GGCATCACCTGGCGAACCCTGGGCGGCAGCATTGACCTGTACTTCTACTCCGGCCCTACCCAG}< / annotation>< / semantics>< / com:Math> <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{\tt GCTGACGTCACCCGATCTTACCAGACCAGCACCGTTGGCCTGCCT <com:Math><semantics>𝙰𝙲𝙲𝚃𝚃𝙲𝙶𝙶𝙲𝚃𝙰𝙲𝙲𝙰𝙲𝙲𝙰𝙶𝚃𝙶𝙲𝙲𝙶𝙰𝚃𝙶𝙶𝙶𝙶𝙲𝚃𝙰𝙲𝙲𝙰𝙶𝙰𝙰𝙲𝚃𝙶𝙶𝚃𝙲𝚃𝙶𝚃𝙲𝙰𝚃𝙶𝙶𝙲𝚃𝙶𝙰𝙲𝙶𝚃𝚃𝙶𝚃𝚃<annotation encoding="application / x-tex">\texttt{ACCTTCGGCTACCACCAGTGCCGATGGGGCTACCAGAACTGGTCTGTCATGGCTGACGTTGTT}< / annotation>< / semantics>< / com:Math> AGCTCTTTCGCCAAGTTCCAGATTCCTCTGGAGACCATTTGGAGCGACATCGACTACATGAAC GCTTACCGAGACTTCGAGAACGACCCTATCCGATTCTCTTACTCTGAGGGCGCTGAGTTCCTC GGCCAGCTGCACGAGAACGGCCAGCACTGGGTTCCTATTGTTGACTCCGCCATCTACATCCCT AACAGCGAGAACGCCAGCGACGCTTACGACGTTTACACCCGAGGCGAGGCCGACGCGTTTGG <com:Math><semantics>𝙰𝚃𝙶𝙰𝙲𝙲𝙰𝙰𝙲𝙲𝙲𝙶𝙰𝙲𝙶𝙶𝙲𝙰𝙶𝙲𝙲𝚃𝙶𝚃𝙰𝙲𝙰𝚃𝚃𝙶𝙶𝙲𝙶𝙲𝙲𝙶𝚃𝚃𝚃𝙶𝙶𝙲𝙲𝙲𝙶𝙶𝙲𝚃𝙰𝙲𝙰𝙲𝙲𝙶𝚃𝚃𝚃𝚃𝙲𝙲𝙲𝙲𝙲<annotation encoding="application / x-tex">\texttt{ATGACCAACCCGACGGCAGCCTGTACATTGGCGCCGTTTGGCCCGGCTACACCGTTTTCCCCC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝙰𝙲𝚃𝙶𝙶𝙲𝙰𝙲𝙰𝙰𝙲𝙲𝙲𝚃𝙰𝙰𝙶𝙶𝙲𝚃𝙲𝙰𝙲𝙶𝙰𝙶𝚃𝚃𝙲𝚃𝙶𝙶𝚃𝙲𝚃𝙰𝙰𝙲𝙶𝙰𝙶𝙰𝚃𝚃𝙶𝙲𝚃𝙰𝙲𝙲𝚃𝙶𝙶𝙲𝙰𝙲𝙲𝙰𝙶𝙰𝙰𝙶<annotation encoding="application / x-tex">\texttt{GACTGGCACAACCCTAAGGCTCACGAGTTCTGGTCTAACGAGATTGCTACCTGGCACCAGAAG}< / annotation>< / semantics>< / com:Math> <Formule mathématique disponible dans le document PDF, Math available in the PDF document>$\tt GTCGCTTTCGACGGCATTTGGATTGACATGTCTGAGGTCAGCTCTTTCTGCGTTGGCTCTTGC$ <com:Math><semantics>𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝙶𝙲𝙰𝙰𝙲𝙲𝚃𝙶𝙰𝙲𝙲𝙲𝚃𝙲𝙰𝙰𝙲𝙲𝙲𝙲𝙶𝚃𝙲𝙲𝙰𝙲𝙲𝙲𝚃𝚃𝙲𝚃𝚃𝚃𝙲𝙲𝚃𝙶𝙲𝚃𝙲𝙲𝙲𝙲𝙶𝙶𝙲𝙶𝙰𝙶𝙲𝙲𝙲𝙶𝙶𝙲<annotation encoding="application / x-tex">\texttt{GGCACCGGCAACCTGACCCTCAACCCCGTCCACCCTTCTTTCCTGCTCCCCGGCGAGCCCGGC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙶𝙲𝚃𝙶𝚃𝚃𝙰𝚃𝙰𝚃𝙰𝚃𝙶𝙰𝙲𝚃𝙰𝙲𝙲𝙲𝚃𝙶𝙰𝙶𝚃𝙲𝚃𝚃𝚃𝙲𝙰𝙰𝙲𝙶𝚃𝙲𝙰𝙲𝙲𝙰𝙰𝙲𝚃𝙲𝚃𝙰𝙲𝙲𝙶𝙰𝙶𝙶𝙲𝚃𝙶𝙲𝚃𝚃𝙲𝚃𝙶𝙲𝚃<annotation encoding="application / x-tex">\texttt{GCTGTTATATATGACTACCCTGAGTCTTTCAACGTCACCAACTCTACCGAGGCTGCTTCTGCT}< / annotation>< / semantics>< / com:Math> TCAGCCGCATCGGTCTCCCAGGCTGCTGCTACCGCTTCTGCCTCTGCCTCAACCACCACCTCT <com:Math><semantics>tTACCTGCGAACCACCCCTACCCCGGCGTCCGAGACGTTAACCACCCTCCTTACGTTATTAAC<annotation encoding="application / x-tex">{ t TACCTGCGAACCACCCCTACCCCGGCGTCCGAGACGTTAACCACCCTCCTTACGTTATTAAC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙰𝙰𝙲𝙶𝚃𝙲𝙲𝙰𝙶𝙲𝙲𝚃𝙶𝙶𝙲𝙲𝙰𝙲𝙶𝙰𝙲𝙲𝚃𝙲𝙶𝙲𝙲𝙶𝚃𝙲𝙲𝙰𝙲𝙶𝙲𝙲𝙶𝚃𝚃𝙰𝙶𝙲𝙲𝙲𝙲𝙰𝙰𝙲𝙶𝙲𝚃𝙰𝙲𝙲𝙲𝙰𝙲𝙰𝚃𝚃𝙶𝙰𝙲<annotation encoding="application / x-tex">\texttt{AACGTCCAGCCTGGCCACGACCTCGCCGTCCACGCCGTTAGCCCCAACGCTACCCACATTGAC}< / annotation>< / semantics>< / com:Math> GGCGTTAGCGAGTACGACGTCCACAACCTGTGGGGGCTACCAGATTCTGAACGCTACCTAC <com:Math><semantics>𝙶𝙶𝙲𝙲𝚃𝙲𝙲𝚃𝙲𝙰𝙰𝙶𝙶𝚃𝚃𝚃𝙶𝙶𝙶𝙰𝙶𝙶𝙰𝙲𝙰𝙰𝙶𝙲𝙶𝙲𝙲𝙲𝚃𝚃𝚃𝙲𝙰𝚃𝚃𝙰𝚃𝚃𝙶𝙶𝙲𝙲𝙶𝙰𝙰𝙶𝙲𝙰𝙲𝙲𝚃𝚃𝙲𝙶𝙲𝚃𝙶𝙶𝙲<annotation encoding="application / x-tex">\texttt{GGCCTCCTCAAGGTTTGGGAGGACAAGCGCCCTTTCATTATTGGCCGAAGCACCTTCGCTGGC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙰𝙶𝙲𝙶𝙶𝙲𝙰𝙰𝙶𝚃𝙶𝙶𝙶𝙲𝚃𝙶𝙶𝙲𝙲𝙰𝙲𝚃𝙶𝙶𝙶𝙶𝙲𝙶𝙶𝙲𝙶𝙰𝙲𝙰𝙰𝙲𝙰𝙲𝙲𝚃𝙲𝚃𝙲𝚃𝙶𝚃𝙶𝙶𝙶𝙲𝚃𝚃𝙰𝙲𝙰𝚃𝙶𝚃𝚃𝙲𝚃𝚃𝙲<annotation encoding="application / x-tex">\texttt{AGCGGCAAGTGGGCTGGCCACTGGGGCGGCGACAACACCTCTCTGTGGGCTTACATGTTCTTC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙰𝙶𝙲𝙰𝚃𝚃𝙲𝙲𝚃𝙲𝙰𝙶𝙶𝙲𝚃𝙲𝚃𝙶𝚃𝙲𝚃𝚃𝙲𝙰𝙶𝙲𝙲𝚃𝙶𝚃𝚃𝙲𝙶𝙶𝙲𝙰𝚃𝙲𝙲𝙲𝙲𝙰𝚃𝙶𝚃𝚃𝙲𝙶𝙶𝙲𝙶𝚃𝚃𝙶𝙰𝙲𝙰𝙲𝙲𝚃𝙶𝙲<annotation encoding="application / x-tex">\texttt{AGCATTCCTCAGGCTCTGTCTTCAGCCTGTTCGGCATCCCCATGTTCGGCGTTGACACCTGC}< / annotation>< / semantics>< / com:Math> <Formule mathématique disponible dans le document PDF, Math available in the PDF document>{\tt GGCTTCAACGGCAACTCCGACGAAGAACTGTGCAACCGATGGATG <com:Math><semantics>𝙲𝙲𝚃𝚃𝚃𝙲𝚃𝙰𝙲𝙲𝙶𝙰𝙰𝙰𝙲𝙲𝙰𝙲𝙰𝙰𝙲𝙶𝚃𝙲𝙲𝚃𝙲𝙰𝙶𝙲𝙶𝙲𝚃𝙰𝚃𝚃𝙰𝙶𝙲𝙲𝙰𝙰𝙶𝙰𝙰𝙲𝙲𝚃𝚃𝙰𝙲𝙶𝚃𝚃𝚃𝙶𝙶𝙶𝙲𝙲𝚃𝙲𝙲<annotation encoding="application / x-tex">\texttt{CCTTTCTACCGAAACCACAACGTCCTCAGCGCTATTAGCCAAGAACCTTACGTTTGGGCCTCC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>tGTCATTGACGCTTCTAAGGCCGCCATGAAGATTCGATACGCTCTGCTGCCTTACATATACACCC<annotation encoding="application / x-tex">{ t GTCATTGACGCTTCTAAGGCCGCCATGAAGATTCGATACGCTCTGCTGCCTTACATATACACCC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝚃𝙶𝚃𝚃𝙲𝚃𝙰𝙲𝙲𝚃𝙲𝙶𝙲𝚃𝙲𝙰𝙲𝙰𝙲𝙲𝙰𝙲𝙲𝙶𝙶𝙲𝙰𝙶𝙲𝙰𝙲𝙲𝙶𝚃𝙲𝙰𝚃𝙶𝙲𝙶𝙰𝙶𝙲𝚃𝙶𝚃𝙲𝚃𝙲𝚃𝚃𝙶𝙶𝙶𝙰𝙶𝚃𝚃𝙲𝙲𝙲𝚃<annotation encoding="application / x-tex">\texttt{CTGTTCTACCTCGCTCACACCACCGGCAGCACCGTCATGCGAGCTGTCTCTTGGGAGTTCCCT}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙰𝙰𝙲𝙶𝙰𝙲𝙲𝙲𝚃𝙰𝙶𝙲𝙲𝚃𝙲𝙶𝙲𝙲𝙶𝙲𝙲𝙰𝚃𝚃𝙶𝙰𝙲𝙰𝙲𝙲𝙲𝙰𝙶𝚃𝚃𝙲𝙲𝚃𝙲𝙲𝚃𝙶𝙶𝙶𝙲𝙲𝙲𝚃𝙰𝙶𝙲𝙲𝚃𝙲𝙰𝚃𝙶𝙶𝚃𝚃𝙶𝚃𝙲<annotation encoding="application / x-tex">\texttt{AACGACCCTAGCCTCGCCGCCATTGACACCCAGTTCCTCCTGGGCCCTAGCCTCATGGTTGTC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝙲𝙲𝙶𝚃𝙲𝙲𝚃𝙶𝙶𝙰𝙶𝙲𝙲𝚃𝙲𝙰𝙶𝙶𝚃𝚃𝙶𝙰𝙲𝚃𝙰𝙲𝙶𝚃𝚃𝙰𝙰𝙶𝙶𝙶𝙲𝙶𝚃𝚃𝚃𝚃𝙲𝙲𝙲𝙲𝙶𝙶𝙲𝙶𝚃𝚃𝙶𝙶𝙲𝙰𝙰𝙲𝙶𝙶𝙲𝙶𝙰𝙶<annotation encoding="application / x-tex">\texttt{CCCGTCCTGGAGCCTCAGGTTGACTACGTTAAGGGCGTTTTCCCCGGCGTTGGCAACGGCGAG}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>tGTTTGGTACGACTGGTACACCCAGTCTGTTTTCGACGCCAAGCCCGGCGTTAACACCACCATC<annotation encoding="application / x-tex">{ t GTTTGGTACGACTGGTACACCCAGTCTGTTTTCGACGCCAAGCCCGGCGTTAACACCACCATC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙰𝙶𝙲𝙶𝙲𝚃𝙲𝙲𝚃𝙲𝚃𝙲𝙶𝙶𝙲𝙲𝙰𝙲𝙰𝚃𝙲𝙲𝙲𝙲𝙶𝚃𝚃𝚃𝚃𝙲𝙶𝚃𝙲𝙲𝙶𝙰𝙶𝙶𝙲𝙶𝙲𝙰𝙶𝙲𝙰𝚃𝚃𝙲𝚃𝙶𝙲𝙲𝚃𝙰𝚃𝙶𝙲𝙰𝙰𝙶𝙰𝙶<annotation encoding="application / x-tex">\texttt{AGCGCTCCTCTCGGCCACATCCCCGTTTTCGTCCGAGGCGCAGCATTCTGCCTATGCAAGAG}< / annotation>< / semantics>< / com:Math> <Formule mathématique disponible dans le document PDF, Math available in the PDF document>$\tt CCCGCTCTGACCACCCGCGACGCTCGAAAGACCCCTTGGGCTCTCCTGACCGCTCTGGGCGGC$ <com:Math><semantics>𝙰𝙰𝙲𝙶𝙶𝙲𝙰𝙲𝙲𝙶𝙲𝚃𝚃𝙲𝚃𝙶𝙶𝙲𝙲𝙰𝙶𝙲𝚃𝙶𝚃𝙰𝙲𝙰𝚃𝙲𝙶𝙰𝙲𝙶𝙰𝙲𝙶𝙶𝙲𝙶𝙰𝙶𝚃𝙲𝚃𝙰𝚃𝚃𝙰𝙲𝙲𝙲𝙲𝚃𝙰𝙰𝙲𝙶𝙲𝙲𝙰𝙲𝙲<annotation encoding="application / x-tex">\texttt{AACGGCACCGCTTCTGGCCAGCTGTACATCGACGACGGCGAGTCTATTACCCCTAACGCCACC}< / annotation>< / semantics>< / com:Math> <com:Math><semantics>𝙲𝚃𝙶𝙰𝙰𝙲𝙶𝚃𝙶𝙶𝙰𝙲𝚃𝚃𝙲𝙶𝚃𝚃𝙶𝙲𝚃𝚃𝙲𝙲𝙰𝙰𝙲𝚃𝙲𝚃𝙰𝙰𝙲𝙲𝚃𝙲𝙶𝚃𝚃𝙶𝙲𝙶𝚃𝙲𝙰𝙲𝙲𝚃𝙲𝙶𝙶𝙶𝙶𝙲𝚃𝙲𝚃𝚃𝙶𝙶𝙶𝚃𝚃<annotation encoding="application / x-tex">\texttt{CTGAACGTGGACTTCGTTGCTTCCAACTCTAACCTCGTTGCGTCACCTCGGGGCTCTTGGGTT}< / annotation>< / semantics>< / com:Math> GAGAAGAACCCTCTCGCTAACGTTACCGTCCTCGGCGTCCCTACCGCTCCTAGCTCCGTTACC TTCAACGGCGCCGCTGTTCCTCGCGCTTCCGTCGCTTACAACTCTACCTCCAAGACCCTGTTC GTTGGCGGCCTGCAAGACTTCACCAAGACCGGCGCTTGGGCTGACAAGTGGGTTCTCAAGTGG

[0045] The foregoing gene was inserted into the pTTT expression vector (described in WO2015 / 017256), resulting in plasmid pTTT-PvaGlu1 (Figure 1). pTTT-PvaGlu1 was transformed into a quad-deleted Trichoderma reesei strain (described in WO 05 / 001036) using protoplast transformation (Te'o et al. (2002) J. Microbiol. Methods 51:393-99). Transformants were selected and grown using the methods described in WO 2016 / 138315. Supernatants from these cultures were used to confirm the protein expression by SDS-PAGE analysis and in assays to measure enzyme activity.

[0046] A seed culture of the transformed cells mentioned was subsequently grown in a 2.8 L fermenter in defined medium. Fermentation broth was sampled at elapsed times of 42 hours, 65 hours, and 95 hours for SDS-PAGE analysis and measurements of dry cell weight, residual glucose and extracellular protein concentration. Following centrifugation, filtration and concentration, 500 mL of concentrated sample having a protein concentration of 10.7 g / L was obtained. PvaGlu1 was purified via hydrophobic interaction chromatography (HIC) followed by ammonium sulfate precipitation, and then stored at -20°C until needed. Example 3. Substrate specificity of PvaGlu1

[0047] Substrate specificity of PvaGlu1 was assayed based on the release of glucose following incubation with isomaltose, maltose, trehalose, panose, maltulose, sucrose, leucrose, nigerose, kojibiose, maltotriose or maltoheptaose. The rate of glucose release was measured using a coupled glucose oxidase / peroxidase (GOX / HRP) method (Ngo, T.T. and Lenhoff, H.M. (1980) Anal. Biochem. 105:389-397). Glucose was quantified as the rate of oxidation of 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) by peroxide which was generated from coupled GOX / HRP enzymes reacted with glucose.

[0048] Substrate solutions were prepared by mixing 9 mL of each substrate mentioned above (1% in water, w / v), 1 mL of 0.5 M pH 5.0 sodium acetate buffer, and 40 μL of 0.5 M calcium chloride in a 15-mL conical tube. Coupled enzyme (GOX / HRP) solution with ABTS was prepared in 50 mM sodium acetate buffer (pH 5.0), with the final concentrations of 2.74 mg / mL ABTS, 0.1 U / mL HRP, and 1 U / mL GOX.

[0049] Serial dilutions of α-glucosidase samples and glucose standard were prepared in Milli Q water. Each α-glucosidase sample (10 μL) was transferred into a new microtiter plate (Corning 3641) containing 90 µL of substrate solution preincubated at 50 °C for 5 min at 600 rpm. The reactions were carried out at 50°C for 10 min (for isomaltose, maltose, panose, maltulose, nigerose, and kojibiose) and for 60 min (for sucrose and leucrose) with shaking (600 rpm) in a thermomixer (Eppendorf). 10 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>L of reaction mixtures as well as 10 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>L of serial dilutions of glucose standard were quickly transferred to new microtiter plates (Corning 3641), followed by the addition of 90 µL of ABTS / GOX / HRP solution. The microtiter plates containing the reaction mixture were immediately measured at 405 nm at 11 seconds intervals for 5 min on SoftMax Pro plate reader (Molecular Device). The output was the reaction rate, Vo, for each enzyme concentration. Linear regression was used to determine the slope of the plot Vo versus enzyme dose. The specific activity of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase was calculated based on the glucose standard curve using the following equation: [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document]

[0050] Except for isomaltose and maltose, the value of the reaction rate with enzyme dosage at 5 ppm was directly used to indicate the enzyme activity on other substrates listed. Using the method mentioned above, substrate specificity of PvaGlu1 was determined and compared with the benchmarks, oligo-1,6-glucosidase (a product purchased from Megazyme) and purified TrTG (transglucosidase from Trichoderma reesei) as shown in Table 1. PvaGlu1 showed broader substrate specificity than the other enzymes, with high activity towards isomaltose, maltose, nigerose, and kojibiose and moderate activity towards panose, maltotriose and maltoheptaose. Table 1. Substrate specificity of purified PvaGlu1compared with Oligo-1,6-glucosidase and TrTG. [Image disponible dans le document PDF, Image available in the PDF document] Example 4. pH profile of PvaGlu1

[0051] The effect of pH (from 3.0 to 10.0) on α-glucosidase activity of PvaGlu1 was monitored using isomaltose as a substrate. Buffer working solutions consisted of the combination of glycine / sodium acetate / HEPES (250 mM), with pH varying from 3.0 to 10.0. Substrate solutions were prepared by mixing isomaltose (1% in water, w / v) with 250 mM buffer solution at a ratio of 9:1, containing calcium chloride at a final concentration of 2 mM. Enzyme working solutions were prepared in water at a certain dose (showing signal within linear range as per dose response curve). All the incubations were carried out at 50°C for 10 min following the same protocol as described in Example 3 for activity of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase towards isomaltose. Enzyme activity at each pH was reported as relative activity compared to enzyme activity at optimum pH. The results are shown in Table 2. PvaGlu1 was found to have an optimum pH at 5.0 and retain greater than 70% of maximum activity between 3.4 and 6.6. Table 2. pH profile for PvaGlu1. [Image disponible dans le document PDF, Image available in the PDF document] Example 5. Temperature profile of PvaGlu1

[0052] The effect of temperature (from 40 °C to 85 °C) on α-glucosidase activity of PvaGlu1 was monitored using isomaltose as a substrate. Substrate solutions were prepared by mixing 9 mL of isomaltose (1% in water, w / v), 1 mL of 0.5 M pH 5.0 sodium acetate buffer, and 40 μL of 0.5 M calcium chloride in a 15-mL conical tube. Enzyme working solutions were prepared in water at a certain dose (showing signal within linear range as per dose response curve). Incubations were performed at temperatures from 40 °C to 85 °C for 10 min following the same protocol as described in Example 3 for activity of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase towards isomaltose. Activity at each temperature was reported as relative activity compared to enzyme activity at optimum temperature. The results are shown in Table 3. PvaGlu1 was found to have an optimum temperature at 55-60°C and was able to keep higher than 70% of maximum activity between 49°C and 68 °C. Table 3. Temperature profile of PvaGlu1 [Image disponible dans le document PDF, Image available in the PDF document] Example 6. Glucose release from corn fiber in the presence of a-glucosidase

[0053] Whole stillage, the by-product of a starch ethanol fermentation and distillation, was prepared for a secondary saccharification by treatment with acid (see, e.g., U.S. Patent No. 8,633,003) and addition of enzymes. Specifically, about 0.4 grams of material containing approximately 20% dry solids was placed in a reaction vessel along with the commercial enzymes ACCELERASE TRIO<com:Sup>TM< / com:Sup> and STARGEN<com:Sup>TM< / com:Sup> 002 (DuPont Industrial Biosciences).

[0054] ACCELERASE TRIO™ includes several enzymatic activities, including 2000-2600 CMC U / g endoglucanase activity, >3000 ABX U / g xylanase activity and >2000 pNPG U / g β- glucosidase activity. One carboxy methyl cellulose (CMC) unit is defined as the amount of activity required to liberate 1 µmol of reducing sugars (expressed as glucose equivalents) in one minute at 50°C (122°F) at pH 4.8. One ABX unit is defined as the amount of enzyme required to generate 1 µmol of xylose reducing sugar equivalents per minute at 50°C (122°F) and pH 5.3. One pNPG unit denotes 1 µmol of nitro-phenol liberated from paranitrophenyl-B- D-glucopyranoside per minute at 50°C (122°F) and pH 4.8. ACCELERASE TRIO™ has negligible, if any, α-glucosidase activity. ACCELERASE TRIO<com:Sup>TM< / com:Sup> was dosed at 0.07% wt / vol relative to stillage.

[0055] STARGEN<com:Sup>TM< / com:Sup> 002 enzyme contains the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-amylase from Aspergillus kawachi and the glucoamylase from Trichoderma reesei. One glucoamylase Unit (GAU) is the amount of enzyme that will liberate one gram of reducing sugars calculated as glucose per hour from soluble starch substrate under the conditions of the assay. STARGEN<com:Sup>TM< / com:Sup> 002 has minimal <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidase activity, which may be attributable to the small amount <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase activity associated with α-amylases. STARGEN<com:Sup>TM< / com:Sup> 002 was dosed at 0.017 GAU / gram dry solid stillage.

[0056] To the above reactions several different concentrations of purified <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase was added and the reaction vessels placed in an incubator at 50°C for 18-24 hr with shaking, followed by termination by the addition of 0.2 M H<com:Sub>2< / com:Sub>SO<com:Sub>4< / com:Sub>. Following centrifugation, the supernatant was collected and analyzed by HPLC to measure the concentration of glucose. The name, Carbohydrate Active Enzyme (CAZy) classification, and SEQ ID NO of the enzymes tested is shown in Table 4, below. Note that E3 is PvaGlu1 described in Examples 1- 5. Table 4. α-glucosidases tested [Image disponible dans le document PDF, Image available in the PDF document]

[0057] The results are shown in Figure 2. The addition of both GH31 and GH97 <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidases results in increased glucose release in a dose-dependent manner. However, while the increase observed with GH97 enzymes is only about 1%, the increase observed with GH31 enzymes is as much as 60-70%, with a significant amount of the increase being observed at below 50, and even below 25 µg enzyme per g dry solids.

Claims

CLAIMS What is claimed is:

1. A method for increasing the production of fermentable sugars in an ethanol fermentation process, comprising: chemically-pretreating corn fiber material derived from a predominantly corn kernel feedstock; and contacting the pretreated corn fiber with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidase to release fermentable sugars from the corn fiber; wherein the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidase releases fermentable sugars from the corn fiber material derived from a predominantly corn kernel feedstock that otherwise would not be available for fermentation, wherein the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase is a GH31 enzyme and wherein the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase is performed in the presence of one or more additional enzymes.

2. The method of claim 1, wherein the corn fiber material is the stillage resulting from distillation of a fermentation product, or a derivative, thereof.

3. The method of claim 1, wherein the corn fiber material is produced by physically separating corn fiber from starch in a substrate for ethanol fermentation.

4. The method of claim 1, wherein the corn fiber material is exposed to a temperature of at least 80°C prior to the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase.

5. The method of claim 1, wherein the corn fiber material is subjected to an alcohol distillation step prior to the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase.

6. The method of claim 1, wherein the corn fiber material contains between about 1- 12% corn fiber (wt / wt).

7. The method of any one of claims 1-6, wherein the pretreatment is acid pretreatment.

8. The method of any one of claims 1-6, wherein the pretreatment is alkaline pretreatment.

9. The method of any one of claims 1-8, wherein the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase has at least 70% amino acid sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4.

10. A method for increasing the production of fermentable sugars from chemically- pretreated stillage, comprising: fermenting a predominantly corn kernel feedstock with an ethanolagen to produce ethanol; evaporating at least a portion of the ethanol and recovering stillage; chemically-pretreating the stillage; contacting the stillage with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase to release fermentable sugars; fermenting the released fermentable sugars with an ethanolagen to produce additional ethanol; and recovering the additional ethanol; wherein the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase releases fermentable sugars from the corn fiber material derived from the predominantly corn kernel feedstock that otherwise would not be available for fermentation wherein the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase is a GH31 enzyme and wherein the contacting with <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>- glucosidase is performed in the presence of one or more additional enzymes.

11. The method of claim 10, wherein the pretreatment is acid pretreatment.

12. The method of claim 10, wherein the pretreatment is alkaline pretreatment.

13. The method of any one of claims 10-12, wherein the corn fiber material contains between about 1-12% corn fiber (wt / wt).

14. The method of any one of claims 10-13, wherein the <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase has at least 70% amino acid sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4.

15. A polypeptide having <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase activity, selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 97% identity to the polypeptide of SEQ ID NO: 3; (b) a mature polypeptide produced by the processing of the polypeptide of SEQ ID NO: 6 by a signal peptidase or post translational modification during secretion from an expression host; and (c) a fragment of a polypeptide of (a) or (b) that has <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-glucosidase activity.

16. A polynucleotide comprising a nucleotide sequence that encodes the polypeptide of claim 15.