Introduction

β-glucosidβ-glucosidases (EC. 3.2.1.21) asre represents a category of heterologous enzymes that break down glyenzymes that catalyze the hydrolysis of the glucosidic bonds inof disaccharides, oligosaccharides, and so on. The applications of β-realizing glucosidasese extend^[1]. to dThiverse biotechnological industries, such as enhancing aroma in juices and wine, hydrolyzing soybean isoflavone glycosides, reducing toxicity in animal feed, and facilitating cellulose degradation for biofuel production ^[5]. Cellulose, comprising s kind of enzyme is used in many industrial approximately 40 to 50% of plant biomass weight, stands as the predominant biomass source on Earth. It consists of glucose monomers linked by β-1,4 glucosidic bonds. The glucose derived from cellulose breakdown holds the potential for fermentation to yield bioethanol, offering a sustainable and eco-friendly alternative for fuel ications, such as food and biofuel production. ^[5].

In second-generation biofuel production, they act in synergy with other classes of enzymes, such as endo-glucanases and exo-glucanases ^[2][3], to obtain fermentable sugars from biomass. Endoglucanases function within the cellulose chain, generating oligosaccharides of varying lengths, while exoglucanases operate on these oligosaccharides, primarily yielding cellobiose ^[5]. β-glucosidases play a crucial role in this enzymatic system by breaking β-1,4 glucosidic bonds with the assistance of a water molecule, resulting in the production of two glucose molecules. It is widely recognized that β-glucosidases are pivotal in removing cellobiose, a potent inhibitor of both endoglucanases and exoglucanases within this enzymatic process. However, the literature has described that most β-glucosidase enzymes are inhibited by glucose, which has been considered one of the most impacting bottlenecks for highly -efficient industrial production ^[4].

Recently, a more efficient class of β-glucosidases hasve been described in the literature. They are called glucose-tolerant, due to their high resistance to inhibition, even in high glucose concentrations ^[5]. This class of enzymes has excellent potential for use in industrial applications such as biofuel production. Hence, glucose-tolerant β-glucosidase enzymes have been an essential target of several studies to detect mutations that transform non-tolerant enzymes into glucose-tolerant enzymes ^[6].

β-glucosidases have been organized into glycoside hydrolase families 1, 3, 5, 9, and 30, with many of them falling into families 1 and 3 ^[5]. Most glucose-tolerant β-glucosidases are obtained from glycoside-hydrolase family 1 (GH1). GH1 family enzymes belong to the GH-A clan, characterized by proteins with a TIM barrel fold and conserved catalytic amino acids on β-strands 4 and 7 of the barrel ^[5]. GH1 β-glucosidases employ a retention mechanism on the anomeric carbon, utilizing two glutamates as a catalytic nucleophile and catalytic acid/base. These enzymes have garnered attention for their remarkable resistance to product inhibition, making them highly valuable for cellulose degradation. 

In the next section, you will learn a little more about the sequences and structures of beta-glucosidases available in public databases.

Sequence and structure

The primary structure of a protein corresponds to its sequence. We can generally obtain protein sequences from our own databases, such as UniProt (http://www.uniprot.com.br). However, due to their biotechnological importance, beta-glucosidases have a series of databases of both sequences and three-dimensional structures. Two examples are the BETAGDB and Glutantbase databases.

Below, you can see the sequence of the beta-glucoseins from thidase from a metagenome sample collected in the South China Sea. This sequence was obtained in the BETAGDB database (available at http://bioinfo.dcc.ufmg.br/betagdb/). GH1 beta-glucosidases present approximatel family 400 residues (each letter represents an amino acid residue). 

>Yang_Y et al. (2015) tr|D5KX75|D5KX75_9BACT Beta-glucosidase OS=uncultured bacterium PE=3 SV=1 
MTKISLPTCSPLLTKEFIYGVATASFQIEGGSAHRLPCIWDTFCDTPGKIADNSNGHVAC 
DHYNNWKQDIDLIESLGVDAYRLSISWPRVITKSGELNPEGVKFYTDILDELKKRNIKAF 
VTLYHWDLPQHLEDEGGWLNRETAYAFAHYVDLITLAFGDRVHSYATLNEPFCSAFLGYE 
IGIHAPGKVGKQYGRKAAHHLLLAHGLAMTVLKQNSPTTLNGIVLNFTPCYSISEDADDI 
AATAFADDYLNQWYMKPIMDGTYPAIIEQLPSAHLPDIHDGDMAIISQSIDYLGINFYTR 
QFYKAHPTEIYEPIEPTGPLTDMGWEIYPKSFTELLVTLNNTYTLPPIFITENGAAMPDS 
YNNGEINDVDRLDYYNSHLNAVHNATEQGVRIDGYFAWSLMDNFEWAEGYLKRFGIVYVD 
YSTQQRTIKNSGLAYKALISNR

Another example is the β-glucosidase structures obtained from fungi, such as Humicola grisea and Humicola insolens. The sequence below illustrates the primary structure of a fungi β-glucosidase structure.

>de Giuseppe et al. (2014); Benoliel et al. (2010) tr|O93784|O93784_HUMGT Beta-glucosidase OS=Humicola grisea var. thermoidea GN=bgl4 PE=1 SV=1 PDB=4mdo
MSLPPDFKWGFATAAYQIEGSVNEDGRGPSIWDTFCAIPGKIADGSSGAVACDSYKRTKE
DIALLKELGANSYRFSISWSRIIPLGGRNDPINQKGIDHYVKFVDDLIEAGITPFITLFH
WDLPDALDKRYGGFLNKEEFAADFENYARIMFKAIPKCKHWITFNEPWCSAILGYNTGYF
APGHTSDRSKSPVGDSAREPWIVGHNILIAHARAVKAYREDFKPTQGGEIGITLNGDATL
PWDPEDPADIEACDRKIEFAISWFADPIYFGKYPDSMRKQLGDRLPEFTPEEVALVKGSN
DFYGMNHYTANYIKHKTGVPPEDDFLGNLETLFYNKYGDCIGPETQSFWLRPHAQGFRDL
LNWLSKRYGYPKIYVTENGTSLKGENDMPLEQVLEDDFRVKYFNDYVRAMAAAVAEDGCN
VRGYLAWSLLDNFEWAEGYETRFGVTYVDYANDQKRYPKKSAKSLKPLFDSLIRKE

Below, you can see a comparison between both sequences generated using the Clustal Omega web tool (aavailable at https://www.ebi.ac.uk/Tools/msa/clustalo/). Humicola insolens β-glucosidase is shown above, and Uncultured bacteria's β-glucosidase sequence from the South China Sea is shown below. The asterisks (*) represent conserved amino acid residues, while the punctuation mark (:) represents a modification for an amino acid residue with similar characteristics. Take note that β-glucosidase sequences from the same family present similar sequences.

Humicola        ----------MSLPPDFKWGFATAAYQIEGSVNEDGRGPSIWDTFCAIPGKIADGSSGAV	50
uncultured      MTKISLPTCSPLLTKEFIYGVATASFQIEGGS--AHRLPCIWDTFCDTPGKIADNSNGHV	58
                            *  :* :*.***::****.     * *.******  ******.*.* *

Humicola        ACDSYKRTKEDIALLKELGANSYRFSISWSRIIPLGGRNDPINQKGIDHYVKFVDDLIEA	110
uncultured      ACDHYNNWKQDIDLIESLGVDAYRLSISWPRVITKSGE---LNPEGVKFYTDILDELKKR	115
                *** *:. *:** *::.**.::**:**** *:*  .*.   :* :*:..*..::*:* : 

Humicola        GITPFITLFHWDLPDALDKRYGGFLNKEEFAADFENYARIM-FKAIPKCKHWITFNEPWC	169
uncultured      NIKAFVTLYHWDLPQHLEDE-GGWLNRETA-YAFAHYVDLITLAFGDRVHSYATLNEPFC	173
                .*. *:**:*****: *:.. **:**:*     * :*. :: :    : : : *:***:*

Humicola        SAILGYNTGYFAPGHTSDRSKSPVGDSAREPWIVGHNILIAHARAVKAYREDFKPTQGGE	229
uncultured      SAFLGYEIGIHAPGKVGKQYGR----K------AAHHLLLAHGLAMTVLKQNSPTTLN--	221
                **:***: * .***:...:       .      ..*::*:**. *:.. :::   * .  

Humicola        IGITLNGDATLPWDPEDPADIEACDRKIEFAISWFADPIYFGKYPDSMRKQLGDRLPEFT	289
uncultured      -GIVLNFTPCY-SISEDADDIAATAFADDYLNQWYMKPIMDGTYPAIIEQLPSAHLPDIH	279
                 **.**         **  ** *     ::  .*: .**  *.**  :.:  . :**:: 

Humicola        PEEVALVKGSNDFYGMNHYTANYIKHKTGVPPEDDFLGNLETLFYNKYGDCIGPETQSFW	349
uncultured      DGDMAIISQSIDYLGINFYTRQFYKAHPTEI-------------YEP-IEPTGPLTDMGW	325
                  ::*::. * *: *:*.** :: * :                 *:   :  ** *:  *

Humicola        LRPHAQGFRDLLNWLSKRYGYPKIYVTENGTSLKGENDMPLEQVLEDDFRVKYFNDYVRA	409
uncultured      E-IYPKSFTELLVTLNNTYTLPPIFITENGAAMPDSYNN---GEINDVDRLDYYNSHLNA	381
                   : :.* :**  *.: *  * *::****::: .. :      ::*  *:.*:*.::.*

Humicola        MAAAVAEDGCNVRGYLAWSLLDNFEWAEGYETRFGVTYVDYANDQKRYPKKSAKSLKPLF	469
uncultured      VH-NATEQGVRIDGYFAWSLMDNFEWAEGYLKRFGIVYVDYSTQQRTIKNSG-LAYKALI	439
                :   .:*:* .: **:****:********* .***:.****:.:*:   :..  : * *:

Humicola        DSLIRKE	476
uncultured      ----SNR	442
                     :.

Also, proteins from the GH1 family have a conserved foldinserved folding structure called TIM-barrel ^[7]. Figure 1 illustrates the three-dimensional structure of GH1 β-glucosidase from the fungus Humicola insolens complexed with a glucose molecule ^[8]. 

Figure 1. GH1 β-glucosidase from the fungus Humicola insolens. Obtained from PDB ID: 4MDP. Figure generated using ChimeraX ^[9].

Glucose tolerance mechanisms

Some beta-glucosidases have beeIn described in the literature as resistant to inhibition by glucose. They are called glucose-tolerant. Several recent studies have addressed the reasons for this resistance to inhibition. Recently, a study ^[2] demonstrated that glucoseonclusion, β-tolerant beta-glucosidases have a mechanism for removing glucose from the catalytic pocket region. This was termed the "slingshot mechanism". This mechanism involves the participation of an amino acid residue, usually an aspartate, which performs hydrogen bond interactions with the glucose structure, acting as a type of catapult that pulls the glucose out of the site. Studies have shown that replacing this aspartate with histidine increases the inhibition effect, possibly by removing this ability to remove glucose from the site. It is worth mentioning that the catalytic pocket entry of glucose-tolerant beta-glucosidases is narrower, being formed by a series of hydrophobic residues. These residues are responsible for positioning glucose so the slingshot mechanism can release it. At the same time, the interactions performed by the catalytic residues with glucose are replaced by bonds between them ^[2].

Conclusion

In conclusion, β-glucosidase enzymes have enzymes have an excellent potential for second-generation biofuel production. However, genetic engineering applications are still necessary to improve the activity of non-tolerant enzymes. Additionally, bioinformatics applications have been successfully used to bring new insights to detect sites for mutations that could improve their activity.