Structure and substrate recognition by the Ruminococcus bromii amylosome pullulanases.

Pullulanases are glycoside hydrolase family 13 (GH13) enzymes that target α1,6 glucosidic linkages within starch and aid in the degradation of the α1,4- and α1,6- linked glucans pullulan, glycogen and amylopectin. The human gut bacterium Ruminococcus bromii synthesizes two extracellular pullulanases, Amy10 and Amy12, that are incorporated into the multiprotein amylosome complex that enables the digestion of granular resistant starch from the diet. Here we provide a comparative biochemical analysis of these pullulanases and the x-ray crystal structures of the wild type and the nucleophile mutant D392A of Amy12 complexed with maltoheptaose and 63-α-D glucosyl-maltotriose. While Amy10 displays higher catalytic efficiency on pullulan and cleaves only α1,6 linkages, Amy12 has some activity on α1,4 linkages suggesting that these enzymes are not redundant within the amylosome. Our structures of Amy12 include a mucin-binding protein (MucBP) domain that follows the C-domain of the GH13 fold, an atypical feature of these enzymes. The wild type Amy12 structure with maltoheptaose captured two oligosaccharides in the active site arranged as expected following catalysis of an α1,6 branch point in amylopectin. The nucleophile mutant D392A complexed with maltoheptaose or 63-α-D glucosyl-maltotriose captured ß-glucose at the reducing end in the -1 subsite, facilitated by the truncation of the active site aspartate and stabilized by stacking with Y279. The core interface between the co-crystallized ligands and Amy12 occurs within the -2 through + 1 subsites, which may allow for flexible recognition of α1,6 linkages within a variety of starch structures.

Novel carbohydrate binding modules in the surface anchored α-amylase of Eubacterium rectale provide a molecular rationale for the range of starches used by this organism in the human gut.

Gut bacteria recognize accessible glycan substrates within a complex environment. Carbohydrate binding modules (CBMs) of cell surface glycoside hydrolases often drive binding to the target substrate. Eubacterium rectale, an important butyrate-producing organism in the gut, consumes a limited range of substrates, including starch. Host consumption of resistant starch increases the abundance of E. rectale in the intestine, likely because it successfully captures the products of resistant starch degradation by other bacteria. Here, we demonstrate that the cell wall anchored starch-degrading α-amylase, Amy13K of E. rectale harbors five CBMs that all target starch with differing specificities. Intriguingly these CBMs efficiently bind to both regular and high amylose corn starch (a type of resistant starch), but have almost no affinity for potato starch (another type of resistant starch). Removal of these CBMs from Amy13K reduces the activity level of the enzyme toward corn starches by ∼40-fold, down to the level of activity toward potato starch, suggesting that the CBMs facilitate activity on corn starch and allow its utilization in vivo. The specificity of the Amy13K CBMs provides a molecular rationale for why E. rectale is able to only use certain starch types without the aid of other organisms.

The Sus operon: a model system for starch uptake by the human gut Bacteroidetes.

Resident bacteria in the densely populated human intestinal tract must efficiently compete for carbohydrate nutrition. The Bacteroidetes, a dominant bacterial phylum in the mammalian gut, encode a plethora of discrete polysaccharide utilization loci (PULs) that are selectively activated to facilitate glycan capture at the cell surface. The most well-studied PUL-encoded glycan-uptake system is the starch utilization system (Sus) of Bacteroides thetaiotaomicron. The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. In this review, we discuss what is known about the eight Sus proteins of B. thetaiotaomicron that define the Sus-like paradigm of nutrient acquisition that is exclusive to the Gram-negative Bacteroidetes. We emphasize the well-characterized outer membrane proteins SusDEF and the α-amylase SusG, each of which have unique structural features that allow them to interact with starch on the cell surface. Despite the apparent redundancy in starch-binding sites among these proteins, each has a distinct role during starch catabolism. Additionally, we consider what is known about how these proteins dynamically interact and cooperate in the membrane and propose a model for the formation of the Sus outer membrane complex.

Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale.

Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute-binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, whereas the membrane-associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute-binding proteins display a range of glycan-binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6-branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch-degrading Clostridium cluster XIVa organisms in the human gut.

Lysozyme activity of the Ruminococcus champanellensis cellulosome.

Ruminococcus champanellensis is a keystone species in the human gut that produces an intricate cellulosome system of various architectures. A variety of cellulosomal enzymes have been identified, which exhibit a range of hydrolytic activities on lignocellulosic substrates. We describe herein a unique R. champanellensis scaffoldin, ScaK, which is expressed during growth on cellobiose and comprises a cohesin module and a family 25 glycoside hydrolase (GH25). The GH25 is non-autolytic and exhibits lysozyme-mediated lytic activity against several bacterial species. Despite the narrow acidic pH curve, the enzyme is active along a temperature range from 2 to 85°C and is stable at very high temperatures for extended incubation periods. The ScaK cohesin was shown to bind selectively to the dockerin of a monovalent scaffoldin (ScaG), thus enabling formation of a cell-free cellulosome, whereby ScaG interacts with a divalent scaffodin (ScaA) that bears the enzymes either directly or through additional monovalent scaffoldins (ScaC and ScaD). The ScaK cohesin also interacts with the dockerin of a protein comprising multiple Fn3 domains that can potentially promote adhesion to carbohydrates and the bacterial cell surface. A cell-free cellulosomal GH25 lysozyme may provide a bacterial strategy to both hydrolyze lignocellulose and repel eventual food competitors and/or cheaters.

Clostridium butyricum Prazmowski can degrade and utilize resistant starch via a set of synergistically acting enzymes.

Resistant starch is a prebiotic fiber that is best known for its ability to increase butyrate production by the gut microbiota. This butyrate then plays an important role in modulating the immune system and inflammation. However, the ability to use this resistant starch appears to be a rare trait within the gut microbiota, with only a few species such as Ruminococcus bromii and Bifidobacterium adolescentis having been demonstrated to possess this ability. Furthermore, these bacteria do not directly produce butyrate themselves, rather they rely on cross-feeding interactions with other gut bacteria for its production. Here, we demonstrate that the often-used probiotic organism Clostridium butyricum also possesses the ability to utilize resistant starch from a number of sources, with direct production of butyrate. We further explore the enzymes responsible for this trait, demonstrating that they exhibit significant synergy, though with different enzymes exhibiting more or less importance depending on the source of the resistant starch. Thus, the co-administration of Clostridium butyricum may have the ability to improve the beneficial effects of resistant starch.IMPORTANCEClostridium butyricum is seeing increased use as a probiotic, due to potential health benefits tied to its ability to produce butyrate. Here, we demonstrate that this organism can use a variety of resistant starch sources and characterize the enzymes it uses to accomplish this. Given the relative rarity of resistant starch utilizing ability within the gut and the health benefits tied to resistant starch, the combined use of this organism with resistant starch in synbiotic formulations may prove beneficial.

Evaluating the efficacy of non-thermal microbial load reduction treatments of heat labile food components for in vitro fermentation experiments.

Increasingly, in vitro simulated colon fermentations are being used as a pre-clinical step to assess the impacts of foods and drugs on the gut microbiota in a cost-effective manner. One challenge in such systems is that they are potentially susceptible to the influences of contaminating microbes in test materials. Simulated gastric and intestinal digestion can relieve some of these concerns, however, live microbes may remain that can confound analysis. Autoclave treatment of test materials is the surest way to eliminate these microbes but presents problems when using heat labile components such as resistant starch. In this study, liquid chemical sterilant alternatives to moist heat sterilization were explored for treating pulse flours for use during in vitro simulated colon fermentation. Key attributes considered in chemical selection were accessibility, impact on treated food components, and effectiveness of the treatments for reducing microbial load. Three chemicals were selected for evaluation, bleach, alcohol, and hydrogen peroxide, at varying concentrations. Flours chosen for testing were from green lentil, field pea, chickpea, or sprouted green lentil. All treatments significantly reduced microbial loads, though there were still detectable levels of microbes after alcohol treatments. Furthermore, in vitro simulated colon fermentations of the treated pulses showed minimal difference from the untreated control both in terms of microbial composition and short chain fatty acid production. Scanning electron microscopy showed minimal impact of sterilization treatments on the gross structure of the pulse flours. Together these results suggest that bleach and hydrogen peroxide treatments can be effective nonthermal treatments to eliminate contaminating microbes in pulse flours without causing significant damage to starch and other fermentable substrates. This is thus also a promising treatment method for other starchy food substrates, though further testing is required.

Affinity Electrophoresis for Analysis of Catalytic Module-Carbohydrate Interactions.

Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate-binding modules (CBMs). In recent years carbohydrate surface binding sites of proteins, mostly enzymes, have also been investigated by this method. Here we describe a protocol for identifying binding interactions between enzyme catalytic modules and a variety of carbohydrate ligands.

Surface Plasmon Resonance Analysis for Quantifying Protein-Carbohydrate Interactions.

During the past two decades, surface plasmon resonance (SPR) analysis has emerged as an important tool for studying protein-carbohydrate interactions, with several commercial instruments available. Binding affinities in the nM to mM range can be determined; however, there are pitfalls that require careful experimental design to avoid. Here we give an overview of each step in the SPR analysis from immobilization to data analysis, providing key points of consideration that will allow practitioners to achieve reliable and reproducible results.

Impact of mashing protocol on the formation of fermentable sugars from millet in gluten-free brewing.

The production of fermentable sugars (FS) in gluten-free (GF) brewing is hindered by the high starch gelatinization temperatures of GF malts and lower diastatic power compared to barley malt. Our previous work has demonstrated that starch gelatinization was the primary hurdle, and when decoupled from a single mash phase, high concentrations of FS could be produced. However, more research was required to improve the applicability of GF brewing. In this study, millet was used as a model GF malt demonstrating that despite the low α-amylase and ß-amylase activities compared to barley malt âˆ¼ 90 % of the FS (∼110 g/L) could be produced within 40 min. Limitations to enzyme extraction and separation due to coarse milling and lautering initially limited FS by âˆ¼ 30 g/L, requiring additional processing or exogenous enzyme supplements that improved fermentable sugar generation by âˆ¼ 20 g/L. Overall, millet is a promising brewing ingredient, provided appropriate mashing procedures are implemented.

Microscopic assessment of the degradation of millet starch granules by endogenous and exogenous enzymes during mashing.

The high gelatinization temperature (GT) of millet starch prevents the usage of infusion or step mashes as an effective means to generate fermentable sugars (FS) in brewing because the malt amylases lack thermostability at GT. Here, we investigate processing modifications to determine if millet starch can be efficiently degraded below GT. We determined that producing finer grists through milling did not introduce enough granule damage to markedly change gelatinization characteristics, though there was improved liberation of the endogenous enzymes. Alternatively, exogenous enzyme preparations were added to investigate their ability to degrade intact granules. At the recommended dosages (0.625 µL/g malt), significant FS concentrations were observed, although at lower concentrations and with a much-altered profile than possible with a typical wort. When exogenous enzymes were introduced at high (10×) addition rates, significant losses of granule birefringence and granule hollowing were observed well below GT, suggesting these exogenous enzymes can be utilized to digest millet malt starch below GT. The exogenous maltogenic α-amylase appears to drive the loss of birefringence, but more research is needed to understand the observed predominate glucose production.

Additional Resistant Starch from One Potato Side Dish per Day Alters the Gut Microbiota but Not Fecal Short-Chain Fatty Acid Concentrations.

The composition of the gut microbiota and their metabolites are associated with cardiometabolic health and disease risk. Intake of dietary fibers, including resistant starch (RS), has been shown to favorably affect the health of the gut microbiome. The aim of this research was to measure changes in the gut microbiota and fecal short-chain fatty acids as part of a randomized, crossover supplemental feeding study. Fifty participants (68% female, aged 40 ± 13 years, BMI 24.5 ± 3.6 kg/m2) completed this study. Potato dishes (POT) contained more RS than refined grain dishes (REF) (POT: 1.31% wet basis (95% CI: 0.94, 1.71); REF: 0.73% wet basis (95% CI: 0.34, 1.14); p = 0.03). Overall, potato dish consumption decreased alpha diversity, but beta diversity was not impacted. Potato dish consumption was found to increase the abundance of Hungatella xylanolytica, as well as that of the butyrate producing Roseburia faecis, though fecal butyrate levels were unchanged. Intake of one potato-based side dish per day resulted in modest changes in gut microbiota composition and diversity, compared to isocaloric intake of refined grains in healthy adults. Studies examining foods naturally higher in RS are needed to understand microbiota changes in response to dietary intake of RS and associated health effects.

Effect of processing on the anti-inflammatory efficacy of cocoa in a high fat diet-induced mouse model of obesity.

Obesity causes inflammation which may lead to development of co-morbidities like cardiovascular diseases. Cocoa is a popular food ingredient that has been shown to mitigate obesity and inflammation in preclinical models. Cocoa typically undergoes fermentation and roasting prior to consumption, which can affect the polyphenol content in cocoa. The aim of this study was to compare the effect of fermentation and roasting protocols on the ability of cocoa to mitigate obesity, gut barrier dysfunction, and chronic inflammation in high fat (HF)-fed, obese C57BL/6J mice. We found that treatment of mice with 80 mg/g dietary cocoa powder for 8 weeks reduced rate of body weight gain in both male and female mice (46-57%), regardless of fermentation and roasting protocol. Colonic length was increased (11-24%) and gut permeability was reduced (48-79%) by cocoa supplementation. Analysis of the cecal microbiome showed that cocoa, regardless of fermentation and roasting protocol, reduced the ratio of Firmicutes to Bacteroidetes. Multivariate statistical analysis of markers of inflammation and body weight data showed sex differences in the effect of both the HF diet as well as cocoa supplementation. Based on this data there was strong protective efficacy from cocoa supplementation especially for the more processed cocoa samples. Overall, this study shows that anti-obesity and anti-inflammatory efficacy of cocoa is resilient to changes in polyphenol content and composition induced by fermentation or roasting. Further, this study shows that although cocoa has beneficial effects in both males and females, there are significant sex differences.

In vitro Fermentation Reveals Changes in Butyrate Production Dependent on Resistant Starch Source and Microbiome Composition.

One of the primary benefits associated with dietary resistant starch (RS) is the production of butyrate by the gut microbiome during fermentation of this fiber in the large intestine. The ability to degrade RS is a relatively rare trait among microbes in the gut, seemingly confined to only a few species, none of which are butyrate producing organisms. Thus, production of butyrate during RS fermentation requires a network of interactions between RS degraders and butyrate producers. This is further complicated by the fact that there are multiple types of RS that differ in their structural properties and impacts on the microbiome. Human dietary intervention trials with RS have shown increases in fecal butyrate levels at the population level but with individual to individual differences. This suggests that interindividual differences in microbiome composition dictate butyrate response, but the factors driving this are still unknown. Furthermore, it is unknown whether a lack of increase in butyrate production upon supplementation with one RS is indicative of a lack of butyrate production with any RS. To shed some light on these issues we have undertaken an in vitro fermentation approach in an attempt to mimic RS fermentation in the colon. Fecal samples from 10 individuals were used as the inoculum for fermentation with 10 different starch sources. Butyrate production was heterogeneous across both fecal inocula and starch source, suggesting that a given microbiome is best suited to produce butyrate only from a subset of RS sources that differs between individuals. Interestingly, neither the total amount of RS degraders nor butyrate producers seemed to be limiting for any individual, rather the membership of these sub-populations was more important. While none of the RS degrading organisms were correlated with butyrate levels, Ruminococcus bromii was strongly positively correlated with many of the most important butyrate producers in the gut, though total butyrate production was strongly influenced by factors such as pH and lactate levels. Together these results suggest that the membership of the RS degrader and butyrate producer communities rather than their abundances determine the RS sources that will increase butyrate levels for a given microbiome.

Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi.

Cellulase A (CenA) from Cellulomonas fimi is an inverting glycoside hydrolase and a member of family 6 of the CAZy database classification system. We replaced its putative catalytic base aspartyl residues, Aps392 and Asp216, with cysteinesulfinate using a combination of site-directed mutagenesis and chemical modification to investigate the applicability of this approach for the modulation of enzymatic properties. The substituted cysteinyl residues were oxidized to cysteinesulfinic acid with hydrogen peroxide, and the resulting protein products were demonstrated to retain their native structure. Oxidation of the Asp392Cys mutant enzyme restored 52% of wild-type activity when assessed at pH 7.5, whereas Asp216Cys CenA remained inactive. This suggests that Asp216 is not the catalytic base and provides further support for Asp392 performing this role. Similar substitution of the catalytic acid residue Asp252 or the catalytic nucleophile of the retaining enzyme Cel5A from Thermobifida fusca failed to produce active enzymes. This indicates a potential utility of this approach for uniquely identifying catalytic base residues. The replacement of Asp392 with cysteinesulfinate induced an acidic shift in the pH profile of the enzyme such that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining site-directed mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.

Resistant starch: impact on the gut microbiome and health.

Resistant starch has received a lot of attention for its potential to exert a healthy impact on the gut and certain members of its resident microbiota, particularly through enhanced butyrate production. However, resistant starch is a broad category that encompasses several structurally different starches. While all resist digestion by human enzymes, they differ in their effects on the microbiota. Individual variation in microbiota composition also has a substantial influence on butyrate production. Research on this interaction between resistant starch and the microbiota is using in vitro fermentations, cross-over design clinical trials and mouse studies with isotopically labeled starch. These studies are demonstrating that more personalized approaches are needed for finding resistant starch or other fiber that will promote a healthy gut.

Characterization of Microbial Dynamics and Volatile Metabolome Changes During Fermentation of Chambourcin Hybrid Grapes From Two Pennsylvania Regions.

Microbial diversity present on grapes in wineries, and throughout fermentation has been associated with important metabolites for final wine quality. Although microbiome-metabolome associations have been well characterized and could be used as indicators of wine quality, the impact of regionality on the microbiome and metabolome is not well known. Additionally, studies between microbiome and metabolome have been conducted on single species grape such as Vitis vinifera instead of other species and interspecific hybrids. Although the Pennsylvania wine industry is relatively young compared to California, the industry has been experiencing rapid growth over the past decade and is expected to continue to grow in the future. Pennsylvania's climate of cold winters and high levels of rainfall throughout the growing season favors cultivation of interspecific hybrid grapes such as Vitis ssp. Chambourcin, one of the most commonly grown hybrid varieties in the state. Chambourcin is a prime candidate for studying the impact of regionality on microbiome-metabolome interactions as interspecific hybrid varieties could shape the future of winemaking. Here, we identify for the first time the regional distribution of microbial communities and their interactions with volatile metabolome during fermentation (0-20 days) by integrating high throughput Illumina sequencing (16S and ITS) and headspace-solid phase microextraction-gas chromatography-mass spectrometry. Analyzing 88 samples from nine wineries in the Central and East Pennsylvania regions, we observed high microbial diversity during early stages of fermentation (1-4 days) where non-Saccharomyces yeasts such as Starmerella and Aureobasidium and non-Oenococcus bacteria, Sphingomonas, likely contribute to microbial terroir to the resulting wines. Furthermore, key differentiators between two regions in Pennsylvania, as identified by LEfSe analysis, include the fungal genera Cladosporium and Kazachstania and the bacterial genera Lactococcus and Microbacterium. Moreover, 29 volatile fermentation metabolites were discriminated significantly (variable importance in projection > 1) between the two regions as shown by Partial Least Squares-Discriminant Analysis. Finally, Spearman's correlation identified regional differences of microbial-metabolite associations throughout fermentation that could be used for targeted microbiome manipulation to improve wine quality and preserve regionality. In summary, these results demonstrate the microbial signatures during fermentation and differential microorganisms and metabolites further support impact of regionality on Chambourcin wines in Pennsylvania.

Structural basis for the flexible recognition of α-glucan substrates by Bacteroides thetaiotaomicron SusG.

Bacteria that reside in the mammalian intestinal tract efficiently hydrolyze dietary carbohydrates, including starch, that escape digestion in the small intestine. Starch is an abundant dietary carbohydrate comprised of α1,4 and α1,6 linked glucose, yet mammalian intestinal glucoamylases cannot effectively hydrolyze starch that has frequent α1,6 branching as these structures hinder recognition and processing by α1,4-specific amylases. Here we present the structure of the cell surface amylase SusG from Bacteroides thetaiotaomicron complexed with a mixed linkage amylosaccharide generated from transglycosylation during crystallization. Although SusG is specific for α1,4 glucosidic bonds, binding of this new oligosaccharide at the active site demonstrates that SusG can accommodate α1,6 branch points at subsite -3 to -2, and also at subsite+1 adjacent to the site of hydrolysis, explaining how this enzyme may be able to process a wide range of limit dextrins in the intestinal environment. These data suggest that B. thetaiotaomicron and related organisms may have a selective advantage for amylosaccharide scavenging in the gut.

Polysaccharide Degradation by the Intestinal Microbiota and Its Influence on Human Health and Disease.

Carbohydrates comprise a large fraction of the typical diet, yet humans are only able to directly process some types of starch and simple sugars. The remainder transits the large intestine where it becomes food for the commensal bacterial community. This is an environment of not only intense competition but also impressive cooperation for available glycans, as these bacteria work to maximize their energy harvest from these carbohydrates during their limited transit time through the gut. The species within the gut microbiota use a variety of strategies to process and scavenge both dietary and host-produced glycans such as mucins. Some act as generalists that are able to degrade a wide range of polysaccharides, while others are specialists that are only able to target a few select glycans. All are members of a metabolic network where substantial cross-feeding takes place, as by-products of one organism serve as important resources for another. Much of this metabolic activity influences host physiology, as secondary metabolites and fermentation end products are absorbed either by the epithelial layer or by transit via the portal vein to the liver where they can have additional effects. These microbially derived compounds influence cell proliferation and apoptosis, modulate the immune response, and can alter host metabolism. This review summarizes the molecular underpinnings of these polysaccharide degradation processes, their impact on human health, and how we can manipulate them through the use of prebiotics.

Modulating the pH-activity profile of cellulase A from Cellulomonas fimi by replacement of surface residues.

One industrial process for the production of cellulosic ethanol and or value-added products involves exposing the cellulose content of plant materials by steam explosion in the presence of strong acid, followed by its neutralization and subsequent digestion with a cocktail of cellulolytic enzymes. These enzymes typically have activity optima at slightly acidic or neutral pH and so generating enzymes that are more active and tolerant in more acidic conditions would help to reduce associated costs. Here, we describe the engineering of cellulase A from Cellulomonas fimi as a model to replace residues that were identified as potentially influencing the pH-activity profile of the enzyme based on sequence alignments and analysis of the known three-dimensional structures of other CAZy family 6 glycoside hydrolases with the aim to lower its pH optimum. Twelve specific residues and a sequence of eight were identified and a total of 30 mutant enzymes were generated. In addition to being replaced with natural amino acids, some of the identified residues were substituted with cysteine and subsequently oxidized to cysteinesulfinate. Of the four single amino acid replacements that produced enhancements of activity at acidic pH, three involved the removal of charged groups from the surface of the enzyme. The generation of double mutations provided mixed results but the combination of Glu407 → Ala and Tyr321 → Phe replacements had an additive effect on the enhancement, reaching a total activity that was 162% of the wild-type level. This study thus illustrated the utility of altering the surface charge properties of the family 6 glycoside hydrolases to enhance activity at low pH and thereby an avenue for further protein engineering.