Glycosyl Hydrolases

This review examines the enzymes of 4-chlorobenzoate to 4-hydroxybenzoate converting pathway found in certain soil bacteria. This pathway consists of three enzymes: 4-chlorobenzoate: Coenzyme A ligase, 4-chlorobenzoyl-Coenzyme A dehalogenase and 4-hydroxybenzoyl-Coenzyme A thioesterase. Recent progress made in the cloning and expression of the pathway genes from assorted bacterial strains is described. Gene order and sequence found among these strains are

Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D16...

There have been many attempts to understand the mechanisms that determine the activity and stability of enzymes at extreme temperatures. Previous studies often compared enzymes from mesophiles and thermophiles and speculated that the observed differences were factors involved in adaptation to a certain temperature range. Most of these studies concluded that gross structural changes such as the quantity of certain amino acids or the total number of hydrogen bonds are responsible for the adaptation of optimal activity to temperature extremes. However, these studies have compared similar enzymes from distantly related organisms leaving one to wonder if the differences noted are from an adaptation to temperature or merely the result of genetic drift.
In this dissertation, I present an analysis of the cold-active beta-galactosidase (BgaS) from the Arthrobacter sp. SB. A study of the primary sequence and modeled structure of BgaS showed that many of the proposed adaptations for optimal activity in the cold did not hold true for this enzyme. Consequently, I decided to alter the BgaS enzyme to determine the contributions of specific amino acids to activity and stability at low temperatures.
I first examined the area of BgaS that aligned with the domain five mobile loop of the LacZ beta-galactosidase of Escherichia coli. In LacZ this area (residues 794-803) aids the binding of substrate and alterations at residue 794 increased the catalytic efficiency of the enzyme with lactose. However, when similar mutations were made in bgaS, they caused either a complete loss or a decrease of activity showing that although this area is also important for BgaS function the alterations affect the enzymes differently.
To further explore the low temperature activity of BgaS, I screened for second site revertants of a null mutant resulting from a G803D change. Restoration of activity was accomplished with the addition of only two mutations (E229D and V405A). Separation of these two mutations into a wild type background yielded an enzyme with a three fold increase in catalytic efficiency with little effect on the thermostability. This shows that small and subtle changes to the enzyme can further increase the activity at low temperatures.
I also explored the thermostability of BgaS through directed mutagenesis and directed evolution studies. The rational mutagenesis targeted the C-terminal portion of BgaS, an area in LacZ known to affect thermostability. From this study, I discovered that a single cysteine to glycine or cysteine to serine mutation resulted in an increase in thermal optimum for BgaS and in another closely related enzyme. The directed evolution study primarily targeted the active site in an attempt to create a more active version of BgaS. One mutant obtained from this screen, with a single alteration in amino acid sequence, created an enzyme with more activity and a 3.3 fold increase in the time it remained active at 30C, compared to BgaS.
Through the combination of mutational analysis and biochemical characterization, I have shown that the introduction of a limited number of amino acid changes are sufficient to alter the activity and/or thermal properties of an enzyme whereas previous studies have suggested that multiple alterations would be required. I have also increased the thermal optimum of three closely related enzymes by altering one amino acid. Considering the large size of the beta-galactosidase subunit, the finding that one change in the C-terminal 25 residues has any effect on the enzyme, not to mention an up to 20C increase in temperature optimum, is quite interesting. Taken as a whole, this work illustrates that small, unpredicted changes in the amino acid sequence of even large enzymes can have dramatic effects on their thermostability and/or activity.

Enzymes from extreme thermophiles that grow above 70 °C have a number of attractions in industrial applications. They are often highly resistant to denaturing conditions and are stable at elevated temperatures and over a range of pH values. There has been a widespread search for micro-organisms producing novel enzymes and where found,most publications (and research grant applications) promise that their superior properties would be suited to particular industries that operate at elevated temperatures - for example, bleaching of kraft pulp in the pulp and paper industry. Yet examination of the academic and patent literature reveals few of these proteins adopted in industrial enzymology. Most employed successfully have been as laboratory reagents, particularly Thermus aquaticus DNA polymerase, which made the polymerase chain reaction possible and revolutionized gene manipulation at a laboratory level. Extremozymes marketed for the laboratory are lower volume / higher value products and have been cloned and expressed (usually) in Escherichia coli. This review examines the characterization and application of thermophilic enzymes for several activities that have been identified and (usually) produced in recombinant bacteria. DNA polymerases, glycosyl hydrolases, lipases and proteases from extreme thermophiles are described and evaluated for their potential and actual applications in biotechnology. Some of the barriers to widespread industrial acceptance are described. Emphasis is placed on a number of examples illustrated by the anaerobic extreme thermophiles Caldicellulosiruptor sp. and Dictyoglomus sp. with which the authors are familiar. A number of attractive enzymes are not scalable economically in Escherichia coli or the organism from which the gene has been isolated is an obligate anaerobe and enzyme yields from the native organism are low. Consequently, attention has turned to commonly used ‘cell factories’ to provide suitable yields of enzymes used as bulk chemicals. These 'factories' are usually fungi such as Saccharomyces cerevisiae and Trichoderma reesei or bacteria such as Bacillus sp. A number of challenges, such as codon usage incompatibility, must be overcome to achieve economic yields.

Abstract: Sixteen mesophilic fungal strains of Aspergilli (10) and Penicilli (3) and Cochliobolus lunatus isolated from deciduous green forests and wetlands were characterized at molecular level and screened for cellulases (endoglucanase, β- glucosidase (cellobiase), cellobiohydrolase (CBHI/EGI) and FPase) and hemicellulase (xylanase, α -Larabinofuranosidase, β -xylosidase, acetyl xylan esterase and feruloyl esterase) production on rice straw & wheat bran containing solidified substrate. Penicillium janthinellum (3CHP) was found to produce high levels of β-glucosidase and α- L-arabinofuranosidase activities, whereas, Aspergillus tubingensis (8 DAL) produced maximal levels of endoglucanase, Fpase, xylanase and β-xylosidase. The cultures extracts from different strains were analysed for expression of multiple isoforms of endoglucanases, β-glucosidases, xylanases, α-L-arabinofuranosidases, β-xylosidases and acetyl xylan esterase.
The enzyme cocktails were used for saccharification of alkali treated rice straw at substrate loading rate of (7%). The maximal saccharification (56%) was observed in enzymatic hydrolysates from Aspergillus terreus.

The synergistic protective effect of Nicorandil (KATP channel opener) and Amlodipine (calcium channel blocker) on heart tissue antioxidant defense system and lipid profile were examined on isoproterenol induced myocardial infarction in rats. The rats given isoproterenol (150 mg kg−1 daily, i.p.) for 2 days showed significant changes in antioxidant defense system and lipid profile levels. Pretreatment with Nicorandil (2.5 mg kg−1 daily, p.o.) and Amlodipine (5.0 mg kg−1 daily, p.o.) for 3 days significantly prevented these alterations and restored the enzyme activities to near normal. These findings indicate the synergistic protective effect of Nicorandil and Amlodipine on tissue defense system and lipid metabolism during isoproterenol induced cardiac damage.

The constant need to develop environmental friendly processes using renewable raw materials and to reduce harmful waste production motivates the present investigation focusing on biocatalyst development for enzymatic synthesis, a biotechnological approach in the field of green chemistry.
At first, structural functional experiments were made for a β-glucosidase from T. neapolitana (TnBgl3B). As a result the first three-domain thermostable member of a glycoside hydrolase (GH) from family 3 was reported, giving insights on substrate specificity and some structural explanations for its use in synthesis of alkyl glucosides. Moreover, efficiency in hexyl glucoside synthesis was compared between two thermostable β-glucosidases from T. neapolitana, belonging to family 1 (GH1) and 3 (GH3), TnBgl1A and TnBgl3B, respectively. For this purpose, a novel direct screening method in 96-well format was developed, using glucose, a cheap substrate from renewables, together with hexanol for reverse hydrolysis in a two-phase system.
The GH1 β-glucosidase TnBgl1A was, in a following study, used as biocatalyst in hydrolysis reactions, connected to flavonoid extractions from onion waste using pressurized hot water, to obtain a uniform deglycosylated product. Structural homology models and mutagenesis around the aglycone in the active site, was in this case used to identify important residues that led to increased hydrolytic activity towards flavonoid 3-glucosides.
The second part of the project focused on thermostable glycosynthases, which were engineered and tested using oligosaccharide synthesis as main model reaction. Two methodologies were used to obtain synthesis products, one using fluorinated sugar as donor, and the second using an exogenous nucleophile. The product specificity was shown to be towards synthesis of β-1,3-linkages for both TnBgl1A and TnBgl3B.
Structural examination of theTnBgl1A glycosynthase, after docking with the substrate, showed flexibility at the active site with catalytic residues located on loops, a statement verified using molecular dynamics.
The construction of a glycosynthase from TnBgl3B required an extra mutation next to the nucleophile. A residue that stabilized the product at the +1 subsite was also identified, together with one more hydrophobic residue located on the loop
from the second domain, suggested to be important for the accommodation of acceptor molecules.
A natural product (antioxidant), such as the flavonoid quercetin-3-glucoside was also used as acceptor molecule in glycosynthase reactions at high temperature (70° C), in an attempt to look at an alternative acceptor and to expand the glycosynthase application to selective glycosylation of antioxidants. This assay used the exo-glycosynthases from TnBgl1A and TnBgl3B plus a new endo- glycosynthase constructed of a cellulase belonging to GH12 from R. marinus (RmCel12A). The main product of the thermostable glycosynthases was quercetin-3,4’-diglucoside when quercetin-3-glucoside was used as acceptor. Hence the 4’-hydroxyl of the acceptor was selected in the transglycosylation reactions, when the molecule was fitted in the active site.
In conclusion, thermostable glycoside hydrolases and glycosynthases can be useful biocatalysts for synthesising chemicals from renewable resources, such as alkyl glucosides, oligosaccharides or flavonoids with modified glycosylation. Thanks to the structural and functional information, the biocatalysts can be developed to harbour new or altered activities relevant for use to process renewable resources, such as in the modification of flavonoids combined with extraction form onion waste. Structural data also aided the construction of new glycosynthases coming from less investigated families like GH3. New interesting applications in antioxidant stabilization were assayed using thermostable glycosynthase for the glucosylation of flavonoids.

A novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon Thermococcus kodakarensis (TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The Km value toward pullulan was 2 mg ml(-1), with a Vmax of 109 U mg(-1). Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.

Aims:  Grey mould caused by Botrytis cinerea is an economically important disease of strawberries in Tunisia and worldwide. The aim of this study was to select effective halophilic bacteria from hypersaline ecosystems and evaluate the abilities of antifungal bacteria to secrete extracellular hydrolytic enzymes, anti-Botrytis metabolites and volatiles.Methods and Results:  Grey mould was reduced in strawberry fruits treated with halophilic antagonists and artificially inoculated with B. cinerea. Thirty strains (20·2%) were active against the pathogen and reduced the percentage of fruits infected after 3 days of storage at 20°C, from 50% to 91·66%. The antagonists were characterized by phenotypic tests and 16S rDNA sequencing. They were identified as belonging to one of the species: Virgibacillus marismortui, B. subtilis, B. pumilus, B. licheniformis, Terribacillus halophilus, Halomonas elongata, Planococcus rifietoensis, Staphylococcus equorum and Staphylococcus sp. The effective isolates were tested for antifungal secondary metabolites.Conclusions:  Moderately halophilic bacteria may be useful in biological control against this pathogen during postharvest storage of strawberries.Significance and Impact of the study:  The use of such bacteria may constitute an important alternative to synthetic fungicides. These moderate halophiles can be exploited in commercial production and application of the effective strains under storage and greenhouse conditions.

Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a β-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology.

Dehalospirillum multivorans is a strictly anaerobic bacterium that is able to dechlorinate tetrachloroethene (perchloroethylene; PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) as part of its energy metabolism. The present communication describes some features of the dechlorination reaction in growing cultures, cell suspensions, and cell extracts of D. multivorans. Cell suspensions catalyzed the reductive dechlorination of PCE with pyruvate as electron donor at specific rates of up to 150 nmol (chloride released) min-1 (mg cell protein)-1 (300 microM PCE initially, pH 7.5, 25 degrees C). The rate of dechlorination depended on the PCE concentration; concentrations higher than 300 microM inhibited dehalogenation. The temperature optimum was between 25 and 30 degrees C; the pH optimum at about 7.5. Dehalogenation was sensitive to potential alternative electron acceptors such as fumarate or sulfur; nitrate or sulfate had no significant effect on PCE reduction. Propyl iodide (50 microM) almost completely inhibited the dehalogenation of PCE in cell suspensions. Cell extracts mediated the dehalogenation of PCE and of TCE with reduced methyl viologen as the electron donor at specific rates of up to 0.5 mumol (chloride released) min-1 (mg protein).-1 An abiotic reductive dehalogenation could be excluded since cell extracts heated for 10 min at 95 degrees C were inactive. The PCE dehalogenase was recovered in the soluble cell fraction after ultracentrifugation. The enzyme was not inactivated by oxygen.