Comparative Analysis of the Recombinant α-Glucosidases from the Thermotoga neapolitana and Thermotoga maritima Maltodextrin Utilization Gene Clusters

Abstract

The hyperthermophilic bacterium Thermotoga maritima contains an amylolytic gene cluster with two adjacent α-glucosidase genes, aglB and aglA. We have now identified a similar pair of α-glucosidase genes on a 5,451 bp fragment of T. neapolitana genomic DNA. Like in T. maritima, aglA of T. neapolitana is located downstream of aglB. The deduced AglB primary structure allows its assignment to glycoside hydrolase family 13 (GHF13), whereas AglA belongs to GHF4. The aglB gene of T. neapolitana and the corresponding gene from T. maritima were expressed in E. coli, and the recombinant enzymes were characterized. Both enzymes hydrolyzed cyclomaltodextrins and linear maltooligosaccharides to yield glucose and maltose. Evidence from the hydrolysis of non-natural oligosaccharides and the pseudo-tetrasaccharide acarbose suggests that linear malto-oligosaccharides are progressively degraded by T. neapolitana and T. maritima AglB from the reducing end, which is highly uncommon for α-glucosidases. AglB, in contrast to the cofactor-dependent (NAD⁺, Mn²⁺) α-glucosidase AglA, does not cleave maltose. The recent elucidation of the crystal structure of T. miritima AglA indicates that AglA and AglB employ different catalytic mechanisms for glycosidic bond cleavage. Possible reasons for the presence of two α-glucosidase genes in the same amylolytic gene cluster of Thermotoga species are discussed.     

Thermotoga neapolitana gene clusters participating in degradation of starch and maltodextins: molecular structure of the locus

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from Thermotoga maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the E. coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the alpha-glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the Thermotoga maritima cofactor-dependent alpha-glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.     

A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Expression of the aglB and aglA Genes in E. coli and the Properties of the Recombinant Enzymes

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85°. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with -glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent -amylase and -glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl₂, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the -glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent -glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with -links. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrare. For the enzyme to be active, the presence of NAD⁺, DTT, and Mn²⁺ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.     

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase

The gene for the α-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hy-drolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritimaα-glucosidase has the unusual property of requiring NAD⁺ and Mn²⁺ for activity. Co²⁺ and Ni²⁺ also activated AglA, albeit less efficiently than Mn²⁺. T. maritima AglA represents the first example of a maltodextrin-degrading α-glucosidase with NAD⁺ and Mn²⁺ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or β-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD⁺ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of d-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90°C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50°C and 70°C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h. Received: October 6, 1999 / Accepted: February 9, 2000     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Fractionation of cellulase and β-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning

An aqueous two-phase system based on the two polymers poly(ethylene glycol) and dextran has been used for the fractionation of cellulase enzymes present in culture liquid obtained by fermentation with Trichoderma reesei. The activities of -glucosidase and glucanases were separated to high degree by using the two-phase systems for a counter-current distribution process in nine transfer steps. While the glucanases had high affinity to the poly(ethylene glycol) rich top phase the -glucosidase was enriched in the dextran-containing bottom phase. Multiple counter-current distribution performed indicates the heterogeneity of -glucosidase activities assuming at least four isoenzyme forms. One step concentration of -glucosidase by using system with 46:1 phase volume ratio resulted in 16 times higher enzyme activity.This revised version was published online in October 2005 with corrections to the Cover Date.     

Genes of the R-phycocyanin II locus of marine Synechococcus spp., and comparison of protein-chromophore interactions in phycocyanins differing in bilin composition

R-phycocyanin II (RPCII) is a recently discovered member of the phycocyanin family of photosynthetic light-harvesting proteins. Genes encoding the and subunits of RPCII were cloned and sequenced from marine Synechococcus sp. strains WH8020 and WH8103. The deduced amino acid sequences of RPCII were compared to two other types of phycocyanin, C-phycocyanin (CPC) and phycoerythrocyanin (PEC). These three types vary in the composition of their covalently bound bilin prosthetic groups. In terms of amino acid sequence identity RPCII is highly homologous to CPC and PEC, suggesting that the known three-dimensional structures of the latter two are representative of RPCII. Thus the amino acid residues contacting the three bilins of RPCII could be inferred and compared to those in CPC and PEC. Certain residues were identified among the three phycocyanins as possibly correlating with specific bilin isomers. In overall sequence RPCII and CPC are more homologous to one another than either is to PEC. This probably reflects functional homology in the roles of RPCII and CPC in the transfer of light energy to the core of the phycobilisome, a function not attributed to PEC. The genomes of Synechococcus sp. strains WH8020, WH8103 and WH7803 share homologous open reading frames in the vicinity of RPCII genes. The nucleotide sequence extending 3 from RPCII genes in strain WH8020 revealed two open reading frames homologous to components of an CPC phycocyanobilin lyase. These open reading frames may encode a lyase specific for the attachment of phycoerythrobilin to RPCII.     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

Nucleotide sequence of the Clostridium thermocellum bgIB gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases

Summary The nucleotide sequence of the bglB gene, coding for the thermostable -glucosidase B of Clostridium thermocellum was determined. The coding region of 2265 bp was identified by comparison with the N-terminal amino acid sequence of -glucosidase B purified from Escherichia coli. The derived amino acid sequence corresponding to a polypeptide of M_r 84100 was confirmed by sequencing of the C-terminal peptide generated by cleavage with cyanogen bromide. The protein bears no resemblance to other bacterial -glucosidase sequences. However, extensive regions of homology were identified between the C. thermocellum enzyme and fungal -glucosidases. The N-terminal homologous region contains an amino acid sequence very similar to the active site of -glucosidase A₃ from Aspergillus wentii. The striking sequence similarities between C. thermocellum -glucosidase B and Kluyveromyces fragilis -glucosidase suggest the possibility of a genetic exchange between thermophilic anaerobic bacteria and yeasts.     

Close genetic relationship between Nitrobacter hamburgensis nitrite oxidoreductase and Escherichia coli nitrate reductases

The nitrite oxidoreductase (NOR) from the facultative nitrite-oxidizing bacterium Nitrobacter hamburgensis X14 was investigated genetically. In order to develop a probe for the gene norB, the N-terminal amino acid sequence of the NOR -subunit (NorB) was determined. Based on that amino acid sequence, an oligo-nucleotide was derived that was used for the identification and cloning of gene norB. Sequence analysis of DNA fragments revealed three adjacent open reading frames in the order norA, norX, norB. The DNA sequences of norX and norB represented complete genes while the open reading frame of norA was truncated by the cloning site. The deduced amino acid sequence of protein NorB contained four cysteine clusters with striking homology to those of iron-sulfur centers of bacterial ferredoxins. NorB shares significant sequence similarity to the -subunits (NarH, NarY) of the two dissimilatory nitrate reductases (NRA, NRZ) of Escherichia coli. Additionally, the derived amino acid sequence of the truncated open reading frame of norA showed striking resemblance to the -subunits (NarG, NarZ) of the E. coli nitrate reductases.     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Kinetics of beta-glucosidase production by Saccharomyces cerevisiae recombinants harboring heterologous bgl genes

The maximum productivity of -glucosidase by Saccharomyces cerevisiaerecombinants under the control of GALI promoter was 100 IU l^–1 h^–1. The highest productivity of -glucosidase by a S. cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea. The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1 g ethanol g^–1 cellobiose and g ethanol l^–1 h^–1, respectively.     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Sequence structure and expression of a cloned beta-glucosidase gene from an extreme thermophile

Summary The gene for a -glucosidase from the extremely thermophilic bacterium Caldocellum saccharolyticum has been isolated from a genomic library and sequenced. An open reading frame identified by computer analysis of the sequence could encode a protein of M_r 54400, which is close to the size of the polypeptide experimentally determined using maxicells. Analysis of the amino-terminal residues of the protein produced in Escherichia coli suggests that it is processed by a methionine aminopeptidase. A sequence within C. saccharolyticum DNA upstream of the -glucosidase gene was found to act as a promoter for expression of the thermophile gene in E. coli. The protein has been overproduced in E. coli and Bacillus subtilis where it retains its enzymatic activity and heat stability. There appears to be a single copy of the gene in Caldocellum DNA.     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

The nucleotide sequence of the lipo-penicillinase gene of alkalophilic Bacillus sp. strain 170

The lipo-penicillinase (LIPEN) gene from an alkalophilic Bacillus sp. strain 170 was cloned in Escherichia coli using the vector pHSG399. A plasmid, pFAP121, was isolated from an ampicillin resistant transformant and the cloned LIPEN gene was found to be in a 2.2 kb DNA fragment. The nucleotide sequence of a 1.9 kb segment encoding the LIPEN was determined. This segment showed an open reading frame which would encode a polypeptide of 310 amino acids. The amino acid sequence of this LIPEN gene product has strong homology with those of the Bacillus cereus -lactamase III and Bacillus licheniformis penicillinase.     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.