Cloning, sequencing and expression of an alpha-L-arabinofuranosidase from Aspergillus sojae

The arabinofuranosidase gene was cloned from the cDNA of Aspergillus sojae. It was found to contain an open reading frame composed of 984 base pairs (bp) and to encode 328 amino acid residues (aa). The cDNA sequence suggested that the mature enzyme is preceded by a 26-aa signal sequence and the molecular mass was predicted to be 32,749 Da. The A. sojae arabinofuranosidase consists of a single catalytic domain; it does not have a specific substrate-binding domain such as the xylan-binding domain reported in an arabinofuranosidase from Streptomyces lividans (Vincent, P. et al.: Biochem. J., 322, 845-852, 1997). The deduced amino acid sequence of the catalytic domain of the mature enzyme exhibits extensive identity with the catalytic domains of Streptomyces coelicolor (74%), Aspergillus niger (75%), S. lividans (74%), and Aspergillus tubingensis (75%), which are enzymes that belong to family 62 of the glycosyl hydrolases. The cloned AFdase gene was expressed in Escherichia coli BL21 (DE3) pLysS as a cellulose-binding domain tag fusion protein. The specific activity of the purified recombinant enzyme was 18.6 units/mg protein, which is one-fourth that of the enzyme purified from a solid-state culture of A. sojae.     

Molecular cloning and characterization of a glucan synthase gene from the human pathogenic fungus Paracoccidioides brasiliensis

1,3-beta-D-glucan is a fungal cell wall polymer synthesized by the multi-subunit enzyme 1,3-beta-D-glucan synthase. A subunit of this integral membrane protein was first described as the product of the FKS1 gene from Saccharomyces cerevisiae using echinocandin mutants. Other FKS1 genes were also reported for Candida albicans, Aspergillus nidulans and Cryptococcus neoformans. Here, we report the nucleotide sequence of the first homologous FKS gene cloned from the pathogenic fungus Paracoccidioides brasiliensis. An open reading frame of 5942 bp was identified in the complete sequence, interrupted by two putative introns, the first close to the 5' end and the second close to the 3' end of the gene. A promoter region is also described containing consensus sequences such as canonical TATA and CAAT boxes and, possibly, multiple sites for glucose regulation by creA protein. The deduced sequence of 1926 amino acid show more than 85% similarity to FksAp from A. nidulans, and 71% to Fks1p and Fks2p from S. cerevisiae. Computational analysis of P. brasiliensis Fks1p suggests a similar structure to transmembrane proteins, such as FksAp, with the presence of two domains composed by hydrophobic helices that limit the putative highly hydrophilic catalytic domain within the cytoplasm.     

Purification, characterization and gene analysis of exo-cellulase II (Ex-2) from the white rot basidiomycete Irpex lacteus

A new exo-type cellulase, named exo-cellulase II (Ex-2), was purified from the crude enzyme preparation of Irpex lacteus. Ex-2 was very similar to the previously characterized exo-cellulase I (Ex-1) with respect to enzymatic features such as optimal pH, temperature, heat stability, and catalytic activity. However, Ex-2 exhibited greater pH stability than Ex-1. The molecular mass and carbohydrate content of Ex-2 (56,000, 4.0%) were different from those of Ex-1 (53,000, 2.0%). A cellulase gene (named cel2) encoding both Ex-2 and Ex-1 was isolated from an I. lacteus genomic library. The cel2 gene was found to consist of 1569 bp with an open reading frame encoding 523 amino acids, interrupted by two introns. The deduced amino acid sequences revealed that cel2 ORF has a modular structure consisting of a catalytic domain and a fungal-type cellulose-binding domain (CBD) separated by a serine-rich linker region. The catalytic domain was homologous to those of fungal cellobiohydrolases belonging to family 7 of the glycosyl hydrolases. Northern blot analysis showed that expression of the cel2 gene was induced by various cellulosic substrates and repressed by glucose, fructose, and lactose.     

Structural analysis of glucoamylase encoded by the STA1 gene of Saccharomyces cerevisiae (var. diastaticus)

The sequence of the STA1-encoded glucoamylase of amylolytic Saccharomyces cerevisiae (var. diastaticus) strains shows two well-defined regions: an amino-terminal part rich in serine and threonine residues and a carboxy-terminal part very similar to the catalytic domain of other fungal glucoamylases. A version of the enzyme in which most of the amino-terminal region was deleted still has glucoamylase activity, indicating that the remaining carboxy-terminal part forms a functional catalytic domain. Homology-based models of the two parts of the protein have been obtained. As expected, the shortened form of the enzyme is very similar to the catalytic domain of related glucoamylases of known structure. However, the amino-terminal part yielded a structure revealing an unexpected similarity to bacterial invasins, suggesting functional connections between several yeast proteins homologous to STA1-encoded glucoamylase and invasins. A characteristic of Saccharomyces glucoamylase in its native form is its extreme degree of glycosylation. Despite its high molecular mass (about 300 kDa), and in contrast with what occurs with other extracellular glycoproteins produced by yeast, the enzyme does not remain attached to the cell wall, being fully and efficiently secreted into the medium, even when it is produced in large amounts by overexpression of its gene.     

Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile

In this paper, we present the expression and characterization of two novel enzymes from the alpha-amylase family exhibiting cyclomaltodextrinase specificity. The nucleotide sequences encoding the enzymes were isolated from the genomic DNA of two thermophilic bacterial strains originating from Icelandic hot springs and belonging to the genera Anoxybacillus (AfCda13) and Laceyella (LsCda13). The genes were amplified using a consensus primer strategy utilizing two of the four conserved regions present in glycoside hydrolase family 13. No identifiable signal peptides were present in open reading frames encoding the enzymes, indicating an intracellular location of both enzymes, and their physiological function to be intracellular cyclodextrin degradation. The domain structures of both enzymes were also similar, including an N-terminal domain, the catalytic module composed of the A- and B-domains, and a C-terminal domain. Despite the similarity in domain composition, the two enzymes displayed differences in the oligomeric state with AfCda13 being a dimeric protein, whereas LsCda13 was monomeric. The two enzymes also displayed significantly different activity profiles, despite being active on the same range of substrates. It was shown that the enzyme displaying the highest activity on cyclodextrin was dimeric (AfCda13). Moreover, a fraction of the dimeric enzyme could be converted to a monomeric state in the presence of KCl and this fraction retained only 23% of its activity on alpha-cyclodextrin while its activity on starch was not significantly affected, indicating that the oligomeric state is an important factor for a high activity on cyclodextrin substrates.     

产甲壳素酶菌株的筛选、鉴定及其酶解产物分析

以甲壳素为唯一碳源,从自然发酵的麻虾酱中筛选到1 株产甲壳素酶的放线菌X1。通过形态学、分子生物学以及生理生化实验鉴定为淀粉酶链霉菌（Streptomyces diastaticus）,命名为S. diastaticus strain CS 1801。在30 ℃条件下培养7 d,该菌株所产甲壳素酶活力为117.4 U/L。利用超高效液相色谱-四极杆-飞行时间串联质谱仪对发酵液中成分进行分析,确定甲壳素在淀粉酶链霉菌CS1801作用下分解成壳二糖、壳三糖、壳四糖、壳五糖和壳六糖。     

Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.     

Hexokinase PII: structural analysis and glucose signalling in the yeast Saccharomyces cerevisiae.

Hexokinase PII (Hxk2) is a yeast glucose phosphorylating enzyme that, besides its role in glycolysis, seems to have an additional role in glucose signalling. To study the domains in Hxk2 that may participate in this latter process, we have constructed 11 mutant alleles using site-directed mutagenesis. Six of them were clustered charged-to-alanine mutants in which clusters of charged residues were changed to alanine residues. Two of them contained substitutions in Ser15 to either alanine or glutamic acid and three of them had deletions at either the N-terminus or the C-terminus of the protein. In most of them, the catalytic activity correlated directly with their functionality in glucose signalling. However, we found two mutants (Delta1-15 and Delta476-486) that, having low catalytic activity, were still fully functional in glucose signalling. This may indicate that other factors and not just the catalytic activity of the enzyme may be important for the functionality of the protein in glucose signalling.     

淀粉酶链霉菌几丁质酶克隆表达及催化功能分析

目的:提高几丁质酶降解几丁质生产几丁寡糖产量。方法:利用基因工程方法对淀粉酶链霉菌几丁质酶进行克隆、原核表达、酶学性质探究,并通过同源建模、催化域氨基酸比对、定点突变等方法探究几丁质酶活性位点。结果:克隆和原核表达后,产酶周期由7 d缩短至24 h,酶活可达132 U/L,较原始菌酶活性(100 U/L)提高了32%。决定几丁质酶活性的氨基酸为催化域的128,130位的天冬氨酸和132位的谷氨酸。结论:对几丁质酶基因克隆表达后,其产酶周期缩短、酶活性提高。     

Stable expression of a thermostable xylanase of Clostridium thermocellum in cultured tobacco cells

Two distinct domains of the xynA gene from Clostridium thermocellum encoding a xylanase catalytic domain (XynAl) and a xylanase catalytic domain with a cellulose binding domain (XynA2) under the control of the cauliflower mosaic virus 35S promoter were electroporated into cultured tobacco BY-2 cells. Transgenic BY -2 calli expressing xylan-hydrolyzing activity were obtained at high frequency for both genes. Western blot analysis using an anti-XynA antibody indicated that XynAl and XynA2 were produced in these calli.     

Immobilisation and stabilisation of β-galactosidase from Kluyveromyces lactis using a glyoxyl support

β-galactosidase from Kluyveromyces lactis was covalently immobilised on a Glyoxyl Sepharose (GS) support by multi-point attachment. The enzyme immobilisation process was very efficient; the supports immobilised almost all the protein responsible for the catalytic activity in a short period of time, retaining approximately 82% of the activity in the case of the optimal immobilised preparations. Stability of the GS derivatives varied as a function of enzyme-support incubation time. The optimal immobilised preparation was produced after 2 h of incubation with the support at alkaline pH. This derivative, obtained by multi-point covalent attachment, was 100-fold more stable at pH 7 and 50 °C than the cyanogen bromide Sepharose derivative obtained by a one-point covalent immobilisation method. Stabilisation was also observed under a wide range of experimental conditions. This method allowed the immobilisation of 9000 IU enzyme g^?1 of support, resulting in highly active and stable derivatives suitable for industrial processes.     

Thermal and Molecular Characterization of Aspergillus awamori Glucoamylase Catalytic and Starch‐Binding Domains

Aspergillus awamori glucoamylase catalytic domain, linker, and starch‐binding domain, the first and third expressed from yeast, have molecular masses of 56.2, 12.6 and 12.9 kDa, respectively, as determined by MALDI‐TOF mass spectroscopy, and have 10.2, 73.2 and 7.0 % (w/w) carbohydrate, respectively, showing overglycosylation by yeast. Unfolding of the starch‐binding domain monitored by circular dichroism is reversible at pH 6.0—8.0, with the unfolding T_m and ΔH increasing from 49.7 to 58.5 °C and from 284 to 351 kJ/mol, respectively, as pH decreases from 8.0 to 6.0. The catalytic domain unfolds irreversibly at pH 7.5, producing a single asymmetric endotherm by differential scanning calorimetry, with T_m and ΔH at a 1 °C/min heating rate being 60.9 °C and 1720 kJ/mol, but with both increasing as the heating rate increases. This suggests that unfolding is partially under kinetic control, while various tests show that it does not follow a simple two‐state irreversible model. Values of ΔH from calculated solvent‐accessible surface areas of unglycosylated catalytic and starch‐binding domains are about 100 kJ/mol lower than experimentally determined ΔH values of the corresponding glycosylated domains, showing the effect of glycosylation on unfolding enthalpies.     

Novel bifidobacterial glycosidases acting on sugar chains of mucin glycoproteins

Bifidobacterium bifidum was found to produce a specific 1,2-alpha-L-fucosidase. Its gene (afc A) has been cloned and the DNA sequence was determined. The Afc A protein consisting of 1959 amino acid residues with a predicted molecular mass of 205 kDa can be divided into three domains; the N-terminal function-unknown domain (576 aa), the catalytic domain (898 aa), and the C-terminal bacterial Ig-like domain (485 aa). The recombinant catalytic domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and sugar chains of glycoproteins. The primary structure of the catalytic domain exhibited no similarity to those of any glycoside hydrolases but showed similarity to those of several hypothetical proteins in a database, which resulted in establishment of a novel glycoside hydrolase family (GH family 95). Several bifidobacteria were found to produce a specific endo-alpha-N-acetylgalactosaminidase, which is the endoglycosidase liberating the O-glycosidically linked galactosyl beta1-->3 N-acetylgalactosamine disaccharide from mucin glycoprotein. The molecular cloning of endo-alpha-N-acetylgalactosaminidase was carried out on Bifidobacterium longum based on the information in the database. The gene was found to comprise 1966 amino acid residues with a predicted molecular mass of 210 kDa. The recombinant protein released galactosyl beta1-->3 N-acetylgalactosamine disaccharide from natural glycoproteins. This enzyme of B. longum is believed to be involved in the catabolism of oligosaccharide of intestinal mucin glycoproteins. Both 1,2-alpha-L-fucosidase and endo-alpha-N-acetylgalactosaminidase are novel and specific enzymes acting on oligosaccharides that exist mainly in mucin glycoproteins. Thus, it is reasonable to conclude that bifidobacteria produce these enzymes to preferentially utilize the oligosaccharides present in the intestinal ecosystem.     

MULTIPLE FORMS OF α-AMYLASE IN MALTING BARLEY VAR. EMMA.

Multiple forms of α-amylase arise from translation of separate messages and post-translational proteolytic modification during malting. α-amylase activity is tolerant of proteolysis. The enzyme appears to have two domains one of which is associated with starch cleavage. The second site which binds cyclodextrin is destroyed by proteolysis. Cleavage yields a low molecular weight form which retains α-amylase activity.     

Function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase

Amylomaltase from Thermus aquaticus catalyzes three types of transglycosylation reaction, as well as a weak hydrolytic reaction of alpha-1,4 glucan. From our previous study [Fujii et al., Appl. Environ. Microbiol., 71, 5823-5827 (2005)], tyrosine 54 (Y54) was identified as an amino acid controlling the reaction specificity of this enzyme. Since Y54 is not located around the active site but in the proposed second glucan binding site that is 14 A away from catalytic residues, the functions of Y54 and the second glucan binding site are of great interest. In this study, we introduced mutations into another tyrosine (Y101) in the second glucan binding site. The obtained mutated enzymes were subjected to all four types of enzyme assay and the effects of mutations on the reaction specificities of these enzymes were comprehensively investigated. These studies indicated that the amino acid substitution at Y54 or Y101 for removing their aromatic side chain increases cyclization activity (intra-molecular transglycosylation reaction) but decreases disproportionation, coupling and hydrolytic activities (inter-molecular reactions). The superimposition of the reported structures of the enzyme with and without substrate analog revealed the occurrence of a conformational change in which a donor binding site becomes open. From lines of evidence, we conclude that the binding of glucan substrate to the second glucan binding site through an interaction with the aromatic side chains of Y54 and Y101 is a trigger for the enzyme to take a completely active conformation for all four types of activity, but prevents the cyclization reaction to occur since the flexibility of the glucan is restricted by such binding.     

Characterization and crystal structure of the thermophilic ROK hexokinase from Thermus thermophilus

We characterized and determined the crystal structure of a putative glucokinase/hexokinase from Thermus thermophilus that belongs to the ROK (bacterial repressors, uncharacterized open reading frames, and sugar kinases) family. The protein possessed significant enzymatic activity against glucose and mannose, with V(max) values of 260 and 68 μmol·min(-1)·mg(-1) protein, respectively. Therefore, we concluded that the enzyme is a hexokinase. However, the hexokinase showed little catalytic capacity for galactose and fructose. Circular dichroism measurements indicated that the enzyme was structurally stable at 90°C. The crystal structure of the enzyme was determined at a resolution of 2.02 ?, with R(cryst) and R(free) values of 18.1% and 22.6%, respectively. The polypeptide structure was divided into large and small domains. The ROK consensus sequences 1 and 2 were included in the large domain. The cysteine-rich consensus sequence 2 folded into a zinc finger, and the bound zinc was confirmed by both electron density and X-ray absorption fine structure (XAFS) spectrum. The overall structure was a homotetramer that consisted of a dimer of dimers. The accessible surface area buried by the association of the dimers into the tetrameric structures was significantly higher in the T. thermophilus enzyme than in a homologous tetrameric ROK sugar kinase.     

A new type of beta-N-Acetylglucosaminidase from hydrogen-producing Clostridium paraputrificum M-21

A beta-N-acetylglucosaminidase gene (nag84A) was cloned from Clostridium paraputrificum M-21 in Escherichia coli. The nag84A gene consists of an open reading frame of 4647 by encoding 1549 amino acids, with a deduced molecular weight of 174,311, which have a catalytic domain belonging to family 84 of the glycoside hydrolases. Nag84A was purified from a recombinant E. coli and characterized. Although Nag84A exhibited high homology to the hyaluronidase from Clostridium perfringens, it did not degrade hyluronic acid. The enzyme hydrolyzed chitooligomers such as di-, tri-, tetra-, penta- and hexa-N-acetylchitohexaose, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta-D-glucosaminide [4-MU-(G1cNAc)], but did not hydrolyze 4-MU-beta-D-glucoside, 4-MU-alpha-D-glycoside, 4-MU-alpha-D-GlcNAc, 4-MU-alpha-D-galactoside, 4-MU-beta-D-xyloside, PNP-beta-D-galactoside, and PNP-alpha-D-xyloside. The enzyme was optimally active at 50 degrees C and pH 6.5, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 8.5 microM and 1.39 micromol/min/mg of protein, respectively. SDS-PAGE, zymogram, and immunological analyses suggested that Nag84A was inducible by ball-milled chitin. Since Nag84A has a high molecular weight with a family 84 catalytic domain with high homology to hyaluronidases but no hyaluronidase activity, the enzyme is a novel beta-N-acetylglucosaminidase different from others reported having low molecular weights and belonging to family 3 and family 18.     

Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.     

Comparative sequence analysis of spa gene of Staphylococcus aureus isolated from bovine mastitis: characterization of an unusual spa gene variant

The protein A encoding gene spa of four Staphylococcus aureus strains isolated from bovine clinical mastitis was amplified by PCR and sequenced. The four strains were selected after an initial screening of spa gene of 41 strains isolated from mastitic cows and were subjected to detailed investigations. According to the sequencing results the spa gene of three strains (M1, M2, M3) appeared with gene segments encoding five (E, D, A, B and C) and four (E, A, B and C) IgG binding domains for two (M1, M3) and one (M2) strain, respectively and with gene segments encoding four, two and two repeats of the octapeptide Xr-repeats for the strains M1, M2 and M3, respectively. For the remaining Staph. aureus strain (M4) gene segments encoding IgG binding domains E, D and A and a new domain BC with a size of 219 bp could be observed. The BC domain appears, with a deletion of a 123 bp segment from the border region between both domains, as fused domain of both previously characterized domains. The Xr-region of this strain had 11 octapeptide repeats.