Bifunctional recombinant fusion enzyme between maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase of thermophilic microorganism Metallosphaera hakonensis

MhMTS and MhMTH are trehalose (alpha-D-glucopyranosyl- [1,1]-alpha-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused inframe in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around 70 degrees and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at 70 degrees for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above 80 degrees . The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.     

Molecular cloning of maltooligosyltrehalose trehalohydrolase gene from Nostoc flagelliforme and trehalose-related response to stresses

A genomic DNA fragment encoding a putative maltooligosyltrehalose trehalohydrolase (NfMTH) for trehalose biosynthesis was cloned by the degenerate primer- PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTH is 1,848 bp in length and encodes 615 amino acid residues, constituting a 70 kDa protein. The deduced amino acid sequence of NfMTH contains 4 regions highly conserved for MTHs. By expression of NfMTH in E. coli, the function of this protein was demonstrated, where the recombinant protein catalyzed the hydrolysis of maltooligosyl trehalose to trehalose. The expressions of MTH and maltooligosyltrehalose synthase in the filaments of N. flagelliforme were upregulated significantly under dehydration stress, NaCl stress, and high temperature-drought stress. The accumulations of both trehalose and sucrose in the filaments of N. flagelliforme were also improved significantly under the above stresses. Furthermore, trehalose accumulated in smaller quantities than sucrose did when under NaCl stress, but accumulated in higher quantities than sucrose did when under temperature-drought stress, indicating that both trehalose and sucrose were involved in N. flagelliforme adapted to stresses and different strategies conducted in response to various stress conditions.     

Molecular cloning of the gene encoding thermostable endo-1,5-alpha-L-arabinase of Bacillus thermodenitrificans TS-3 and its expression in Bacillus subtilis

The gene that encodes a thermostable endo-arabinase (called ABN-TS) from Bacillus thermodenitrificans TS-3 was cloned, sequenced, and expressed in the mesophilic B. subtilis. The gene contained an open reading frame consists of 939 bp, which encodes 313 amino acids. The deduced amino acid sequence of the enzyme showed 50, 46, and 36% similarity with endo-arabinase from B. subtilis IFO 3134 (PPase-C), Pseudomonas fluorescens (ArbA), and Aspergillus niger (ABNA), respectively. The hydrophobic and acidic amino acids making up ABN-TS outnumbered those in PPase-C. The gene product expressed in B. subtilis, as the host, had substantially the same characteristics, and was stable up to 70 degrees C, and the reaction was optimal around 70 degrees C, as well as native ABN-TS.     

Molecular cloning, DNA sequence, and expression of the gene encoding for thermostable pectate lyase of thermophilic Bacillus sp. TS 47

The gene that encodes a thermostable pectate lyase (called PL 47), from Bacillus sp. TS 47, was cloned, sequenced, and expressed in mesophilic B. subtilis. The gene contained an open reading frame consisting of 1326 bp, which encoded 441 amino acids. The deduced amino acid sequence of the mature enzyme (416 amino acids with a calculated molecular mass of 47,262 Da), showed 52% similarity with PL (BsPel) from mesophilic B. subtilis SO113. The structure-based alignment of the deduced amino acid sequence of PL 47 with that of BsPel suggested that PL 47 might have a parallel beta-helix structure with three long loops. The amino acids making up PL 47 are richer in hydrophobic amino acids and glutamic acid than BsPel. The hydropathy profile of PL 47 indicated that the amino acid sequences around putative calcium binding sites are more hydrophobic than the same region of BsPel. The gene product expressed in B. subtilis as the host was stable up to 70 degrees C and the reaction was optimal around 70 degrees C, as well as native PL 47.     

Gene cloning, sequencing, and biochemical characterization of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

The gene encoding endoxylanase (xynA) from Thermoanaerobacterium saccharolyticum B6A-RI was cloned and expressed in Escherichia coli. A putative 33-amino-acid signal peptide, which corresponded to the N-terminal amino acids, was encoded by xynA. An open reading frame of 3,471 bp, corresponding to 1,157 amino acid residues, was found, giving the xynA gene product a molecular mass of 130 kDa. xynA from T. saccharolyticum B6A-RI had strong similarity to genes from family F beta-glycanases. The temperature and pH optimum for the activity of the cloned endoxylanase were 70 degrees C and 5.5, respectively. The cloned endoxylanase A was stable at 75 degrees C for 60 min and displayed a specific activity of 227.4 U/mg of protein on oat spelt xylan. The cloned xylanase was an endo-acting enzyme.     

Purification, characterisation and cDNA sequencing of pyruvate decarboxylase from Zygosaccharomyces bisporus

Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the non-oxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha4) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent Km value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.     

Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethyl cellulose. The Km and Vmax values for xylan were 3.9 mg/ml and 611 micromol/min/mg of protein, respectively. This enzyme was optimally active at 80 degrees C and was stable up to 70 degrees C at neutral pHs and over the pH range of 4 to 11 at 25 degrees C.     

Trehalose synthesis by sequential reactions of recombinant maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase from Brevibacterium helvolum

A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium Brevibacterium helvolum. The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides. Recombinant BvMTS, BvMTH, and fusion gene BvMTSH, constructed by insertion of an adenylate in the overlapping region, were expressed in Escherichia coli. Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose. The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose. Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units. The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes. The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production. Addition of alpha-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.     

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus

Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.     

Identification of maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase enzymes catalysing trehalose biosynthesis in Anabaena 7120 exposed to NaCl stress

Anabaena 7120 cells were exposed to NaCl (25-175 mM) stress. Maximum growth was recorded in media containing 150mM NaCl. Short-term exposure (48h) of the cyanobacterial biomass to 150mM NaCl, induced highest trehalose level (37mM). Control cells lacking NaCl did not show any trace of trehalose as ascertained by NMR and HPLC analysis. Trehalose biosynthesis observed with NaCl plus high temperature (40 degrees C) indicated that its production was specifically triggered by NaCl, not temperature. The increase in trehalose level during NaCl stress was the result of overexpression of the trehalose-forming enzymes maltooligosyltrehalose synthase (MTSase), EC 5.4.99.15 (114kDa) and maltooligosyltrehalose trehalohydrolase (MTHase), EC 3.2.1.141 (68 kDa) as evidenced by SDS-PAGE analysis. To our knowledge this is the first report of induced trehalose biosynthesis in Anabaena 7120 during salt-stress, accompanied by identification of MTSase and MTHase enzymes on gel. It is suggested that Anabaena 7120 cells synthesize the osmolyte trehalose to withstand osmotic fluctuations.     

Trehalose Synthesis by Sequential Reactions of Recombinant Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase from Brevibacterium helvolum

A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium Brevibacterium helvolum. The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides. Recombinant BvMTS, BvMTH, and fusion gene BvMTSH, constructed by insertion of an adenylate in the overlapping region, were expressed in Escherichia coli. Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose. The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose. Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units. The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes. The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production. Addition of α-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.     

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80 degrees C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80 degrees C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80 degrees C with a half-life of 4 h at 90 degrees C and 1.5 h at 100 degrees C.     

Gene encoding a trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047

A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa.     

Cloning of the cel8Y gene from Pectobacterium chrysanthemi PY35 and its comparison to cel genes of soft-rot Pectobacterium

The phytopathogenic Pectobacterium chrysanthemi (Pch) PY35 secretes multiple isozymes of plant cell wall degrading enzyme cellulases. We cloned a second cel gene that encodes cellulase in Pch PY35. The inserted 2 kb fragment was subcloned in order to geneate pPY710 (cel8Y). The structural organization of the cel8Y gene consists of an open reading frame (ORF) of 999 bp that encodes 332 amino acid residues with a signal peptide of 23 amino acids. The predicted amino acid sequence of Cel8Y was very similar to that of Cellulomonas uda, but completely different from that of the Cel5Z of Pch PY35. It belonged to the glycoside hydrolase family 8, based on amino acid sequence similarities in contrast to Cel5Z of Pch PY35, which was confirmed as family 5. Cel8Y was not closely related to the known cellulases of Pectobacterium. It had the conserved region of the glycoside hydrolase family 8, ASDGDVLIAWALLKAGNKW. The apparent molecular mass of the Cel8Y protein was calculated to be approximately 34 kDa by a carboxymethylcellulosesodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). The Cel8Y had a calculated pl of 6.49. It was optimally active at pH 7 with an approximate optimal temperature around 40 degrees C. The cellulase activity of Cel8Y was lower than that of Cel5Z.     

来源于玫瑰微球菌的麦芽寡糖基海藻糖水解酶基因在大肠杆菌中的融合表达

设计引物克隆玫瑰微球菌QS412中麦芽寡糖基海藻糖水解酶(MTHase)的基因treZ,通过与pET-28a( )载体相连,转化入宿主菌E.coli BL21,进行发酵诱导。通过SDS-PAGE检测到外源基因在大肠杆菌中有很高的MTHase表达量,但大部分都以不溶性包含体形式存在。对菌体超声破碎全菌液检测酶活,结果显示了水解酶酶活。这是来源于微球菌属的麦芽寡糖基海藻糖水解酶首次获得基因克隆和活性表达,为进一步提高酶活、增大海藻糖产量奠定了基础。     

Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization

Three thermostable lactose-hydrolases, namely, two beta-glycosidases (bglA and bglB) and one beta-galactosidase (bgaA) genes were cloned from the genomic library of Thermus sp. IB-21. The bglA, bglB, and bgaA consisted of 1311 bp (436 amino acid residues), 1296 bp (431 aa), and 1938 bp (645 aa) of nucleotides with predicted molecular masses of 49,066, 48,679, and 72,714 Da, respectively. These enzymes were overexpressed in Escherichia coli BL21(DE3) using pET21b(+) vector system. The recombinant enzymes were purified to homogeneity by a heat precipitation (70 degrees C, 40 min) and a Ni2+-affinity chromatography. The molecular masses of the purified enzymes estimated by SDS-PAGE agreed with their predicted values. All the purified enzymes showed their optimal pH at around 5.0-6.0. In contrast, the temperature profiles for activity and thermostability patterns were different for each enzyme. BglB beta-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70 degrees C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70 degrees C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose. A novel treS gene with a length of 3,369 bp, encoding a protein of 1,122 amino acid residues with a predicted molecular mass of 126 kDa, was cloned from a marine Pseudomonas sp. P8005 (CCTCC: M2010298) and expressed in Escherichia coli. The amino acid sequence identities between this novel TreS and other reported TreS is relatively low. The purified recombinant TreS showed an optimum pH and temperature of 7.2 and 37 °C, respectively. The enzyme displayed a high conversion rate (70 %) of maltose to trehalose during equilibrium and had a higher catalytic efficiency (k _cat/K _m) for maltose than for trehalose, suggesting its application in the production of trehalose. In addition to maltose and trehalose, this enzyme can also act on sucrose, although this activity is relatively low. Mutagenesis studies demonstrated that enzymatic activity was reduced dramatically by individually substitution with alanine for D78, Y81, H121, D219, E261, H331 or D332, which implied that these residues might be important in P8005-TreS. Experiments using isotope-labeled substrates showed that [²H₂]trehalose combined with unlabeled trehalose was converted to [²H₂]maltose and maltose, but without any production of [²H]maltose or [²H]trehalose and with no incorporation of exogenous [²H₇]glucose into the disaccharides during the conversion catalyzed by this enzyme. This finding indicated the involvement of an intramolecular mechanism in P8005-TreS catalyzing the reversible interconversion of maltose and trehalose.