Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Developmental regulation of the maize Zm-p60.1 gene encoding a β-glucosidase located to plastids

A β-glucosidase that cleaves the biologically inactive hormone conjugates cytokinin-O- and kinetin-N3-glucosides is encoded by the maize Zm-p60.1 gene. The expression of the Zm-p60.1 gene was analyzed by Northern blot analysis and in-situ hybridization. It was found that the expression levels of the Zm-p60.1-specific mRNA changed after pollination of carpellate inflorescences. The Zm-p60.1 cDNA was expressed in E. coli and antibodies were raised against this protein. An antibody was used to determine the tissue-specific localization of this protein. By in situ immunolocalization experiments, this protein was found to be located in cell layers below the epidermis and around the vascular bundles of the coleoptile. In the primary leaf, the Zm-p60.1 protein was detected in cells of the outermost cell layer and around the vascular tissue. In floral tissue, Zm-p60.1 was present in the glumes, the carpels and in the outer cell layer of the style. In coleoptiles, as determined by immuno-electronmicroscopy, the Zm-p60.1 protein was located exclusively in the plastids. Received: 11 August 1998 / Accepted: 30 December 1998     

Solubilization of a novel isoflavone glycoside-hydrolyzing β-glucosidase from Lactobacillus casei subsp. rhamnosus

A novel isoflavone glycoside-hydrolyzing β-glucosidase produced by Lactobacillus casei subsp. rhamnosus IFO 3425 was solubilized by ultrasonic disruption of the cells in the presence of 2-mercaptoethanol and sorbitol as stabilizer. The β-glucosidase from L. casei subsp. rhamnosus specifically hydrolyzed soybean isoflavone glycosides, namely, daidzin and genistin, converting them to daidzein and genistein, respectively. By contrast, a commercial preparation of almond emulsin β-glucosidase could not hydrolyze these soybean isoflavone glycosides. The undesirably bitter and astringent isoflavone glycosides in soybean were decomposed for the first time with this novel β-glucosidase, an enzyme which has hitherto been considered difficult to solubilize, produced by a lactic acid bacterium.     

Cloning of a gene encoding β-glucosidase from Chaetomium thermophilum CT2 and its expression in Pichia pastoris

A new thermostable β-glucosidase gene (bgl) from Chaetomium thermophilum CT2 was cloned, sequenced and expressed. The full-length DNA of bgl was 3,101 bp and included three introns. The full-length cDNA contained an open reading frame of 2,604-bp nucleotides, encoding 867 amino acids with a potential secretion signal. The C. thermophilum CT2 β-glucosidase gene was functionally expressed in Pichia pastoris. The purified recombinant β-glucosidase was a 119-kDa glycoprotein with an optimum catalytic activity at pH 5.0 and 60°C. The enzyme was stable at 50°C, and retained 67.7% activity after being kept at 60°C for 1 h; the half-time of the enzyme at 65°C was approximately 55 min, and even retained 29.7% activity after incubation at 70°C for 10 min.     

Evolution and expression analysis of the β-glucosidase (GLU) encoding gene subfamily in maize

The maize ??-glucosidase (ZmGLU1) hydrolyzes cytokinin-conjugates for releasing active cytokinins and thus plays important roles in cytokinin regulatory processes. ZmGLU1 belongs to glycosyl hydrolases 1 (GH1) gene family with a large number of members, and the gene function of other homologs remains to be investigated. In this study, 47 Arabidopsis, 34 rice, 31 brachypodium, 28 sorghum and 26 maize GH1 protein sequences were collected and subsequently used to construct a phylogenetic tree by Neighbor-Joining method. ZmGLU1 together with its 7 paralogs and 4 sorghum homologs were assigned into a distinct group (named GLU subfamily) with far evolutionary distance to other GH1 members. None of the Arabidopsis, rice and brachypodium gene falling into this group indicated a recent evolutionary emergence of GLU subfamily in some Poaceae plants after the divergence of Poaceae species. Phylogeny and comparative genome analysis revealed that GLU subfamily members of maize and sorghum evolved from a common ancestor, and expanded independently in each species by several duplications after maize-sorghum split. Ka/Ks analysis showed that purifying selection played important roles in maintenance of similar functions among the maize GLU paralogs. In addition, the similar protein properties and cytokinin-dependent gene expressions further suggested the similar functions of ZmGLUs in cytokinin activation. However, the organ-dependent expression of ZmGLUs exhibited diverse patterns, which might contribute to their diverse roles in cytokinin homeostasis. Taken together, this work put new insights into the evolution and expression of ZmGLU genes, and provided the foundation for future functional investigations.     

Simultaneous expression of genes encoding endoglucanase and β-glucosidase in Zymomonas mobilis

Summary As a step towards constructing strains of Z. mobilis capable of converting cellulose to ethanol, DNA fragments encoding endoglucanase (from Xanthomonas albilineans) and -glucosidase (from either X.albilineans or Pseudomonas sp.) were linked on the same vector and transferred to Z. mobilis. All clones expressed endoglucanase. -Glucosidase was only produced by clones containing the Xanthomonas gene, and when two copies of this gene were present the -glucosidase activity was higher.     

Cloning and characterization of a novel cellobiase gene, cba3, encoding the first known β-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea

A novel cellobiase gene, designated cba3, was cloned from Cellulomonas biazotea. Although cellobiase genes of C. biazotea were previously cloned, published and/or patented, they encoded β-glucosidases all belonging to glycoside hydrolase family 3 (GH3); the new Cba3 cellobiase was identified to be a glycoside hydrolase family 1 (GH1) member, which represents the first discovered GH1 β-glucosidase of C. biazotea. Escherichia coli transformants expressing recombinant Cba3 were shown to grow readily in minimal media using cellobiose as the sole carbon source, supporting the conclusion that Cba3 is a genuine cellobiase. The full-length cba3 gene was revealed by sequencing to be 1344 bp long. Cba3 deletants lacking either the N-terminal 10 amino acids or the C-terminal 10 residues were found to be biologically inactive, supporting the importance of both ends in catalysis. Like other GH1 β-glucosidases, Cba3 was shown to contain the highly conserved NEP and ENG motifs, which are crucial for enzymatic activity. Despite lacking a classical N-terminal signal peptide, Cba3 was demonstrated to be a secretory protein. The findings that Cba3 is a cellobiase, and that it was expressed well as an extracellular protein in E. coli, support the potential of Cba3 for use with other cellulases in the hydrolysis of cellulosic biomass.     

Characterization of a novel β-glucosidase from a Stachybotrys strain

An improved mutant was isolated from the cellulolytic fungus Stachybotrys sp. after nitrous acid mutagenesis. It was fed-batch cultivated on cellulose and its extracellular cellulases (mainly the endoglucanases and β-glucosidases) were analyzed. One β-glucosidase was purified to homogeneity after two steps, MonoQ and gel filtration and shown to be a dimeric protein. The molecular weight of each monomer is 85 kDa. Besides its aryl β-glucosidase activity towards salicin, methyl-umbellypheryl-β-d-glucoside (MUG) and p-nitrophenyl-β-d-glucoside (pNPG), it showed a true β-glucosidase activity since it splits cellobiose into two glucose monomers. The V_max and the K_m kinetics parameters with pNPG as substrate were 78 U/mg and 0.27 mM, respectively. The enzyme shows more affinity to pNPG than cellobiose and salicin whose apparent values of K_m were, respectively, 2.22 and 37.14 mM. This enzyme exhibits its optimal activity at pH 5 and at 50 °C. Interestingly, this activity is not affected by denaturing gel conditions (SDS and β-mercaptoethanol) as long as it is not pre-heated. The N-terminal sequence of the purified enzyme showed a significant homology with the family 1 β-glucosidases of Trichoderma reesei and Humicola isolens even though these two enzymes are much smaller in size.     

The nucleotide sequence of the β-glucosidase gene from Cellvibrio gilvus

A gene encoding β-glucosidase from Cellvibrio gilvus, a cellobiose-producing bacterium, was cloned into Escherichia coli and sequenced. The structural gene consisted of 2565 bp encoding 854 amino acid residues with a characteristic signal peptide. A typical promoter sequence and SD region were located upstream of the initiation ATG codon. A sequence (180 amino acids) having high homology with those of β-glucosidases from several microorganisms was found in the deduced amino acid sequence of C. gilvus β-glucosidase. This sequence contains the aspartic acid residue which was found to be an active site residue in Aspergillus wentii β-glucosidase A3. The β-glucosidase gene of C. gilvus contains a high amount (69.4%) of G+C. These bases are localized not in the 3rd position of the codon, as is usually observed in G+C-rich genes, but rather in the 1st position. This result in a peptide which contains an extremely high amount (48%) of four amino acids (Pro, Ala, Arg, Gly) coded by CCN, GCN, CGN, and GGN.     

Molecular cloning and primary sequence analysis of a gene encoding a putative shitinase gene in Brassica oleracea var.capitata

Chitinase,which catalyzes the hydrolysis of the β-1,4-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin,is involved in inducible plants defense system.By construction of cabbage(Brassica oleracea var. capitata) genomic library and screening the library with pRCH8,a probe of rice chitinase gene fragment,a chitinase genomic sequence was isolated.The complete uncleotide sequence of the putative cabbage chitinase gene (cabch29) was determined,with its longest open reading frame (ORF) encoding a polypeptide of 413 aa.This polypeptide consists of a 21 aa N-terminal signal peptide,two chitin-binding domains different from those of other classes of plant chitinases,and a catalytic domain.Homology analysis illustrated that this cabch29 gene has 58.8% identity at the nucleotide level with the pRCH8 ORF probe and has 50% identity at the amino acid level tiwh the catalytic domains of chitinase from bean,maize and sugar beet.Meanwhile,several kinds of cis-elements,such as TATA box,CAAT box,GATA motif,ASF-1 binding site,wound-response elements and AATAAA,have also been discovered in the flanking region of cabch29 gene.     

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea var.capitata

INTRODUCTIONPlantshavedevelopedseveralbi0chemicaldefensemechanismsinresp0nsetopath0gensandabioticstress.Fo1l0wingpathogenattack,plantsynthesizephenyl-propaniodpr0ductssuchaslignin,l0wm0l.wt.antimicrobia1comp0undsknownasphyt0alexins,andseveraldefense-relatedproteins.Amongthesepr0teinsare"pathogenesis-relatedproteins"includingthefungalcellwalldegradingenzymeschitinaseandP-1,3-glucanase[1].Endochitinasefromhigherplantscatalyzethehydr0lysis0fchitin,aP-1,4-linkedhomop0lymerofN-acetyl-D-glucos…     

Purification,biochemical characterization and gene sequencing of a thermostable raw starch digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov.

This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain Pizzo^T). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from 4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca²⁺, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase.     

N-phenylglucosylamine hydrolysis: a mechanistic probe of β-glucosidase

The spontaneous hydrolysis of glycosylamines, where the aglycone is either a primary amine or ammonia, is over a hundred million-times faster than that of O- or S-glycosides. The reason for this (as pointed out by Capon and Connett in 1965) is that, in contrast to the mechanism for O- or S-glycoside hydrolysis, hydrolysis of these N-glycosides (e.g., glc-NHR) involves an endocyclic C-O bond cleavage resulting in formation of an imine (iminium ion) which then reacts with water. Since ring-opening is kinetically favored with glycosylamines, compounds such as phenylglucosylamine can be a useful probes of enzymes that have been suggested to possibly follow this mechanism. With β-glucosidase from sweet almonds, the enzyme is highly efficient in catalyzing the hydrolysis of phenyl glucoside (k_cat/k_non ∼ 10¹⁴) and phenyl thioglucoside (k_cat/k_non ∼ 10¹⁰) while with either the almond or the Aspergillus niger enzyme or with yeast α-glucosidase, there is no detectable catalysis of phenylglucosylamine hydrolysis (k_cat/k_non < 20). These results are consistent with the generally accepted mechanism involving exocyclic bond cleavage by these enzymes.     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.