Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.
ABSTRACT: beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 220.127.116.11) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei.
Project description:Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS).A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 18.104.22.168), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger beta -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed.This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-beta-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant beta-mannanase will be valuable in various biotechnological applications.
Project description:BACKGROUND: Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 22.214.171.124), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis. RESULTS: The mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose. CONCLUSION: Our study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.
Project description:?-1,4-Mannanase (EC 126.96.36.199) catalyzes the hydrolysis of ?-1,4-glycosidic bonds within mannan, a major constituent group of the hemicelluloses. Bivalves and gastropods possess ?-1,4-mannanase and may degrade mannan in seaweed and/or phytoplankton to obtain carbon and energy using the secreted enzymes in their digestive systems. In the present study, the crystal structure of AkMan, a gastropod ?-1,4-mannanase prepared from the common sea hare Aplysia kurodai, was determined at 1.05?Å resolution. This is the first report of the three-dimensional structure of a gastropod ?-1,4-mannanase. The structure was compared with bivalve ?-1,4-mannanase and the roles of residues in the catalytic cleft were investigated. No obvious binding residue was found in subsite +1 and the substrate-binding site was exposed to the molecular surface, which may account for the enzymatic properties of mannanases that can digest complex substrates such as glucomannan and branched mannan.
Project description:The hydrolysis of polysaccharides containing mannan requires endo-1,4-beta-mannanase and 1,4-beta-mannosidase activities. In the current report, the biochemical properties of two endo-beta-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-beta-mannanase and endo-1,4-beta-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-beta-mannanase activity and little endo-1,4-beta-glucanase activity; however, this enzyme also exhibited 1,4-beta-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of beta-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.
Project description:The barley endo-?-mannanase (MAN) gene family (HvMAN1-6) has been identified and the expression of its members analyzed throughout different plant organs, and upon grain development and germination. The HvMAN1 gene has been found to be highly expressed in developing and germinating grains. The MAN (EC 188.8.131.52) enzymatic activity gets a maximum in grains at 48 h of germination (post-germination event). Immunolocalization of mannan polymers in grains has revealed the presence of these polysaccharides in the endosperm cell walls (CWs). By mRNA in situ hybridization assays, the HvMAN1 transcripts have been localized to the aleurone layer, but not to the dead starchy endosperm cells. These data suggest that MAN1 is synthesized in the aleurone layer during early grain imbibition and moves potentially through the apoplast to the endosperm where the hydrolysis of the mannan polymers takes place after germination sensu stricto. Hence, mannans in the starchy endosperm CWs, besides their structural function, could be used as reserve compounds upon barley post-germination.
Project description:BACKGROUND: Endo-1,4-?-mannanase is an enzyme that can catalyze the random hydrolysis of ?-1, 4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and has a number of applications in different biotechnology industries. Penicillium oxalicum is a powerful hemicellulase-producing fungus (Bioresour Technol 123:117-124, 2012); however, few previous studies have focused on the cloning and expression of the endo-1,4-?-mannanase gene from Penicillium oxalicum. RESULTS: A gene encoding an acidophilic thermostable endo-1,4-?-mannanase (E.C. 184.108.40.206) from Penicillium oxalicum GZ-2, which belongs to glycoside hydrolase family 5, was cloned and successfully expressed in Pichia pastoris GS115. A high enzyme activity (84.4 U mL(-1)) was detected in the culture supernatant. The recombinant endo-1,4-?-mannanase (rPoMan5A) was tagged with 6 × His at its C-terminus and purified using a Ni-NTA Sepharose column to apparent homogeneity. The purified rPoMan5A showed a single band on SDS-PAGE with a molecular mass of approximately 61.6 kDa. The specific activity of the purified rPoMan5A was 420.9 U mg(-1) using locust bean gum as substrate. The optimal catalytic temperature (10 min assay) and pH value for rPoMan5A are 80 °C and pH 4.0, respectively. The rPoMan5A is highly thermostable with a half-life of approximately 58 h at 60 °C at pH 4.0. The K m and V max values for locust bean gum, konjac mannan, and guar gum are 7.6 mg mL(-1) and 1425.5 ?mol min(-1) mg(-1), 2.1 mg mL(-1) and 154.8 ?mol min(-1) mg(-1), and 2.3 mg mL(-1) and 18.9 ?mol min(-1) mg(-1), respectively. The enzymatic activity of rPoMan5A was not significantly affected by an array of metal ions, but was inhibited by Fe(3+) and Hg(2+). Analytical results of hydrolytic products showed that rPoMan5A could hydrolyze various types of mannan polymers and released various mannose and manno-oligosaccharides, with the main products being mannobiose, mannotriose, and mannopentaose. CONCLUSION: Our study demonstrated that the high-efficient expression and secretion of acid stable and thermostable recombinant endo-1, 4-?-mannanase in Pichia pastoris is suitable for various biotechnology applications.
Project description:Mannan endo-1,4-?-mannosidase (commonly known as ?-mannanase) catalyzes a random cleavage of the ?-D-1,4-mannopyranosyl linkage in mannan polymers. The enzyme has been utilized in biofuel production from lignocellulose biomass, as well as in production of mannooligosaccharides (MOS) for applications in feed and food industries. We aimed to obtain a ?-mannanase, for such mannan polymer utilization, from actinomycetes strains isolated in Indonesia. Strains exhibiting high mannanase activity were screened, and one strain belonging to the genus Kitasatospora was selected. We obtained a ?-mannanase from this strain, and an amino acid sequence of this Kitasatospora ?-mannanase showed a 58-71% similarity with the amino acid sequences of Streptomyces ?-mannanases. The Kitasatospora ?-mannanase showed a significant level of activity (944 U/mg) against locust bean gum (0.5% w/v) and a potential for oligosaccharide production from various mannan polymers. The ?-mannanase might be beneficial particularly in the enzymatic production of MOS for applications of mannan utilization.
Project description:A family GH5 (family 5 glycoside hydrolase) (1,4)-beta-D-mannan endohydrolase or beta-D-mannanase (EC 220.127.116.11), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2-6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has K(m) constants of 0.16 mg x ml(-1) and 5.3 mM and kcat constants of 12.9 and 3.9 s(-1) respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-beta-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-beta-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-beta-D-manno-oligosaccharides to Man is a family GH1 beta-D-mannosidase, now designated HvbetaMANNOS1, but previously identified as a beta-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134-11143], which hydrolyses 4NP (4-nitrophenyl) beta-D-mannoside three times faster than 4NP beta-D-glucoside, and has an action pattern typical of a (1,4)-beta-D-mannan exohydrolase.
Project description:The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.
Project description:The microbial degradation of the plant cell wall is a pivotal biological process that is of increasing industrial significance. One of the major plant structural polysaccharides is mannan, a beta-1,4-linked d-mannose polymer, which is hydrolyzed by endo- and exo-acting mannanases. The mechanisms by which the exo-acting enzymes target the chain ends of mannan and how galactose decorations influence activity are poorly understood. Here we report the crystal structure and biochemical properties of CjMan26C, a Cellvibrio japonicus GH26 mannanase. The exo-acting enzyme releases the disaccharide mannobiose from the nonreducing end of mannan and mannooligosaccharides, harnessing four mannose-binding subsites extending from -2 to +2. The structure of CjMan26C is very similar to that of the endo-acting C. japonicus mannanase CjMan26A. The exo-activity displayed by CjMan26C, however, reflects a subtle change in surface topography in which a four-residue extension of surface loop creates a steric block at the distal glycone -2 subsite. endo-Activity can be introduced into enzyme variants through truncation of an aspartate side chain, a component of a surface loop, or by removing both the aspartate and its flanking residues. The structure of catalytically competent CjMan26C, in complex with a decorated manno-oligosaccharide, reveals a predominantly unhydrolyzed substrate in an approximate (1)S(5) conformation. The complex structure helps to explain how the substrate "side chain" decorations greatly reduce the activity of the enzyme; the galactose side chain at the -1 subsite makes polar interactions with the aglycone mannose, possibly leading to suboptimal binding and impaired leaving group departure. This report reveals how subtle differences in the loops surrounding the active site of a glycoside hydrolase can lead to a change in the mode of action of the enzyme.