Beta-mannanase Activity Research Articles

MICROWAVE AND ULTRASONIC TREATMENT AS A PRELIMINARY STAGE OF OBTAINING WATER-SOLUBLE MANNAN FROM COFFEE SLUDGE

In recent years, interest in immunocorrectors of natural origin has increased significantly. Among them is a group of polysaccharides such as mannans. Mannans are polysaccharides composed of D-mannose residues as the main chain. They possess a number of properties: immunomodulatory, cancer-protective, antimicrobial, and normalize blood cholesterol levels. Mannans are present in a number of plants, algae and microorganisms. One of the promising sources of mannans can be coffee sludge, which is accumulated on an industrial scale at instant coffee companies. This article describes a biotechnological method for the production of water-soluble mannan from coffee sludge and investigates the possibility of increasing the yield of water-soluble low-molecular-weight mannan with the maximum content of physiologically active fractions by pretreating the raw material with ultrasound and ultrahigh-frequency radiation. The sludge was pretreated in an aqueous medium using ultrasound at 25, 35 and 40 kHz for 15 minutes and treatment in a 2.45 GHz ultra-high-frequency electric field with a power of 300 W, 600 W and 800 W for 5 minutes. Next, the physical disintegrate was treated with an enzyme preparation with beta-mannanase activity and centrifuged. The resulting water-soluble products were characterized by gel chromatography to determine the molecular weight distribution of the fractions of each sample. It was found that the pretreatment with ultrasound is expedient, since the total amount of fractions with a molecular weight of less than 20 kDa is almost 80%. The treatment of raw materials with microwave rays is inefficient and leads to an increase in fractions of molecular weight less than 1 kDa. Thus, varying the conditions of the preliminary physical treatment of coffee sludge allows to regulate the molecular weight distribution of water-soluble enzymolysis products and obtain products with the required range of molecular weight values.

Obtaining and characterization of modified mannan from the coffee sludge

Mannans are polysaccharides of natural origin. Their main chain consists of residues of D-mannose. They have an immunomodulatory effect, able to induce macrophage activation, inhibit tumor growth and virus development, normalize blood cholesterol, etc. Manooligosaccharides are effective prebiotics The main way to obtain mannan is to extract their alkaline solutions. This makes it impossible to use this polysaccharide in food technologe. This study proposes a biotechnological method of producing water-soluble mannan from coffee sludge. As a source of mannan used a by-product of obtaining instant coffee at the Odessa Combine Food Concentrates which is formed in the processing of coffee beans Arabica. The chemical composition of this coffee sludge was investigated. It is dominated by water-insoluble carbohydrates which are represented by hemicellulose polysaccharides and cellulose. Analysis of the monosaccharide composition of hemicelluloses showed that the hydrolyzate contains mannose, glucose and galactose residues in a ratio of 6: 0,5: 3., which may indicate the presence of galactomannan in their composition. The developed method involves the treatment of the coffee sludge with enzyme preparation with beta-mannanase activity. The process was carried out in aqueous medium at temperature 50 °C, varying the hydromodule in the range of 30… 50, the ratio of enzyme: substrate (1:25, 1:50 and 1:100) for 24…72 hours. This study presents the characteristics of the monomer composition and molecular weight distribution of polysaccharide samples obtained in this way. Only mannose was found in the hydrolyzate of water-soluble mannan. The rational conditions of enzymatic processing of raw materials are determined. The modified mannan is soluble in water. It contains the highest number of target fragments with a molecular weight of about 20 kDa which are considered to be the most physiologically active. Subsequently the modified mannan can be used in nanotechnology functional food ingredients and dietary supplements.

Heterologous Expression and Optimized Production of an Aspergillus aculeatus Endo-1,4-β-mannanase in Yarrowia lipolytica

The Aspergillus aculeatus MRC11624 man1 gene, encoding an endo-beta-1,4-mannanase, was cloned and expressed in the promising heterologous enzyme producer, the ascomycetous yeast Yarrowia lipolytica. Both single- and multi-copy transformants were constructed, and the secretion of the enzyme was evaluated as an in-frame fusion with the LIP2 secretion signal, as well as with its natural secretion signal. In shake-flask analysis, the highest volumetric enzyme activity (13,073 nkat/ml) and specific enzyme activity (1,020 nkat/(mg dcw)) were obtained with a multi-copy integrant utilizing beta-mannanase's own secretion signal. The best beta-mannanase-producing strain was subsequently evaluated in batch fermentation and resulted in a maximum volumetric enzyme activity of 6,719 nkat/ml. Fed batch fermentations resulted in a 3.9-fold increase in volumetric enzyme activity compared with batch fermentation, and a maximum titre of 26,139 nkat/ml was obtained. The results reported in this study indicate that Y. lipolytica is a promising producer of A. aculeatus beta-mannanase, producing higher beta-mannanase activity than that of recombinant Saccharomyces cerevisiae or Aspergillus niger when cultivated in shake flasks, which is encouraging for the use of the enzyme in industrial processes such as extraction of vegetable oil from leguminous seeds and the reduction in viscosity of coffee extracts.

Purification and Characterization of a Low Molecular Weight of β-Mannanase from Penicillium occitanis Pol6

The highest beta-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified beta-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 degrees C and pH 4.0. It was stable for 30 min at 70 degrees C and has a broad pH stability (2.0-12.0). ManIII showed K (m), V (max), and K (cat) values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s(-1) with locust bean gum as substrate, respectively. It was inhibited by mannose with a K (I) of 0.610(-3) mg/ml. ManIII was activated by CuSO4 and CaCl2 (2.5 mM). However, in presence of 2.5 mM Co2+, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.

Antagonistic action of low-fluence and high-irradiance modes of response of phytochrome on germination and beta-mannanase activity in Datura ferox seeds.

Seed germination is often induced by a pulse of red light perceived by phytochrome and cancelled by a subsequent pulse of far-red light. When the pulse of red light is followed by several hours of darkness, a pulse of far-red light is no longer effective and prolonged far-red is necessary to block germination. The aim was to investigate whether the red light pulse and prolonged far-red light act on the same or different processes during germination of Datura ferox seeds. Forty-five hours after the inductive red light pulse, germination could not be blocked by one pulse or six hourly pulses of far-red light, but was significantly reduced by 6 h of continuous far-red light. The pulse of red light increased embryo growth potential and the activities of beta-mannanase and beta-mannosidase extracted from the micropylar region of the endosperm. Continuous far-red light had no effect on embryo growth potential or beta-mannosidase activity, but severely reduced the activity of beta-mannanase. The effect of far-red light had the features of a high-irradiance response of phytochrome. Both germination and beta-mannanase activity were restored by a pulse of red light given after the end of the continuous far-red treatment. It is concluded that the low-fluence response and the high-irradiance response modes of phytochrome have antagonistic effects on seed germination and that the control of beta-mannanase activity is one process where this antagonism is established.

Production of beta-mannanase by B. subtilis from agro-industrial by-products: screening and optimization. Mannanase from wastes.

We have demonstrated that a mixture of wheat bran (35 g l-1), as a main substrate, and palm seed powder (10 g l-1), as a co-substrate, is appropriate for beta-mannanase production by Bacillus subtilis. A 2n factorial experimental design was employed as a primary step for medium optimization. The enzyme activity titters obtained at the optimized growth condition were equivalent to about 319% of the beta-mannanase activity and 114% of the specific activity levels reached by a galactomannan-based culture.

Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp

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.

Induction of alpha‐galactosidase in Penicillium ochrochloron by guar (Cyamopsis tetragonobola) gum

High yields of extracellular alpha-galactosidase from fungal cultures were obtained by inducing enzyme production with guar gum (a galactomannan obtained from the seeds of Cyamopsis tetragonobola) as the sole carbon source. An alpha-galactosidase was isolated from the culture medium of Penicillium ochrochloron culture and purified 867-fold by CM-cellulose and Sephacryl S-200 column chromatography to apparent homogeneity. Gel-filtration data revealed an M(r) of 57,500, which was in close agreement with SDS/PAGE M(r) estimation, for a single band, of 60,200. The alpha-galactosidase activity is strictly dependent upon the pH and temperature of the incubation medium, being maximal at pH 4.5 and 55 degrees C respectively. This enzyme from P. ochrochloron was isolated and purified, devoid of beta-mannanase activity, which cleaves the main beta-mannan backbone of galactomannans and greatly diminishes its gel-promoting capacity. The properties of purified guar-gum-induced alpha-galactosidase activity in P. ochrochloron culture were evaluated in order to ascribe a possible application for alpha-galactosidase in the controlled generation of an improved guar-gum-based gel promoter.

The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme.

The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.

Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum"

A lambda recombinant phage expressing beta-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis. The sequence of a 2.1-kb fragment containing the mannanase gene has been determined. One open reading frame was found which could code for a protein of Mr 38,904. The mannanase gene (manA) was overexpressed in E. coli by cloning the gene downstream from the lacZ promoter of pUC18. The enzyme was most active at pH 6 and 80 degrees C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan. The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the "C. saccharolyticum" genome by homologous recombination.

Two Bacillus beta-mannanases having different COOH termini are produced in Escherichia coli carrying pMAH5.

The nucleotide sequence was determined for the alkalophilic Bacillus sp. strain AM-001 beta-mannanase gene which produced two beta-mannanases (A and B) in Escherichia coli transformants. The putative beta-mannanase gene was 1,539 base pairs long and encoded a mature beta-mannanase protein of 487 amino acids and a signal peptide of 26 amino acids. The COOH-terminal amino acid of beta-mannanase A is an arginine residue located at amino acid 513 of the deduced amino acid sequence, and that of beta-mannanase B is a valine residue located at amino acid 365. Deletion derivatives having 1,098 base pairs from the ATG start codon maintained the beta-mannanase activity of the encoded polypeptide. However, clones harboring DNA fragments (1,051 base pairs) shorter than the gene which encoded beta-mannanase B (1,095 base pairs) did not exhibit the beta-mannanase activity. The simultaneous production of both beta-mannanases A and B in an E. coli transformant was demonstrated by the maxicell procedure.