Agarase Production Research Articles

Production of agarase from a novel Micrococcus sp. GNUM-08124 strain isolated from the East Sea of Korea

The agar degrading bacterial strain GNUM-08124 was isolated from Enteromorpha compressa collected in the East Sea of Korea by using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. GNUM-08124 grows to produce a circular, smooth, yellow-colored, and raised colony. Its ability to hydrolyze agar was confirmed by staining the ASW agar plate with Lugol’s solution. In liquid culture, the cell density (A600) increased exponentially and reached a maximum level on the third day of cultivation. The specific agarase activity also increased in proportion to the cell density and reached maximum agarolytic activity on the third day. The 16S rRNA sequence of GNUM-08124 showed a close relationship to Micrococcus luteus (99.65%) and Micrococcus endophyticus (99.15%), which led us to assign it to the genus Micrococcus. Physiological studies indicated that optimal growth conditions were between 30 and 40°C, pH 4 and 7, using media containing between 5 and 10% NaCl (w/v), respectively. The GNUM-08124 strain was a grampositive, urease-positive, and catalase-positive bacterium. It could not hydrolyze gelatin, cellulose, xylan, or starch, but fermented a broader range of substrates, including Dglucose, D-galactose, D-fructose, D-lactose, D-trehalose, D-mannitol, D-melibiose, D-raffinose, D-xylose, methyl-α-D-glucopyranoside, N-acetyl-glucosamine, and xylitol, than those fermented by M. luteus or M. endophyticus, suggesting GNUM-08124 is a novel agar hydrolyzing microorganism belonging to Genus Micrococcus. Micrococcus sp. GNUM-08124 showed the highest agarase activity when it was cultured in ASW-YP medium supplemented with 0.4% glucose, but demonstrated lower activity in rich media (LB or TSB), in spite of superior cell growth, implying that agarase production is tightly regulated in an agar-dependent manner and repressed in rich conditions.

Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34

Effect of medium composition and culture conditions on agarase production by Agarivorans albus YKW-34 was investigated in shake flasks. The most suitable carbon source, nitrogen source, and culture temperature were agar, yeast extract, and 25 °C, respectively, for agarase production by one-factor-at-a-time design. The nutritional components of the medium and culture conditions were analyzed by Plackett–Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effects on agarase production ( p < 0.05). The optimum levels of these key variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy enhanced the agarase production from 0.23 U/ml to 0.87 U/ml. The economic medium composition and culture condition as well as the dominant occupation of agarase with high activity in culture fluid enlighten the potential application of A. albus YKW-34 for the production of agarase.

Production and purification of agarase from a marine agarolytic bacteriumAgarivoranssp. HZ105

Isolation and characterization of an agarase-producing bacterium Agarivorans sp. HZ105. An agarase-producing bacterium strain HZ105 had been isolated from marine sediment sample. Based on phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, as well as biochemical analyses, this strain was named Agarivorans sp. HZ105. Effect of pH, NaCl on the growth and agarase production of strain HZ105 was studied. Strain HZ105 produced three extracellular agarases which were purified to homogeneity from bands in the PAGE gel. Two agarases of these three had a molecular mass of 54, 58 kDa, respectively. And the MS and MS/MS spectra were used to identify the agarases. The MS spectra result showed that the agarases of strain HZ105 should be beta-agarase and belong to the family 50 of glycosyl hydrolases. The agarases could keep stable activity at room temperature. The strain HZ105 was useful to produce stable agarases. The solution produced by agar's degradation in the agar plates was first reported to be used for purification of agarase. Agarases were purified to homogeneity directly from the PAGE gel without stained by Coomassie brilliant blue.

Optimization of Culture Conditions for the Production of Extracellular Agarases from Newly Isolated Pseudomonas Aeruginosa AG LSL-11

An agar-degrading bacterium capable of utilizing agar as sole source of carbon and energy was isolated from sea water by enrichment culture technique. The bacterium was identified as Pseudomonas aeruginosa and the culture conditions were standardized for the maximal production of extracellular agarases. The bacterium grew in the pH range 5.0–11.0, optimal between pH 7.0 and 8.0; temperature between 25 °C and 37 °C, optimal at 30 °C and sodium chloride concentration 0–8% and optimal at 2% respectively. The agarases secreted by Pseudomonas aeruginosa AG LSL-11 were inducible by agar and not by any other simple sugars tested. Maximal agarase production was observed at pH 8.0, and temperature 30 °C. The bacterium had no requirement for NaCl for both growth and production of agarases. The bacterium did not utilize other polysaccharides like ĸ-carrageenan, alginate, cellulose and CMC. The activity staining of partially purified agarase preparation after native-PAGE revealed the presence of three different agarases, agarase LSL-11a, LSL-11b and LSL-11c, whose molecular weights were estimated to be 76, 64 and 46 kDa respectively.

Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

An agar-liquefying Acinetobacter species capable of utilizing agar as sole source of carbon and energy was isolated from soil samples and the culture conditions were standardized for the maximal production of extracellular agarase. The bacterium was capable of liquefying an agar-plate within 3 days of incubation and produced extracellular agarase within a short period of time (16–18 h) when grown in defined mineral salts medium. Bacterium grew in the pH range 4.0–9.0, optimal at pH 7.0; temperature 25–40 °C and optimal at 37 °C. The agarase secreted by the Acinetobacter strain was inducible by agar and not repressed by other simple sugars when supplemented along with agar in the medium. The bacterium did not require NaCl for growth or production of agarase. The bacterium did not utilize other polysaccharides like κ-carrageenan, alginate, cellulose, and CMC. The activity staining of partially purified agarase preparations after native-PAGE and SDS PAGE revealed the presence of a single zone of clearance corresponding to the molecular weight 100 kDa, suggesting that it is a monomer. Neoagarobiose was the end product of agarose hydrolysis by this enzyme. The agarase was an endo-type glycosidase and belongs to Group-III β-agarase family.

Effects of phosphate limitation on agarase production by Streptomyces lividans TK21

The dagA gene from Streptomyces coelicolor, encoding an extracellular agarase, has been expressed in Streptomyces lividans on a multicopy plasmid. Chemostat cultures were used to investigate the influence of phosphate limitation on the synthesis and secretion of this enzyme. The strain was cultivated with glucose as the carbon source and magnesium or phosphate as growth-limiting substrates and samples used to determine dagA transcription, agarase secretion and alkaline phosphatase activity under the various limitations. The data show that, compared to growth under magnesium limitation, phosphate-limited cells show increases in dagA transcription, the rate of agarase production and secretion, and alkaline phosphatase activity.

Effects of phosphate limitation on agarase production byStreptomyces lividansTK21

The dagA gene from Streptomyces coelicolor, encoding an extracellular agarase, has been expressed in Streptomyces lividans on a multicopy plasmid. Chemostat cultures were used to investigate the influence of phosphate limitation on the synthesis and secretion of this enzyme. The strain was cultivated with glucose as the carbon source and magnesium or phosphate as growth-limiting substrates and samples used to determine dagA transcription, agarase secretion and alkaline phosphatase activity under the various limitations. The data show that, compared to growth under magnesium limitation, phosphate-limited cells show increases in dagA transcription, the rate of agarase production and secretion, and alkaline phosphatase activity.

Effect of glucose on agarase overproduction by Streptomyces.

The Streptomyces coelicolor dagA gene, coding for an extracellular agarase, has been propagated on a multicopy plasmid in S. coelicolor A3(2), the natural agarase producer strain and in S. lividans TK21, a closely related, nonproducer strain. The effect of the carbon source on the production of agarase by both strains, upon cultivation in liquid medium, revealed that the glucose repression affected the synthesis of agarase at the level of secretion, rather than at the level of transcription. In the presence of glucose, the pre-agarase was degraded intracellularly and the overall secretion of proteases decreased considerably in both strains, suggesting a negative regulatory role for glucose in the overall secretion in Streptomyces.

Purification and Properties of an Extracellular Agarase from Alteromonas sp. Strain C-1

A marine bacterial strain isolated from the Bay of San Vicente, Chile, was identified as Alteromonas sp. strain C-1. In the presence of agar, this strain produced high levels of an extracellular agarase. The production of agarase was repressed by glucose, with a parallel decrease in bacterial growth. The enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with an overall yield of 45%. The enzyme has a molecular weight of 52,000, is salt sensitive, and hydrolyzes agar, yielding neoagarotetraose as the main product, with an optimum pH of about 6.5.

The Agarase Gene (dag A) of Streptomyces coelicolor A3(2): Affinity Purification and Characterization of the Cloned Gene Product

The coding and regulatory sequences of the agarase gene of Streptomyces coelicolor A3(2) were cloned in Streptomyces lividans 66 on the plasmid vector pIJ61, resulting in a several hundred-fold increase in the production of the secreted protein. Subcloning experiments localized the sequences required for agarase production and for the mediation of carbon catabolite repression to a segment of about 1.2 kb. A simple protein purification procedure that uses affinity binding of agarase to agarose beads was developed. Preliminary characterization of the enzyme, together with the results of in vitro transcription-translation studies, suggest that the intracellular form of agarase (about 34 kDa) possesses a signal sequence that is cleaved upon secretion across the cell membrane to produce an extracellular protein of about 29 kDa.

Cloning and expression of an extracellular-agarase gene from Streptomyces coelicolor A3(2) in Streptomyces lividans 66

An extracellular agarase gene was cloned from Streptomyces coelicolor A3(2) strain M130 into S. lividans 66 using the multicopy plasmid vector pIJ702. Various deletion derivatives of the initial clone (pMT605) were obtained by in vitro and in vivo methods. This allowed the gene to be localised to a 1.9-kb segment of DNA. The agarase enzyme was overproduced (up to 500 times) and exported efficiently into the medium. The agarase protein was identified as a 28-kDal band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE); in the case of one derivative, pMT608, this band accounted for nearly 50% of the total extracellular protein. Differences in agarase production between the deletion derivatives correlated well with plasmid stability.

A Chromosomal Locus Controlling Extracellular Agarase Production by Streptomyces coelicolor A 3(2), and its Inactivation by Chromosomal Integration of Plasmid SCP1

The properties of various mutants showed that, in the presence of an inorganic nitrogen source, agar utilization by Streptomyces coelicolor requires the action of at least three groups of enzymes: a diffusible or cell-free extracellular agarase, some further degradative step(s), and the enzymes of galactose metabolism. Two unselected mutations (dag), both leading to loss of diffusible agarase and an undefined second agar utilization function, were detected among a small number of S. coelicolor derivatives from a culture collection, and mapped to the 9 o’clock ‘silent’ region of the genetic map. One of these mutations (dagA1) had apparently resulted, directly or indirectly, from the insertion of the plasmid SCP1 in this region during formation of the NF fertility type. The other (dag-2) was 100% linked to dagA1 and the integrated SCP1, but only 98% linked to the site of insertion of SCP1 in an ‘NF-like’ but Dag+ strain. SCP1-chromosome interactions in other regions of the genetic map had no effect on the agar-solubilizing phenotype, nor had three other unstable genetic phenomena in S. coelicolor.

The regulation of agarase production by resting cells of Cytophaga flevenis.

The regulation of the synthesis of extracellular agarase by Cytophaga flevensis was studied in resting-cell suspensions. Enzyme synthesis was strictly dependent on the presence of a suitable inducer. Enzyme production was maximal at 20 C in phosphate buffer pH 6.9 in the presence of 1.3 mM calcium chloride, 0.03% casamino acids and inducer. Enzyme production was virtually the same at 15 and 20 C, reduced to 50% at 25 C and was not detectable at 30 C. It was highly stimulated by the presence of 0.03% of casamino acids in the incubation mixture and was also favoured by the presence of 1.3 mM calcium ions. Of a variety of compounds tested, only melibiose or neoagaro-oligosaccharides were effective inducers. Among the neoagaro-oligosaccharides, neoagarotetraose was the best inducer. At higher concentrations of inducer compounds catabolite repression of enzyme synthesis was apparent. This was also found when glucose was added to the incubation mixture. This repression was not relieved by the addition of cyclic AMP. Indications were found that the excretion process was limiting the rate of production of extracellular enzyme