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PubMed1993

In vitro and in vivo development of mouse morulae encapsulated in 2% sodium alginate or 0.1% poly-l-lysine.

In Experiment 1, development of zona pellucida-intact (ZPI) morulae was measured every 24 hours for 120 hours after encapsulation in 2% sodium alginate (ALG) or 0.1% poly-L-lysine (PLL). Encapsulation significantly reduced development to hatched blastocysts at 48 and 72 hours. Developmental stages and diameters of ZPI and zona pellucida-free (ZPF) unencapsulated and encapsulated morulae were measured every 24 hours for 72 hours in Exeriment 2. At 72 hours, the percentage of ZPI embryos developing to expanded blastocysts, their diameters and their nuclear counts were not different from each other or from ZPF embryos. In Experiment 3, ZPI morulae encapsulated in ALG or PLL were transferred into recipients. Five of six recipients that received unencapsulated embryos (n=71) delivered 16 live pups. None of the recipients of encapsulated embryos delivered offspring; therefore, a final experiment was performed to examine fetal development on Day 10 of gestation. The percentage of pregnant recipients was similar for all 3 treatments: unencapsulated (71.4%), ALG (87.5%) and PLL (87.5%). However, the presence of viable fetuses was higher for unencapsulated embryos (42.1%) than for ALG (17%) and PLL (14.6%) embryos. These results suggest that encapsulation did not detrimentally affect embryonic size or cellular development in vitro; however, mortality occurred in vivo due to an asynchronous condition between the uterine environment and the embryos.

Journal Article↗
PubMed1996

Heterologous expression of full-length and truncated forms of the recombinant guluronate-specific alginate lyase of Klebsiella pneumoniae.

The alyA gene, which encodes a guluronate-specific alginate lyase from Klebsiella pneumoniae, was cloned into the plasmid pCT54 to facilitate heterologous expression of the enzyme in Escherichia coli. The greatest enzyme specific activity was observed when the complete gene and flanking regions of DNA (contained within a 1.95 kb HindIII fragment) were cloned in the correct orientation relative to the vector-encoded trp promoter. Heterologous expression of the intact gene complete with flanking regions resulted in a 20-fold increase in enzyme yield compared to the original construct, pRC5. PCR mutagenesis and/or restriction endonuclease digestion was used to generate a series of fragments containing either the whole or truncated versions of the gene. Active enzyme was produced from constructs in which the region encoding the signal peptide had been deleted, although the recombinant protein was retained within the bacterial cells. An internal methionine (position 74) could be used as a start site for translation but resulted in the accumulation of inactive protein within inclusion bodies.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and heterologous expression of a Klebsiella pneumoniae gene encoding alginate lyase.

The alginate lyase (Aly; guluronate specific)-coding gene of Klebsiella pneumoniae was cloned using the cosmid vector pMMB33, transduced into Escherichia coli and expressed in this host. Four Aly-positive clones with unstable phenotypes were identified out of 700 kanamycin-resistant transductants. A stable derivative of one of the clones was studied further and contained 12.1-kb of insert DNA. The Aly-coding gene (aly), still partially under the control of its native promoter, was localised within a 1.95-kb HindIII fragment by transposon gamma delta mutagenesis and sub-cloning. Most of the Aly produced was secreted into the medium by both the original K. pneumoniae strain (71.7%) and the E. coli recombinant clones (85.1%). The enzyme from both K. pneumoniae and the E. coli clones had a pI of 8.9 and comprised a single 28-kDa polypeptide chain. Other minor bands were also observed on isoelectric focusing and these were attributed to processing intermediates of a single gene product. It is concluded that E. coli can recognise and process the signal peptide of Aly to produce a mature polypeptide that is identical to that synthesised by K. pneumoniae.

Cloning, Molecular↗
PubMed1994

Alginate lyase from Klebsiella pneumoniae, subsp. aerogenes: gene cloning, sequence analysis and high-level production in Escherichia coli.

The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25, has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa, in good agreement with SDS-PAGE analysis, and a calculated pI of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus, probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse a beta-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.

Amino Acid Sequence↗
PubMed2006

Microbiological and sensory quality of dry fermented sausages containing alginate-microencapsulated Lactobacillus reuteri.

The probiotic bacterium Lactobacillus reuteri was added to dry sausage batter, without or after being microencapsulated in alginate using either extrusion or emulsion technology. Pediococcus pentosaceus and Staphylococcus carnosus were added at 7 log cfu/g as starter cultures for fermentation. The sausage batter was stuffed in 55 mm fibrous casings and fermented, with smoking, at <or=26 degrees C and 88% relative humidity (RH) for 72 h. During fermentation as well as drying at 75% RH and 13 degrees C for 25 days, the pH and water activity (a(w)) of the sausages were measured. The numbers of the three organisms in sausages during processing were determined. A consumer taste panel study was conducted to determine if addition of L. reuteri as unencapsulated cells or in microcapsules added at 1% (w/w) affected the sensory quality of the dry fermented sausages. Changes in sausage pH from 5.8 to 4.8, and a(w) from 0.97 to 0.89, were the same for both control and microcapsule-containing sausages. The numbers of unencapsulated L. reuteri cells dropped by 2.6 log units, whereas microencapsulated L. reuteri were reduced by only <or=0.5 log unit after drying. No significant difference in sensory quality was found between control and sausages containing either unencapsulated or microencapsulated L. reuteri. Thus, microencapsulation of probiotics like L. reuteri may be an option for formulation of fermented meat products with viable heath-promoting bacteria.

Animals↗
PubMed2004

NMR relaxation and pulsed field gradient study of alginate bead porous media.

Experiments are presented, which correlate molecular displacement with the multi-exponential T2 relaxation times of water flowing and diffusing through an alginate bead pack. Three systems were studied comprising beads of 3, 1 or < mm in diameter. T2-resolved propagators were obtained through a combined pulsed gradient stimulated echo (PGSTE) and Carr-Purcell-Meiboom-Gill (CPMG) experiment. Fourier transformation with respect to q produces a propagator for each echo in the CPMG train. Inverse Laplace transformation of the CPMG decays for each point (Z) in the propagator produced a two-dimensional propagator. Analysis of these two-dimensional propagators provided insight into the transport and exchange behaviour of water flowing through this system. This experiment has been simulated in a model bead structure and the resulting T2 relaxation time behaviour and T2-resolved propagators were found to be in good agreement with the experimental data. We also present a theoretical analysis of the response to the combined PGSTE/CPMG sequence in the simple model case of Pouseille flow in a cylindrical capillary, where diffusion to a surface sink is assumed to be the dominant relaxation mechanism.

Journal Article↗
PubMed2001

Controlled malolactic fermentation in cider using Oenococcus oeni immobilized in alginate beads and comparison with free cell fermentation.

Cells of Oenococcus oeni (formerly Leuconostoc oenos) immobilized in alginate beads were used as starter culture to conduct malolactic fermentation in cider production. Concentrations of major organic acids and volatile compounds were monitored during the process, and results were compared to those obtained when using free cells in the same conditions. The rates of malic acid consumption were similar but lower ethanoic acid content and higher concentration of alcohols were detected with immobilized cells. These features have beneficial effects on the organoleptic properties of cider. A comparison between the kinetic behavior in immobilized and free cells, based on the data obtained for the malic acid consumption, has been developed solving the homogeneous diffusion model when it is applied to the system with immobilized cells.

Journal Article↗
PubMed2001

An efficient purification process for sweet potato beta-amylase by affinity precipitation with alginate.

beta-amylases are used in production of maltose syrup. It is shown that sweet potato beta-amylase can be purified by affinity precipitation with alginate with 80% activity yield and 44 fold purification. SDS-PAGE of the purified protein showed a single band and a subunit weight of 50 kDa. Preliminary data with soybean and barley enzymes indicate that this may be a general method for purification of beta-amylases.

Journal Article↗
PubMed2000

Immobilization of Nicotiana tabacum plant cell suspensions within calcium alginate gel beads for the production of enhanced amounts of scopolin.

Scopolin-producing cells of Nicotiana tabacum were immobilized within Ca-alginate gel beads. Free cell suspensions accumulated scopolin within cytoplasmic compartments and cell disruption was necessary to recover scopolin. On the contrary, immobilized plant cells excreted considerable amounts of scopolin. Scopolin diffused throughout the gel matrix and reached the culture media. A large fraction of produced scopolin could then be recovered from the culture medium without disrupting cells. Immobilized N. tabacum cells produced more scopolin than free cell suspensions did (3.8 mg/g fresh weight biomass [into the culture media] versus 0.2 mg/g fresh weight biomass [intracellular]). Variation of the immobilization conditions revealed a marked influence on the behavior of N. tabacum plant cells: production of scopolin and enhanced excretion, cell growth, and morphological aspect of plant cell colonies. This excretion phenomenon could be used advantageously at an industrial production level.

Journal Article↗
PubMed2000

Crystallization and preliminary X-ray crystallographic analysis of alginate lyase A1-II from Sphingomonas species A1.

Alginate lyase A1-II of Sphingomonas species A1 was purified and crystallized using the hanging drop vapor-diffusion method in 0.1 M Tris-HCl buffer containing 43% saturated ammonium sulfate, 8% polyethylene glycol 4000 and 0.2 M Li(2)SO(4) at pH 8.5 and 20 degrees C. The crystals are tetragonal and belong to the space group P4(3)2(1)2 or P4(1)2(1)2 with unit cell dimensions of a=b=144.07 and c=296.38 A. The diffraction data up to 2.8 A were collected by a synchrotron radiation source at SPring-8 in Japan.

Bacterial Proteins↗
PubMed2001

Enumeration and factors influencing the relative abundance of a denitrifier, Pseudomonas sp. JR12, entrapped in alginate beads.

The relative abundance of the denitrifier, Pseudomonas sp. JR12, was examined in an alginate-based entrapment complex under non-sterile, denitrifying conditions. Immuno-labeling of the Pseudomonas inoculant followed by flow cytometry (FCM) was used for determination of the relative abundance of this bacterium under the various incubation conditions. Additional information on the relative abundance of the inoculant was obtained by a quantitative enzyme-linked immunosorbent assay (ELISA) and results obtained by FCM and ELISA were compared. Ambient nitrate levels controlled the successful, long-term proliferation of the inoculant. At low ambient nitrate levels, Pseudomonas sp. remained the dominant microorganism during incubation. Higher ambient nitrate concentrations, attained by either decreasing the inoculum size of Pseudomonas sp. or raising inlet nitrate concentrations of the medium supplied to the incubation vessels, resulted in a gradual shift toward other, nitrite-accumulating denitrifiers. Thus far, most studies on the use of entrapped microorganisms for bioremediation purposes have been conducted under controlled laboratory conditions. Based on this study, conducted under non-sterile laboratory conditions, it is concluded that in-situ bioremediation using entrapped target microorganisms is bound to fail without a proper understanding of the factors that cause the target microorganism to out-compete undesired microbial invaders. Furthermore, based on the close agreement between the two detection methods used, it is concluded that flow cytometry provides a rapid and accurate tool for the detection of the relative abundance of immuno-labeled target organisms in heterogeneous microbial populations.

Environmental Monitoring↗
PubMed2000

Invertase production by Saccharomyces cerevisiae protoplasts immobilized in strontium alginate gel beads.

The invertase productivity of Saccharomyces cerevisiae IFO 0309 protoplasts in a static culture was 35 times (extracellular) and 9 times (extracellular + intracellular) higher than those of cells. When S. cerevisiae protoplasts were immobilized in 1.0% strontium alginate gel as an artificial cell wall, the protoplasts could be cultivated in a shake flask without disruption, and invertase was secreted into the broth. However, cell wall regeneration in the immobilized protoplasts was detected at about 24 h of cultivation. This implies that prevention of cell wall regeneration is a prerequisite for long term process with protoplasts. When 0.5 microg/ml aculeacin A (inhibitor of beta-1,3 glucan synthesis) was added to the broth, active protoplasts were maintained without cell wall regeneration for more than 24 h and invertase was produced extracellularly. Immobilized S. cerevisiae T7 protoplasts (this strain produces twice as much invertase as S. cerevisiae IFO 0309) were used for invertase production in a bubble column reactor, and a high and stable level of invertase (45 U/ml) was maintained for 72 h.

Journal Article↗
PubMed1998

Asymmetric reduction of acetophenone with calcium-alginate-entrapped Baker's yeast in organic solvents

Baker's yeast cells entrapped in alginate beads are shown to catalyze reactions in organic solvents when a cofactor regeneration scheme is implemented. This study focused on the reduction of acetophenone to 1-phenylethanol, using baker's yeast as well as a cosubstrate to regenerate the cofactor. The product is a chiral alcohol, and it was desired to maintain a high enantiomeric excess. The effects of parameters such as the addition of a cosubstrate, water content, fermentation time, buffer pH, and bead diameter have been investigated. Such a general process may be quite useful when single enantiomers are needed, as well as for the production of other chemicals.

Journal Article↗
PubMed1999

Survival of Lactobacillus helveticus entrapped in Ca-alginate in relation to water content, storage and rehydration.

Lactobacillus helveticus CNRZ 303 entrapped in Ca-alginate gel beads was investigated for improved survival and stability during fluidized-bed drying, storage and rehydration. Addition of protective solutes was very important. Studies of the conditions showed that inactivation of entrapped L. helveticus started when the water content exceeded 0.3-0.4 g H2O (g dry wt)-1 for adonitol, glycerol and reconstituted non fat milk solids (NFSM). With Ringer's solution (control) and betaine, the fall in viability was evident above 1 g H2O (g dry wt)-1. Drying down to 0.2 g H2O (g dry wt)-1 required the removal of 98.5-98.9% of the water. The best survival rate with the least injured cells among survivors was experienced with adonitol and NFMS, respectively, 71% and 57% (compared to the initial) immediately after dehydration. Adonitol and NFMS were also best for survival during storage. The highest cell recovery was obtained by rehydrating the cells in cheese whey permeate between 20-30 degrees C done at pH 6.0-7.0, satisfying the demands for cell survival, repair and slow swelling (adaptions).

Journal Article↗
PubMed2004

Final report of the amended safety assessment of Glyceryl Laurate, Glyceryl Laurate SE, Glyceryl Laurate/Oleate, Glyceryl Adipate, Glyceryl Alginate, Glyceryl Arachidate, Glyceryl Arachidonate, Glyceryl Behenate, Glyceryl Caprate, Glyceryl Caprylate, Glyceryl Caprylate/Caprate, Glyceryl Citrate/Lactate/Linoleate/Oleate, Glyceryl Cocoate, Glyceryl Collagenate, Glyceryl Erucate, Glyceryl Hydrogenated Rosinate, Glyceryl Hydrogenated Soyate, Glyceryl Hydroxystearate, Glyceryl Isopalmitate, Glyceryl Isostearate, Glyceryl Isostearate/Myristate, Glyceryl Isostearates, Glyceryl Lanolate, Glyceryl Linoleate, Glyceryl Linolenate, Glyceryl Montanate, Glyceryl Myristate, Glyceryl Isotridecanoate/Stearate/Adipate, Glyceryl Oleate SE, Glyceryl Oleate/Elaidate, Glyceryl Palmitate, Glyceryl Palmitate/Stearate, Glyceryl Palmitoleate, Glyceryl Pentadecanoate, Glyceryl Polyacrylate, Glyceryl Rosinate, Glyceryl Sesquioleate, Glyceryl/Sorbitol Oleate/Hydroxystearate, Glyceryl Stearate/Acetate, Glyceryl Stearate/Maleate, Glyceryl Tallowate, Glyceryl Thiopropionate, and Glyceryl Undecylenate.

The safety of 43 glyceryl monoesters listed as cosmetic ingredients was reviewed in a safety assessment completed in 2000. Additional safety test data pertaining to Glyceryl Rosinate and Glyceryl Hydrogenated Rosinate were received and served as the basis for this amended report. Glyceryl monoesters are used mostly as skin-conditioning agents--emollients and/or surfactant--emulsifying agents in cosmetics. The following 20 glyceryl monoesters are currently reported to be used in cosmetics: Glyceryl Laurate, Glyceryl Alginate, Glyceryl Arachidonate, Glyceryl Behenate, Glyceryl Caprylate, Glyceryl Caprylate/Caprate, Glyceryl Cocoate, Glyceryl Erucate, Glyceryl Hydroxystearate, Glyceryl Isostearate, Glyceryl Lanolate, Glyceryl Linoleate, Glyceryl Linolenate, Glyceryl Myristate, Glyceryl Oleate/Elaidate, Glyceryl Palmitate, Glyceryl Polyacrylate, Glyceryl Rosinate, Glyceryl Stearate/Acetate, and Glyceryl Undecylenate. Concentration of use data received from the cosmetics industry in 1999 indicate that Glyceryl Monoesters are used at concentrations up to 12% in cosmetic products. Glyceryl Monoesters are not pure monoesters, but are mostly mixtures with mono-, di-, and tri-esters. The purity of commercial and conventional Monoglyceride (Glyceryl Monoester) is a minimum of 90%. Glyceryl Monoesters (monoglycerides) are metabolized to free fatty acids and glycerol, both of which are available for the resynthesis of triglycerides. Glyceryl Laurate enhanced the penetration of drugs through cadaverous skin and hairless rat skin in vitro and has been described as having a wide spectrum of antimicrobial activity. A low-grade irritant response was observed following inhalation of an aerosol containing 10% Glyceryl Laurate by test animals. Glyceryl monoesters have little acute or short-term toxicity in animals, and no toxicity was noted following chronic administration of a mixture consisting mostly of glyceryl di- and mono- esters. Glyceryl Laurate did have strong hemolytic activity in an in vitro assay using sheep erythrocytes. Glyceryl Laurate, Glyceryl Isostearate, or Glyceryl Citrate/Lactate/Linoleate/Oleate were not classified as ocular irritants in rabbits. Undiluted glyceryl monoesters may produce minor skin irritation, especially in abraded skin, but in general these ingredients are not irritating at concentrations used in cosmetics. Glyceryl monoesters are not sensitizers, except that Glyceryl Rosinate and Hydrogenated Glyceryl Rosinate may contain residual rosin, which can cause allergic reactions. These ingredients are not photosensitizers. Glyceryl Citrate/Lactate/Linoleate/Oleate was not mutagenic in the Ames test system. Glyceryl Laurate exhibited antitumor activity and Glyceryl Stearate was negative in a tumor promotion assay. At concentrations higher than used in cosmetics, Glyceryl Laurate did cause moderate erythema in human repeat-insult patch test (RIPT) studies, but the other glyceryl monoesters tested failed to produce any significant positive reactions. Glyceryl Rosinate was irritating to animal skin at 50%, but did not produce sensitization in clinical tests at concentrations up to 10% and covered with semioccluded patches. There is reported use of Glyceryl Rosinate at 12%in mascara, which is somewhat higher than the concentration in the clinical testing. It was reasoned that the available data do support the safety of this use because there would be minimal contact with the skin and no occlusion. The safety of Arachidonic Acid was not documented and substantiated for cosmetic product use in an earlier safety assessment and those same safety questions apply to Glyceryl Arachidonate. Based on these data, the Cosmetic Ingredient Review (CIR) Expert Panel found that these glyceryl monoesters are safe as cosmetic ingredients in the present practices of use and concentration: except that the available data are insufficient to support the safety of Glyceryl Arachidonate. Additional data needed to support the safety of Glyceryl Arachidonate include (1) dermal absorption data; and, based on the results of the absorption studies, there may be a need for (2) immunomodulatory data; (3) carcinogenicity and photocarcinogenicity data; and (4) human irritation, sensitization, and photosensitization data.

Animals↗
PubMed2006

Enhancing the drug metabolism activities of C3A--a human hepatocyte cell line--by tissue engineering within alginate scaffolds.

In this study, we investigated the applicability of C3A--a human hepatocyte cell line--as a predicting tool for drug metabolism by applying tissue-engineering methods. Cultivation of C3A cells within alginate scaffolds induced the formation of spheroids with enhanced drug metabolism activities compared to that of two-dimensional (2-D) monolayer cultures. The spheroid formation process was demonstrated via histology, immunohistochemistry, and transmission electron microscope (TEM) analyses. The C3A spheroids displayed multilayer cell morphology, characterized by a large number of tight junctions, polar cells, and bile canaliculi, similar to spheroids of primary hepatocytes. Spheroid formation was accompanied by a reduction in P-glycoprotein (Pgp) gene expression and C3A cell proliferation was limited mainly to cells on the spheroid outskirt. The 3-D constructs maintained a nearly constant cell number according to MTT assay. Drug metabolism by the two most important cytochrome p-450 (CYP) enzymes in human liver, CYP1A2 and CYP3A4, was tested using preferred drugs. With CYP1A2, 3-fold enhancement in activity per cell was seen for converting ethoxyresorufin to resorufin compared to C3A cell monolayers. The spheroids responded to the inducer beta-naphthoflavone and to the inhibitor furafylline of CYP1A2. Enhanced metabolizing activity of CYP3A4, measured by the amount 6beta-testosterone formed from testosterone, and that of the phase II enzyme glucuronosyltransferases (UGT) further indicated that the tissue-engineered C3A spheroids may provide an efficient experimental tool for predicting drug activities by these CYPs. Moreover, the maintenance of constant cell number, as well as the elevated hepatocellular functions and drug metabolism activities, suggest that the tissue-engineered C3A may be applicable in replacement therapies.

Androgens↗
PubMed2006

Platelet-rich plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads.

STUDY DESIGN: In vitro assessment of the effects of platelet-rich plasma on the extracellular matrix metabolism of porcine intervertebral disc cells. OBJECTIVES: To determine whether platelet-rich plasma is effective in stimulating cell proliferation and extracellular matrix metabolism by porcine disc cells cultured in alginate beads. SUMMARY OF BACKGROUND DATA: Platelet-rich plasma is used to accelerate wound healing and tissue regeneration. Activated platelets release multiple growth factors that regulate cell proliferation, differentiation, and morphogenesis. Individual growth factors present in platelet-rich plasma have been demonstrated to affect the metabolism of intervertebral disc cells. METHODS: Platelet-poor and platelet-rich plasma was isolated from fresh porcine blood using a commercially available platelet concentration system. After preculture for 7 days and serum starvation for 24 hours, the beads containing nucleus pulposus and anulus fibrosus cells were then cultured for another 72 hours in serum-free medium, 10% fetal bovine serum, 10% platelet-poor plasma, or 10% platelet-rich plasma. The synthesis of proteoglycans and collagen, the accumulation of proteoglycans, and the DNA content were biochemically assessed. RESULTS: Platelet-rich plasma had a mild stimulatory effect on cell proliferation of intervertebral disc cells. Platelet-rich plasma treatment significantly upregulated proteoglycan and collagen synthesis and proteoglycan accumulation when compared with platelet-poor plasma. CONCLUSIONS: Platelet-rich plasma was effective in stimulating cell proliferation and extracellular matrix metabolism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleuspulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimulating cells to engineer intervertebral disc tissues.

Animals↗
PubMed1993

Cloning, sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp. OS-ALG-9.

A gene (aly) encoding alginate lyase (ALY; EC 4.2.2.3) was isolated from a library constructed with the cosmid vector pHC79 and Sau3AI-digested genomic DNA of Pseudomonas sp. OS-ALG-9. Successive subcloning of the aly-containing cosmid enabled us to locate the gene on a 2.3 kb HpaI fragment. Nucleotide sequencing of this fragment revealed a single open reading frame (ORF) of 1365 bp. The directly determined N-terminal amino acid sequence of the ALY protein purified from Pseudomonas sp. OS-ALG-9 was found in the amino acid sequence deduced from this ORF between nucleotides 282 and 366. Expression of aly was induced by IPTG in Escherichia coli and leakage of the enzyme into the extracellular milieu was significantly enhanced by addition of glycine to the growth medium. The ALY enzyme had a greater specificity for the homopolymer of mannuronate than for that of guluronate.

Amino Acid Sequence↗