EP3844292A1 - Methods for enzymatic processing of polysaccharides from brown algae - Google Patents

Abstract

A cell-free one-pot enzymatic process for producing 2-keto-3-deoxy-gluconate (KDG) from alginate, comprising bringing alginate in contact with at least one alginate lyase enzyme for the conversion of alginate to monomeric uronate (DEH), bringing the obtained DEH in contact with a DEH reductase enzyme and NADPH for the conversion of DEH to KDG, whereby NADPH is oxidized to NADP+, and bringing obtained NADP+ in contact with a glucose dehydrogenase (GDH) enzyme and glucose, for the regeneration of NAD(P)H from NAD(P)+, whereby glucose is oxidized to gluconic acid.The glucose can advantageously be provided in situ by enzymatic conversion of laminarin. The gluconic acid can further be converted in the same one-pot system to additional KDG using a suitable enzyme such as a dihydroxyacid dehydratase enzyme.

Description

Methods for Enzymatic Processing of Polysaccharides from Brown algae

Field of Invention

The invention is within the field of enzymatic processing of biomaterials, specifically alginate, for the production of value-added chemical products, more specifically 2-keto- 3-deoxy-gluconate (KDG).

BackgroundSecond and third generation biomass materials, lignocellulose and algal biomass, respectively, are increasingly seen as alternative sustainable feedstocks for industrial biorefinery processes, that can supplement or supplant fossil-fuel feedstocks and/or 1^st generation biomass from agricultural crops. First generation biomass includes sugars, vegetable oils and such crops that could be used as food or feed source. Biofuels from these sources are referred to as first generation biofuels. Second-generation biomass is a more loosely defined term and refers typically to lignocellulosic biomass or woody crops, agricultural residues or waste, as well as dedicated non-food energy crops grown on marginal land unsuitable for food production. (Some second generation biomass could still be competing with food crops for land use.) Third generation biomass does not compete with other crops for land use. The term typically refers to (but not always limited to) algae biomass. The term is also sometimes used referring to further alternative renewable sources such as utilisation of C02. Much effort is currently put in developing lignocellulose biomass as feedstocks for biorefineries, but the potential of using macroalgal biomass as a feedstock has only recently received attention.

Currently the seaweed processing industry targets mainly the food and feed sectors supplying primary products, hydrocolloids, alginate and carrageenan, seaweed meal and to some extent ingredients to food and feed commodities. These products are based on physical/chemical extraction and fractionation technologies. The full potential of seaweed biomass has, however, not been realized. By further refining of the constituent polysaccharides seaweed biomass could become an important source of bulk precursor chemicals and building blocks for the chemical industry for production of specialty chemicals and biopolymers. At this stage, practical industrial refining tools and processes are lacking.

Brown macroalgae ( Phaeophyceae ), are some of the most promising potential biorefinery feedstock species (especially in the North Atlantic) because of the potential high bulk biomass production and high content of carbohydrates. Their growth rates and productivities far exceed those of terrestrial plants (1).

The major constituent carbohydrates of Brown algae are alginate, fucoidan, laminarin and mannitol can amount up to 60% of dry weight (1). Of these, alginate and laminarin are most suitable for production of platform chemicals. The former, alginate, is a structural hetero-polysaccharide in the cell wall composed of two types of uronic acids, mannuronic and glucuronic acid with alpha- and beta-1,4 linkages (1). The content of alginate is relatively constant and can constitute up to at least than 1/3 of the total carbohydrates of Brown algae (2).

Alginate is highly resistant to physiochemical degradation and large quantity of acid is required for efficient industrial hydrolysis. This also leads to partial degradation of the constituent mono-uronates, guluronic and mannuronic acids and the subsequent neutralization and removal of the hydrolysing chemicals is costly.

The b-glucan laminarin is composed of glucose units linked through beta-1,3 linkages interspersed and with beta-1,6 linkages (1). Laminarin is the major storage poly saccharide in brown algae. The content measured by glucose content varies and depends on species and season but can reach up to 50% of the dry weight in some species under optimal conditions (2)

Cell free synthesis: Cell free systems (CFS) for manufacturing products of interest in vitro using linked enzymatic reactions can in advantageous settings provide more resource-efficient metabolism than cell-based systems. In a CFS process resources are not diverted to sustaining life and growth, cell energetics, metabolic load, and thresholds because of substrate/product transport through cell membranes are bypassed. Each CFS component can then be optimized with respect to concerted activity in a one-pot system. CFS can involve many components and reactions can be synchronized, optimized and concerted in order to obtain a stable and effective industrial process.

KDG : The biochemical compound 2-keto-3-deoxy-gluconate, or KDG, also known as 3- deoxy-2-oxo-D-gluconate, is a potentially useful industrial platform chemical. Its full IUPAC name is (4S,5R)-4,5,6-trihydroxy-2-oxohexanoic acid. It is a keto-aldonic acid of interest to the chemical industry as a precursor in the synthesis of a variety of complex bio-based products, especially furanics such as FDCA (furandicarboxylic acid)(3), and further on to bioplastics/biopolymers (4). FDCA can be used in the synthesis of production of polyesters, e.g. PEF, PPF, biofuels or other high value chemicals. Furanics have been called "Sleeping Giants" because of their enormous market potential and its position in the list of the top 12 of high potential biobased products by the US Department of Energy (5). They have not been commercialized as they cannot be produced in an economic fashion with current technologies. The main companies involved in developing biobased production of FDCA from sugars are DuPont, BASF, Aventium and Corbion. A 60-metric-ton-per-year pilot plant was constructed by DuPont to help develop markets for production of FDCA from enzymatically derived fructose from corn. (See:

The drawbacks are that this process is based on edible feedstock derived from arable land, i.e. conversion of fructose enzymatically derived from corn starch. Furthermore, 5-Hydroxymethylfurfura! (HMF) an intermediate in the pathway is an unstable compound (3).

As regards alginate the synthesis of 2,5-FDCA from uronates goes back almost one and a half centuries, but yields were moderate and intermediates not readily available. This is predicted to change as mono-uronates and derivatives thereof become more widely available especially from macroalgal bio-refineries (3, 6, 7, 8).

KDG is also a starting compound for the synthesis of pharmaceuticals (antiviral, anticancer or antisense agents) via conversion into 2'-deoxynucleoside compounds or 2'-deoxynucleoside precursors which is a patented process (9).

Summary

The present invention relates to production of the biochemical compound 2-keto-3- deoxy-gluconate (KDG) as the single main product and final product and preferably the only product, in a multienzyme synchronized cell-free process which is preferably a "one-pot" process, directly from alginate and glucose or a glucose source that can be laminarin. Alginate and laminarin are two major polysaccharides found in brown seaweed (macroalgae), a 3^rd generation biomass source.

With the new process described and claimed herein, several enzymes are utilized and their enzymatic reactions combined to provide a process for effective and economical conversion of alginate and glucose into KDG, which is a useful and valuable intermediate. The glucose can suitably be provided as a polysaccharide glucose source, converted in situ as part of the overall process, such as laminarin from one and the same feedstock that provides the alginate reagent.

Methods are provided here for the processing of the above-mentioned polysaccharides directly in a one- pot cell-free synthesis comprising a series of enzymatic reactions steps for the production of KDG (Formula I), using a combination of enzymes for catalyzing key reactions for the conversion of the biomass sugars into useful intermediates and end products.

Formula I The process involves regeneration of the cofactor NAD(P)H from NAD(P)⁺ using glucose dehydrogenase that catalyzes the oxidation of glucose to gluconic acid . Gluconic acid is then converted by another enzyme to KDG. Glucose from various sources can be supplied directly to the enzymatic mixture. By adding appropriate enzymes such as laminarinases and beta-glucosidases to the mixture, glucose can also be derived from polysaccharide such as the polysaccharide laminarin, added as substrate to the reaction mixture, e.g . from the same species of brown algae that is the source of the alginate for the process. Other glucans, both a- and b-glucans with appropriate enzymes can substitute for laminarin in the process. For a schematic overview of the process using alginate and laminarin see Figure 1.

Using the methods disclosed, KDG can be generated as the single main product and final product in a one-pot cell-free enzymatic process directly from alginate and laminarin, the two major polysaccharides found in brown seaweed (macroalgae) .

Useful enzymes in the process of the invention and their role in the cell-free enzymatic pathway include the following (see Figures 1 and 2) :

1. Endo-active alginate lyases ( 10, 11) and exo-active alginate lyases such as oligo-alginate lyases ( 12, 13) ; by their combined action alginate is degraded into unsaturated mono-uronic acid (from both mannuronic and guluronic acid) that spontaneously converts to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) .

2. KdgF-enzyme: can be optionally included in the process for accelerating the conversion of unsaturated uronic acid to DEFI ( 14) (-t2) .

3. DEFI-reductase: reduces DEFI to KDG using NAD(P)FI as cofactor which is oxidized to NAD(P)⁺ ( 14, 15) .

4. Glucose dehydrogenase (GDH) : oxidizes glucose in the enzymatic reaction mixture to gluconic acid, concomitantly regenerating NAD(P)FI from NAD(P) ⁺ ( 16) .

5. Dihydroxy-acid dehydratase or gluconate dehydratase : converts gluconic acid to KDG ( 17, 18) .

6. Endo-glucanases hydrolyzing beta-linkages, such as laminarinases or beta- glucosidases or a mixture of such enzymes, for liberating glucose from the polysaccharide laminarin or other beta- 1,3 glucan polysaccharides ( 19,20) .

The enzymes can be obtained and produced as described in the above cited references which are all hereby incorporated in full.

In one aspect, the invention provides a method for the production of 2-keto-3-deoxy- gluconate (KDG) comprising the steps : a) using at least one alginate lyases for the conversion of alginate to monomeric uronate (DEH). The at least one alginate lyase preferably comprises at least one of and more preferably both of an exo- and an endo-type alginate lyase;

b) using DEH reductase and NAD(P)H for the conversion of DEH to KDG whereupon the NAD(P)H is oxidized to NAD(P)⁺;

c) using glucose and a glucose dehydrogenase and/or glucose oxidase (GDH) for the regeneration of NAD(P)H from NAD⁺, whereupon the glucose is oxidized to gluconic acid/gluconate; and

d) converting at least part of said gluconic acid to KDG, by bringing it in contact with a suitable enzyme.

The above method has been experimentally tested and verified as shown in the accompanying Examples.

In one embodiment, the method above further comprises provision of said glucose in situ by enzymatic conversion of a polysaccharide to glucose using one or more suitable enzyme. In situ, in this context means that this conversion takes place in the same reactant medium as the subsequent oxidation of the glucose (step (c)), thus the reagent polysaccharide and suitable enzyme(s) for its conversion are provided in the reaction medium in which the subsequent reaction takes place. The reagent polysaccharide acting as glucose source comprises in preferred embodiments a beta-glucan and/or alpha-glucan polysaccharide. In some embodiments a beta-1, 3-glucan such as laminarin is used for conversion to glucose using one or more suitable enzymes such as laminarinase and beta-glucosidase. In some embodiments, both starting materials, alginate and beta-1, 3-glucans, can be obtained from separate suitable biomass sources. In another embodiment they can both be obtained from the same feedstock, for example from one or more species belonging to the class of Phaeophyceae (Brown algae).

In step (d), in addition to the pathway producing KDG from DEH, KDG is additionally generated from at least part of and preferably most or substantially all of the gluconic acid/gluconate derived from the glucose, using an additional enzyme such as dihydroxy acid dehydratase (17,18). This means that KDG is obtained both from the provided alginate and from the provided glucose, which in preferred embodiments is obtained in situ (in the same reaction medium in which the glucose is converted to gluconate) from laminarin as described further above. A non-limiting example of such double-pathway CFS for generating KDG is illustrated schematically in Figure 1.

The enzymes can conveniently all be in solution or in some embodiment one or more of the enzymes is immobilized on solid support such as bot not limited to beads (rein beads, magnetic beads, etc.), monolithic carriers, etc. Brief description of Figures

Figure 1. Schematic figure showing one-pot enzymatic cell free synthesis of KDG directly from alginate and laminarin according to the invention.

Figure 2. The chemical reactions taking place and the enzymes involved in an embodiment of the process of the invention for the production of KDG from alginate and laminarin.

Figure 3. SDS gels showing expression of the enzymes, Adh51, GDFI 16 and KdgF.

Figure 4. Alginic acid degradation in six different solutions prepared according to Table 1 using two different pH levels, pH 5.5 (A), and pH 6.8 (B). The samples were incubated at 45°C over a time-course of 24, 48 and 72 hours. Complete degradation of alginate to DEFI was observed using a combination of both Alg3 and Alg4 at pH 6.8

Figure 5. The effects of different pH levels ranging from 5.0 to 9.0 on the efficiency of Alg4 (A), Alg3 (B) and the two enzymes combined (C) during alginic acid degradation over a 22-hour time period. Activity was observed in the pH range from pH 5.5 to pH9.

Figure 6. Enzymatic synthesis of KDG from unsaturated mono-uronates (/DEFI) using NADH as a cofactor in a limiting concentration and different amounts of glucose (prepared according to table 2). With higher concentrations of glucose higher KDG yields are observed. This shows that NADFI is regenerated with concomitant glucose oxidation.

Figure 7. Enzymatic synthesis of KDG directly from alginate using enzyme mixtures prepared according to table 3). The reactions were carried out different temperatures, ranging from 40° to 70°C. All reaction mixtures comprised alginate, glucose, NADFI, Alg3, Alg4, Adh51, and GDFI16. Efficient synthesis directly from alginate could be observed at all temperatures tested, from 40° to 70°C.

Figure 8. Production of KDG from mono-uronates, (DEFI). The reaction mixtures differed in concentration of NAD and NADFI according to table 3. They were incubated for 40 minutes at 55°C. DEFI, the mono-uronate substrate used in the reactions, was used as a control (C). Samples were taken at intervals, at the timepoints 0, 5, 10, 20 and 40 minutes. 8. A) 0. 5 mg/mL NAD or NADFI 8. B) 2.5 mg/mL NAD or NADFI. Production of KDG increased during the time-course, using either NAD or NADFI. Description

The invention provides a cell-free process for producing 2-keto-3-deoxy-gluconate (KDG) from alginate. The term "cell-free-system" indicates that the process takes place outside a living cell, such as in a suitable conventional reactor, preferably by linked concerted enzymatic reactions.

Alginate and laminarin are unconventional polysaccharides in the current industrial context. They are relatively new to the chemical industry and product possibilities are largely unexplored. Robust industrial enzymes have been lacking and biocatalytic refining of macroalgae polysaccharides to added-value products in industrial settings is near non-existent. Alginate, especially, is recalcitrant towards physicochemical degradation due to heterogeneous sugar composition, sugar type (uronic acids) and apparent strong glyosidic linkages.

A CFS process based on polysaccharides as provided by this invention requires sequential concerted enzymatic reactions for efficient degradation and further refining of seaweed polysaccharides to added value products. The enzymes should optimally match each other in relevant biochemical properties and they need to be efficient under conditions favouring accessibility of substrate, s e.g . solubility, as well as the stability of products and essential co-enzymes if required. Active forms of a co-enzymes should preferably be recycled in the process to decrease costs. The enzymes should preferably be robust to ensure productivity, stability and long life of processes and be able to be produced in high yields to enhance the economy of the whole system (21,22,23,24) . Thermostable enzymes can be advantageous in CFS process as they have long shelf- life, can be easily recycled and will allow prolonged reaction times if required in bioconversion processes (24)) . Furthermore, high temperature increases solubility and reduces viscosity of feedstock polysaccharides. Thermostability of enzymes also enables purification by heat precipitation of host proteins and therefore lower production costs.

The completed CFS process invented and described herein (Figure 1) fulfils the important criteria for successful industrialization, (i) The process is based on inherently stable enzymes, preferably enzymes from marine thermophiles disclosed herein, that utilize alginate, ensuring stability and long-life of the process and recycling of enzymes is possible, (ii) The enzymes selected are matched in efficiency under the one-pot conditions that favour product stability, and substrate solubility (alkaline pH and temperatures preferably up to and around 50°C or 55°C), by an innovative process engineering approach, (iii) The process in preferred embodiments allows complete utilization of both of the major polysaccharides from Brown algae, alginate and laminarin and this ensures greater resource efficiency and greater output of product (KDG) per unit weight of algal biomass (improving process economy), (iv) The laminarin, depending on availability and processing costs, can be supplemented or supplanted by glucose from other sources ensuring process continuity and economic gain, including adding enzymatic steps to the process for degrading alternative other beta- or alpha-glucans. (v) Substantially a single product is coming from the pathway (this simplifies product recovery and brings down costs), (vi) and the combined processing of laminarin and alginate, additionally enables effective recycling of the co enzyme which further brings down costs.

The process of the invention can be described as a series of steps involving different enzymes that form a pathway of reactions, schematically described in Figure 1. The steps can advantageously take place in one and the same medium and container ("one- pot") which means that all enzymes can be in place together, preferably operating under substantially the same conditions. Alternatively, in some embodiments one or more condition, such as temperature or pH, can be adjusted after a period of time to better suit steps late in the process pathway of reactions. In a "one pot" CFS system used in the present invention al enzymes can be dissolved or suspended in solution or one or more enzyme can be immobilized, e.g. as described above.

Alginate is provided in the process as the main reactant (raw material) for the process. Alginate, also referred to as alginic acid, algin or alginate is a polysaccharide found in the cell walls of brown algae, where through binding of water it forms a viscous gum. Natural alginate is a linear copolymer with homopolymeric blocks of (l-4)-linked b-D- mannuronate (M) monomer residues and residues of its C-5 epimer a-L-guluronate (G), respectively, covalently linked together in different sequences or blocks. The monomers can appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M-blocks) or alternating M and G-residues (MG-blocks). In some embodiments the alginate is pre-treated and converted to sodium alginate prior to the enzymatic process of the present invention.

The alginate is brought in contact with at least one alginate lyase enzyme, preferably in a solution. In some embodiments the at least one alginate lyase enzyme is immobilized on solid substrate such as beads, in other embodiments the enzyme is dissolved. The at least one alginate lyase enzyme is selected so as to convert alginate to unsaturated monomeric uronate that spontaneously converts to (DEH; deoxy-L- erythro-5 hexoseulose uronic acid). The at least one alginate lyase enzyme preferably comprises at least one endo-active alginate lyase and at least one oligo-alginate lyase enzyme. In certain non-limiting embodiments the at least one alginate lyase enzyme is a combination of at least one endo-active alginate lyase and at least one oligo-alginate lyase enzyme. The at least one alginate lyase or combination of enzymes preferably comprises one or more thermostable enzyme, such as an enzyme obtainable from a thermophilic organism. Preferably the alginate lyase enzyme or combination of enzymes is/are stable and active at temperature in the range between 40°C and 70°C and preferably above about 45° and more preferably up to and above about 60°C. In an exemplifying embodiment such enzymes are obtainable from Rhodothermus marinus, examples of such enzymes are disclosed in WO 2015104723 (10) and have the sequences as are shown in SEQ ID NO: l and SEQ ID NO: 2. In some embodiments a combination of an endo-active alginate lyase and an oligo-alginate lyase enzyme are used which have at least 70% sequence identity to the sequences of SEQ ID NO: l and SEQ ID NO: 2, respectively, and more preferably one or both have at least 80% sequence identity and yet more preferably at least 85% sequence identity and more preferably at least 90% sequence identity or at least 95% sequence identity to the sequences of SEQ ID : 1 and SEQ ID NO: 2, respectively. In some embodiments the at least one alginate lyase enzyme is a combination of enzymes substantially similar or identical to the mentioned AlyRM3 and AlyRM4 enzymes.

It applies to the one or more alginate lyase enzyme as well and any or all of other specified useful enzymes, that when useful sequences are provided herein, substantially similar sequences may as well be used in the invention, preferably with sequence identities as above listed, where any amino acid substitutions, deletions or additions should not substantially alter the enzymatic activity but can include terminal alterations (such as but not limited to His-tag or the like modification) and conservative substitutions and/or substitutions or alterations for modifying stability, optimizing for desired conditions and the like.

In an alternative embodiment one or both of said alginases can be replaced in part or fully by other alginases with substantially the same activity, non-limiting embodiments are alginases from another thermophilic bacteria related to Rhodothermus marinus and from the Rhodothermaceae family, such enzymes have been identified by the inventors, having the sequences shown in SEQ ID NO: 6 and SEQ ID NO : 7, respectively. IN some embodiments one or more alginase enzyme is used having at least 70% sequence identity to the sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and more preferably one or both have at least 80% sequence identity and yet more preferably at least 85% sequence identity and more preferably at least 90% sequence identity or at least 95% or at least 97% or at least 99% sequence identity to the sequences of SEQ ID : 6 and SEQ ID NO : 7, respectively. In preferred embodiments of the disclosed invention, the process is carried out at a temperature in the range between 35° and 70°C, such as in the range between 40° and 60°C and mopre preferably in the range between 40° and 55°C, such as at about 35°C, at about 40°C, at about 45°C, at about 50°C, at about 55°C or at about 60°C.

A variety of enzymes can be used in the process of the invention for processing of alginate and production of monomeric uronate/uronic acids and include various alginate lyases including exo-alginate lyases (EC number 4.2.2.-) and oligo-alginate lyases (EC 4.2.2.26) . Examples of enzymes of this type that can be used in certain embodiments of the invention include but are not limited to the following enzymes shown in Table 1. Accordingly, one or more of the listed enzymes, and/or one or more similar variant thereof with similar activity and stability profile, can be used in the present invention.

Table 1 Alginate lyases, showing source, protein family reference, EC number of the enzymes and substrate specificity.

Organism PL family EC Substrate specificity

Pseudomonas sp. E03 5 4.2.2.3 polyM

Stenotrophomonas maltophilia 6 4.2.2. - polyM, polyG

Flavobacterium spS20 7 4.2.2. 11 polyG

Halitos discus hannai 14 4.2.2. - polyM, polyG

Chlorella virusATCV- 1 14 4.2.2. 14 Alginate

Sphingomonas sp. A1 15 4.2.2. - polyM, polyG

Saccharophagus degradans 17 4.2.2.3 polyM, polyG

Pseudoalteromonas sp SM0524 18 4.2.2.3 polyM, polyG

As mentioned, the enzyme used to accomplish step b) is DEH reductase enzyme. One preferred non-limiting example of such enzyme is used in the accompanying examples and referred to as Adh51 which is obtainable from Rhodothermus marinus, having the sequence shown as SEQ ID NO : 3. Corresponding DEH reductase enzyme (i.e. EC 1. 1.1. 126) from another source is used in some other embodiments. Another useful DEH reductase from a related thermophilic organism is shown in SEQ ID NO : 8. The DEH reductase used in the invention is preferably a thermostable enzyme such as an enzyme obtainable from a thermophilic organism. In some embodiments the DEH reductase has at least 70% sequence identity to the DEH reductase having the sequence of SEQ ID NO : l (from Rhodothermus marinus ) or SEQ ID NO : 8 (from a related geothermal bacterium), and more preferably at least 80% sequence identity and yet more preferably at least 85% sequence identity and more preferably at least 90% sequence identity or at least 95% sequence identity or at least 97% or 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO:8. Any such deviation from SEQ ID NO: 1 or SEQ ID NO: 8 preferably comprises conservative substitutions, that do not alter substantially or negatively the activity and stability of the enzyme.

A glucose dehydrogenase (GDH) enzyme (EC 1.1.1.47) is as such known in the art (15, 18). The GDH enzyme used in the present invention is preferably a thermostable enzyme such as an enzyme obtainable from a thermophilic organism. A non-limiting example is a GDH enzyme referred to herein as GDH 16 derived from metagenome obtained from a geothermal source, this particular useful enzyme has the sequence of SEQ ID NO: 5. In some embodiments a similar enzyme is used, with substantially the same activity, having a sequence with at least 70% sequence identity to SEQ ID NO: 5, such as at least 75%, or at least 80% or at least 85% or at least 90% or at least 95% or at least 97% or at least 99% to SEQ ID NO: 5.

The enzymes used for the optional conversion of gluconate to KDG preferably include Dihydroxy acid dehydratase (EC 4.2.1.9) and Gluconate dehydratase (EC 4.2.1.39) (17). This step can advantageously be achieved in the same one-pot system as the above described reactions.

In useful embodiments, the glucose provided in the process is produced in situ by enzymatic processing of laminarin, with a suitable enzyme. The suitable enzyme for this purpose is in some embodiments a laminarase and/or beta-glucosidase enzyme. Such enzymes are generally known to the person skilled in the field. Many alternative sources of glucose can be used in certain embodiments of the invention by enzymatic in situ processing of a suitable oligo- or polysaccharides containing glucose units. A non limiting example of enzymes that can be used for processing of various beta-glucans in the present invention include b-glucosidase (EC 3.2.1.21); l,3-p-glucosidase (EC 3.2.1.58); exo-l,3-l,4-glucanase (EC 3.2.1.-); licheninase (EC 3.2.1.73); glucan endo- l,6-p-glucosidase (EC 3.2.1.75); endo-p-l,4-glucanase (EC 3.2.1.4); reducing end acting cellobiohydrolase (EC 3.2.1.176); endo-p-l,3-l,4-glucanase (EC 3.2.1.73), endo-l,3-p-glucanase (EC 3.2.1.39); endo-l,3(4)-p-glucanase (EC 3.2.1.6); licheninase (EC 3.2.1.73); pustulanase (E.C. 3.2.1.75). A non-limiting example of enzymes that could be used for processing of various alpha-glucans as source of glucose include a-amy!ase (EC 3.2.1,1); puiluianase (EC 3,2,1.41); cydomaitodextrin g!ucano- transferase (EC 2,4.1.19); cydo a!todextrinase (EC 3,2,1,54); o!igo-a-giucosidase (EC 3.2.1,10); maltogenic amylase (EC 3,2,1,133); neopuliulanase (EC 3.2,1,135); a- glucosidase (EC 3.2.1.20); ma!totetraose-forming a-amylase (EC 3.2.1.60); isoamylase (EC 3.2.1.68); g!ucodextranase (EC 3.2,1,70). Examples

Enzymatic production of DEH and KDG from alginate

The examples describe production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) and further conversion of DEH to 2-keto-3-deoxy gluconic acid (KDG) . A procedure was developed using the alginate lyases, Alg4 ("AlyRM4", oligo-alginate lyase) and Alg3 ("AlyRm3", endo-active lyase) (8), for production of DEH. The enzymes were expressed from gene sequences obtained from Rhodothermus marinus, and have the sequences shown as SEQ ID NO : l (Alg3) and SEQ ID NO : 2 (Alg4) . The enzymes have been described in applicant's earlier application WO2015104723, in which Alg3 is referred to as AlyRM3 and Alg4 is referred to as AlyRM4, AlyRM3 is identified therein by SEQ ID NO : 5 and AlyRM4 as SEQ ID NO : 6. A specific suitable reductase, Adh51, was then used for conversion of DEH to KDG.

Enzymes used: In addition to the already produced alginate lyases Alg3 (AlyRm3) and Alg4 (AlyRm4) that in combination degrade alginate to unsaturated mono-uronate, the following enzymes were provided for the method :

1. Adh51 from Rhodothermus marinus. It is a DEH reductase, involved in alginate metabolism and reduces DEH to KDG which is funneled into the Entner Douderoff (ED) glycolysis pathway. The reductase uses NAD(P)H as a co-factor which is oxidized to NAD(P)⁺ . The sequence of the Adh51 enzyme from R. marinus is depicted in SEQ ID NO : 3.

2. GDH 16 (in-house enzyme from a metagenome, having the sequence shown as SEQ ID NO : 5) is a thermophilic glucose dehydrogenase oxidizing glucose to gluconate with concomitant reduction of NAD(P)⁺ to NAD(P)H . It was used for regeneration of NAD(P)H .

3. KdgF is an enzyme that speeds up the spontaneous conversion of unsaturated mono-uronate to DEH ; the specific enzyme used herein was obtained from Rhodothermus marinus and has the sequence shown in SEQ ID N0 : 4.

Example 1. Expression and purification of enzymes used for production of KDG.

A general procedure was followed for all three enzymes, KdgF, Adh51 (DEH reductase) and GDH 16 (Glucose dehydrogenase) . They were recombinantly expressed in E. coli and cells grown in 500mL Luria-Bertani (LB) medium in a shaking incubation with the appropriate antibiotic added and expression induced in early log phase (Fig . 3) . The fully-grown cell culture was centrifuged at 4500 rpm for 30 min at 4°C. The media was poured off and discarded after autoclaving. The cell pellet was transferred to a falcon tube and the weight of the pellet was determined. The pellet was re-suspended in 0.1M Potassium Phosphate buffer pH 7.0 with a concentration of 0.2 g/mL. Cells were lysed by sonication and the crude lysate was then incubated for 30 min at 60°C, cooled down and centrifuged at 13200 rpm for 30 min at 4°C to separate the soluble proteins from the insoluble ones and the cell debris. The supernatant was filtered through a 0.2 pm filter and transferred to a new sterile tube. The enzymes were stored in the fridge at 4°C or for a longer time-period in the freezer at -20°C. Stock solutions of recombinantly produced enzymes were made that contained > 10 mg/mL of the active enzyme or more.

Example 2. Production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from alginate using alginate lyases

The enzymatic production of DEH from alginate was investigated using two alginate lyases Alg3 and Alg4 separately and together, at two different pH values for comparison, 5.5 and 6.8. Reaction solutions were made according to table 1, and the solutions were incubated at 45°C up to 72 hours. Samples were taken at 24, 48 and 72 hours and the results analyzed on TLC plates (Figure 4) .

Table 1. Composition of reaction solutions for enzymatic degradation of alginate

Figure 4 shows that both enzymes degrade alginate, but differently. Alg3 is an endo- alginate lyase and produces short alginate oligosaccharides at both pH values, whereas Alg4 is an exo-enzyme, oligo-alginate lyase (with preference for the mannuronic acid block (M-block) and is only active at pH 6.8. At this pH it only partly degrades alginate, seen by immobile residues at the sample application spot, but it forms only DEH . Complete degradation of Alginate is achieved using both enzymes together. At pH 5.5 all the alginate is degraded but in addition to DEH an unspecified alginate derivative is also formed . At pH 6.8 the alginate is fully degraded using both enzymes, Alg3 and Alg4, and DEH is the only product.

Example 3. The pH range for production of DEH directly from alginate by alg3 and Alg4

The enzymatic production of DEH from alginate at a range of pH values was investigated using the alginate lyases Alg3, Alg4 separately and Alg3 and Alg4 together. The reaction solution ( 1 mL) contained the following concentration of components : 10 mg/mL alginate, 25 mM acetate buffer at different pH and > 1 mg/mL of enzymes, Alg4 (figure 5. A), Alg3 (Figure 5B) and Alg3 and Alg4 together (Figure 5.C) . The reaction solutions were incubated to 22 hours at 45°C, samples were taken at intervals and analyzed on TLC plates (Figure 5) At this temperature activity of Alg4 in forming DEH was observed from pH 6.5 to pH 9, for Alg3 from pH 5.5 to pH 9, and for Alg3 + Alg4 was from pH 6 to pH 9.

Example 4. Enzymatic synthesis of KDG from unsaturated mono-uronates and recycling of NADH

Adding glucose, the GDH 16 enzyme and NADH to the DEH (mono-uronates) (made by using Alg3 and Alg4 prior to experiment) is expected increase to KDG yields by regenerating NADH from NAD+ that is produced in limited quantity from the starting concentration of NADH, according to the scheme presented in Figures 1 and 2.

Unsaturated mono-uronates were obtained by degrading alginate with a mixture of the alginate lyases Alg3 and Alg4 prior to the mixing of the solution.

Reaction solutions were made according to table 2.

Table 2. Composition of reaction solutions for conversion of monouronates (DEH) to KDG

A limiting concentration of starting NADH was used in the reactions, to detect if the regeneration of NADH ("NAD regeneration" in Figure 1) with GDH 16 would result in higher yields of KDG. All samples contained 0.5 mg/mL NADH, 6.4 mg/mL mono- uronates and different concentrations of glucose, which are shown in the bottom part of the figure in each rectangle. This was qualitatively analyzed on TLC and shown that by using increasing concentrations of glucose (Figure 6), an increase in KDG formation was observed .

Example 5. One pot synthesis of KDG from alginate at different temperatures

In this experiment it was investigated if KDG could be produced directly from Alginic Acid in a one-pot reaction. For this reaction the enzymes Alg3 and Alg4, the ADH51 reductase and GDH 16, with a low concentration of Glucose and NADH and a high concentration of alginic acid were all combined. Reaction volumes were 5 ml in Eppendorf tubes

The reaction solutions ( 1 mL) contained the following concentration of components : 10 mg/mL alginate, 25 mM acetic acid buffer pH 6.8 and > 1 mg/mL of each enzyme. a) For DEH production the reaction mixture contained only Alg3 and Alg4 and was incubated at pH 5.5 for up to 24 hours at different temperatures.

Samples were taken at intervals and analyzed on TLC (Figure 7A), DEH was produced in the range of 40 to 70°C, with greatest apparent activity at 70°C. b) For KDG production, the reaction mixture contained additionally an excess of NADH 33 mg/mL, and the reductase Adh51 > 10 mg/mL.

Samples were taken at intervals and analyzed on TLC plate (Figure 7B), activity was observed from 40°C to 70°C, with greatest apparent activity at 60°C. Less apparent yields were observed at 70°C, indicating instability of enzymes or products.

Example 6. Comparison of NAD and NADH in the recycling process

In this experiment it was investigated if the NAD+ form could be used as a starting co enzyme in the enzymatic production of KDG instead of NADH, according to Figure 1. It was expected that NAD+ would be reduced to NADH concomitantly by oxidation of glucose by GDH 16. The mixture was incubated at 55°C for 40 minutes, samples were taken after 0, 5, 10, 20 and 40 minutes. The non-purified, digested alginic acid used in the mixture was also used as the control.

The recycling was catalyzed with bot NAD or NADH (Figure 8) . Table 3. Composition of reaction solutions for conversion of mono-uronates (DEH) to KDG

Conclusion and next steps.

It is shown herein that efficient one pot enzymatic synthesis of KDG from alginate is feasible using alginate lyases, relevant reductase (Adh51) using NADH and regenerating NADH by oxidation of glucose with a specific dehydrogenase (GDH 16) . Gluconate is formed through oxidation catalyzed by GDH 16 and can further be converted to KDG using the enzyme dihydroxyacid dehydratase as described in EP2700714 Al . Furthermore, glucose can be obtained by hydrolysis of laminarin to glucose using beta- glucanases as discussed earlier.

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Claims

1 Claims

1. A cell-free process for producing 2-keto-3-deoxy-gluconate (KDG) from alginate, comprising :

a) bringing alginate in contact with at least one alginate lyase enzyme for the conversion of alginate to monomeric uronate (DEH), b) bringing the obtained DEH in contact with a DEH reductase enzyme and NAD(P)H for the conversion of DEH to KDG, whereby NAD(P)H is oxidized to NAD(P)⁺,

c) bringing obtained NAD(P)⁺ in contact with a glucose dehydrogenase (GDH) enzyme and glucose, for the regeneration of NAD(P)H from NAD(P)⁺, whereby glucose is oxidized to gluconic acid,

d) converting at least part of said gluconic acid to KDG, by bringing it in contact with a suitable enzyme.

2. The process of claim 1, wherein said at least one alginate lyase in step a) comprises at least one endo-active alginate lyase and at least one oligo- alginate lyase enzyme.

3. The process of any claims 1 to 2, wherein said DEH reductase enzyme in step b) has DEH reductase activity and comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO : 3 or SEQ ID NO: 8.

4. The process of claim 1, wherein said glucose in step c) is provided in situ by enzymatic conversion of a polysaccharide to glucose using one or more suitable enzymes.

5. The process of claim 4, wherein said polysaccharide comprises a beta-glucan and/or an alpha-glucan polysaccharide.

6. The process of claim 4, wherein said polysaccharide comprises laminarin and said one or more enzymes suitable for converting polysaccharide to glucose comprise at least one laminarinase and/or beta-glucosidase enzyme.

7. The process of claim 1, wherein said enzyme for converting said gluconic acid comprises at least one dihydroxyacid dehydratase enzyme.

8. The process of claim 1, wherein said enzyme for converting said gluconic acid comprises at least one gluconate dehydratase enzyme. 2

9. The process of any of claims 1 to 8, wherein a Kdgf enzyme is added to the process for enhancing the conversion of unsaturated mono-uronate to DEH .

10. The process according to any of the preceding claims, which is a one-pot process where all said steps a)-d) take place in the same reaction medium and container.

11. The process of any of the preceding claims, wherein said alginate is derived from a renewable biomass such as seaweed.

12. The process of claim 4 or 6, wherein said laminarin is derived from a renewable biomass such as seaweed.

13. The process of claims 11 and 12, wherein said alginate and said laminarin are provided from the same feedstock of seaweed .

14. The process of any of claims 1 to 13 wherein one or more said enzyme is a thermostable enzyme.

15. The process of any of claims 1 to 14 wherein the process is carried out at a temperature in the range from about 40°C to about 60°C and preferably in the range from about 40° to about 55°C.

16. The process of any of claims 1 to 7 wherein produced KDG is used for synthesis of chemical derivatives.