Lactobionic acid production via mutant cellobiose dehydrogenase/laccase continuous enzymatic regeneration of electron acceptors

Abstract Lactobionic acid (LBA) has diverse number of applications in pharmaceutical, food, chemical and nanotechnology fields. Bioproduction of LBA by bi-enzymatic cellobiose dehydrogenase (CDH)/laccase (LAC) is a system where CDH catalyses oxidation of lactose to LBA, whilst LAC has, at the same time, ability to regenerate the redox mediator as electron acceptor for CDH. In this study, CDH mutants derived from Phanerochaete chrysosporium produced in Pichia pastoris were used to determine LBA production rate with varying number of redox mediators. The highest specific productivity was observed for triple mutant CDH (tmCDH) for all redox mediators used in this study. The notable redox mediator was hydroquinone where specific productivity was 29.14 g L−1 kU−1 for tmCDH while specific productivity with DCIP was 27.2 g L−1 kU−1, with ABTS showed 19.1 g L−1 kU−1. Graphic Abstract Key Points Lactobionic acid production using bi-enzymatic system, CDH/LAC. Regeneration of different redox mediators. Recombinant mutants used in reaction system.


Introduction
Lactobionic acid (LBA: 4-O-b-D-galactopyranosyl-D-gluconic acid; C 12 H 22 O 12 ) is an aldolic acid derived from the oxidation of lactose.The chemical structure of lactobionic acid incorporate a galactose moiety linked to a gluconic acid molecule by a linkage.The conversion of lactose to LBA involve the oxidation of the free aldehyde group on the lactose moiety to the carboxyl group (Guti errez et al. 2012b).The first LBA was synthetized by Fischer and Meyer via oxidizing lactose with bromine (Fischer and Meyer 1889).Diverse processes for production of lactobionic acid were used since then, such as chemical synthesis (Gutierrez et al. 2011;Guti errez et al. 2012a), microbial fermentation (Alonso et al. 2011;Pedruzzi et al. 2011;Alonso et al. 2017) or enzymatic synthesis (Ludwig et al. 2004;Van Hecke et al. 2009, 2011).The biocatalytic production of LBA constitutes the oxidation of lactose by means of specific enzymes or by using microorganisms as biocatalysts.
One particular utilization of LBA is its use as an additive in solutions stabilizing organs before transplantation, premised on the outstanding metal-chelating properties of LBA that reduce oxidative damage to tissue during storage and preservation of organs caused by certain metal ions (Guibert et al. 2011).LBA is known for its uses in pharmaceuticals as enhancement of solubility of microlide antibiotics such as erythromycin (Green et al. 2009), as a biodegradable cobuilder in washing powder.In food technology it has several applications such as the reduction of souring and ripening time of cheese and yogurt, the preservation of aroma freshness (Gerling 1998), its mineral salt complexes whom are used to fortify functional drinks with essential minerals or its incorporation in functional food because of its assumed prebiotic effect (Ludwig et al. 2004).
Diverse methods are viable for conversion of lactose to LBA using carbohydrate oxidase (Nordkvist et al. 2007).A bi-enzymatic system relevant to this article is the indirect regeneration of flavoenzymes with laccase (Baminger et al. 2001;Ludwig et al. 2004;Van Hecke et al. 2009).One such flavoenzyme is FAD-containing cellobiose dehydrogenase (CDH; EC 1.1.99.18), an extracellular enzyme produced by diverse number of rot fungi (Henriksson et al. 2000).CDH is able to catalyse the oxidation of di-and oligosaccharides linked by b-1-4-glucosidic bonds, such as cellobiose and lactose, to the corresponding aldoni-1,5-lactones, that spontaneously hydrolyse to aldonic acids in aqueous solution (Sulej et al. 2019).Catalysing the oxidation of lactose to LBA was mostly done using CDH in research studies.The enzyme can transfer electrons to various redox mediators such as 2,6-dichloro-indophenol (DCIP), 1,4-benzoquinone, 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and metal ions (Baminger et al. 2001).A regenerative enzyme, laccase (LAC) needs to be added simultaneously into the system in order to oxidize redox mediators, which serves as an electron acceptor for CDH (Van Hecke et al. 2011;Gangwar et al. 2016).Laccases are a multicopper oxidase detected in various plant species and in diverse number of fungi.Laccase can catalyse the oxidation of manifold of substances such as metal complexes, thiols, and phenols with concurrent reduction of molecular oxygen to water.Despite the fact that laccases strongly prefer oxygen as their oxidizing substrate, they are generally vastly unspecific towards their reducing substrate (Thurston 1994).These features provide laccase as an optimal enzyme for regeneration of a wide range of electron acceptors that can be used during catalytic action by CDH (Baminger et al. 2001).
In this study, we used mutant CDH obtained in previous work (Balaz et al. 2020) for comparison of the bioproduction of lactobionic acid from lactose using different redox mediators.A reaction system consisting of mutant CDH and commercial LAC with different redox mediators was established where the reduced form of CDH is perpetually regenerated by the redox mediator and laccase controlled redox reaction (Splechtna et al. 2001).

Enzyme activity assays
Cellobiose dehydrogenase (CDH) activity was analysed at 22 C with 0.3 mM 2,6-dichloroindophenol (DCIP; k ex 520 nm; e520 ¼ 6.80 mM À1 cm À1 ) as the electron acceptor in 0.1 M sodium acetate buffer pH 4.5, utilizing 30 mM lactose as substrate, and 4 mM sodium fluoride was added when laccase is present in the mixture.One international unit (IU) of enzyme activity is defined as the amount of enzyme that reduces 1 mmol of DCIP per minute under the above-mentioned conditions.

Biotransformation experiments
Oxidation of lactose to lactobionic acid was performed at 22 C in modified enzyme reactors.To establish excess of dissolved oxygen, which is essential for laccase activity, air was introduced through solution with air flow rate of 1 L min À1 .Biotransformation was done for 22h and concentration of produced lactobionic acid was measured with titration (Kankare 1973).The reaction system consists of 200 mM lactose, 0.05 IU ml À1 CDH, and 0.1 IU ml À1 laccase in 10 mM sodium acetate buffer pH 4.5 in 20 mL reaction mixture.The subsequent redox mediators were used in the specified concentrations: 0.2 mM ABTS, 0.6 mM DCIP, 1.75 mM hydroquinone, 0.2 mM K 4 Fe(CN) 6 , and 2 mM guaiacol.

Characterization of biocatalysts
Recombinant cellobiose dehydrogenase from Phanerochaete chrysosporium was characterized catalytically as a key contributor for the conversion of lactose to lactobionic acid.Recombinant enzymes are monomeric protein with glycosylated moieties with molecular mass of 100 kDa (Figure 1).Enzyme oxidizes b-1,4 linked polysaccharides, in particular cellobiose and lactose to equivalent aldolic acids (Baminger et al. 1999).Specific activity of purified recombinant enzymes wt, tm, and H5 mutants CDH when utilizing lactose and DCIP as substrates were 20.4,14.1, and 28.1 IU mg À1 respectively (Balaz et al. 2020).CDH from different fungus strains can reduce a numerous of diverse electron acceptors such as DCIP and ABTS cation radical.Both mutants showed lower Km values for cellobiose and lactose than what was observed for wild type.Kinetic constant was higher for both mutants while using DCIP as electron acceptor in comparison to wt enzyme.For 1,4-benzoquinone, mutants had lower Km values than wt while in case of ABTS cation radical only tmCDH showed lower Km values.For ferricyanide, both mutants showed higher Km values and significantly lower kinetic constants than what was observed with wtCDH.Kinetic information for applied electron acceptors is inscribed in Table 1.
Laccase obtained from T. versicolor is monomeric glycosylated enzyme with molecular mass of $64 kDa.Table 2 presents the kinetic data for different dyes used in biotransformation experiments with oxygen as substrate for laccase.We see that kinetic constant for electron acceptors, except guaiacol, are similar and in the same range.

Lactobionic acid production using coupled enzymes
Regeneration of oxidized electron acceptors used by flavonoenzymes has proved feasible by utilizing laccase (Baminger et al. 2001;Ludwig et al. 2004).Several redox mediators which can alternate electrons between laccase and CDH were used in this work.To investigate how redox mediators influence production of lactobionic acid with different CDH mutant and laccase, biotransformation experiments were conducted in discontinuous manner on a small scale (20 mL) with low range of different redox mediators.Titration with 1 mM NaOH was used to ascertain the concentration of produced lactobionic acid, from processes with blind probe (dye, buffer, and lactose) to  biotransformation with different CDH protein and laccase were shown in Supplementary Figure S1.
In these experiments, lactose conversion to lactobionic acid was done in 22 h.Laccase can be used to proficiently catalyse the continuous conversion of the various reduced redox mediators to their oxidized forms whom cellobiose dehydrogenase can use as the electron acceptor in the course of the oxidation of lactose to lactobionic acid (Ludwig et al. 2004).
A differentiation of productivity of diverse biotransformation experiments utilizing different redox mediators such as DCIP, ABTS, and hydroquinone, is presented in Table 3. Considerable formation of lactobionic acid was detected when using different redox mediators, which shows their potential for regeneration by laccase.Difference in production time can be seen when comparing the time needed for 50% conversion in Table 3.The shortest time for bioconversion as well as the highest productivity was obtained when hydroquinone was used as the redox mediator which is in accord with previous works (Ludwig et al. 2004).The volumetric productivity for this bioconversion was calculated for wtCDH to be 1.36 g lactobionic acid per litre per hour, for tmCDH was 1.46 g L À1 h À1 as for H5 CDH was 1 g L À1 h À1 .The specific productivity for wtCDH, tmCDH, and H5 CDH was calculated to be 27.2 g per kU CDH activity per hour, 29.2 g h À1 kU À1 , and 20 g h À1 kU À1 , respectively.
Lactobionic acid productivity was calculated after 22h of bioconversion is shown in Table 3 (for more extensive comparison with literature, see Supplementary Table S1) with time necessary for conversion of 50% of lactose to lactobionic acid.
Different concentrations of produced lactobionic acid can be seen on Figure 2. tm CDH showed highest production and depending on the redox mediator used H5 CDH variant showed higher production rate in contrast to wt CDH.

Discussion
In this study we report the use of already published CDH mutants (Balaz et al. 2020) as biocatalysts in enzymatic processes for biotransformation of lactobionic acid from lactose.From our two different publications, based on positions of mutations in both mutants and activities obtained, we decided to use both mutants in biotransformation experiments (Bla zi c et al. 2019;Balaz et al. 2020).All three mutations (D20N, A64T, and V529) were located on the surface of CDH protein, where first two were located near the active site containing haem, and third mutation lies in the vicinity of the entrance of the active site containing FAD cofactor.Substitutions A64T and V529M are positioned in the region where there is interaction of   CDHs two domains, which is necessary for the activity of CDH (Bla zi c et al. 2019).In case of H5 mutant, additional mutation V22A was located on the surface of haem domain, in close proximity to D20N substitution.With regarding to previously shown activities of given enzymes we decided to use them in biotransformation experiments.
In order to investigate regeneration of different redox mediators by bi-enzymatic system in more detail, electron acceptors were used in catalytic amounts.Results for the oxidation of 200 mM lactose by 0.05 U mL À1 different mutant of CDH enzyme and 0.1 U mL À1 laccase range of redox mediators are shown in Table 3.
When DCIP was used as redox mediator we can deduce that highest production was observed when tmCDH was employed with specific productivity of 27.2 g L À1 kU À1 which was four times higher than reported 6.8 g L À1 kU À1 for S. rolfsii (Ludwig et al. 2004) where they used 1 IU of CDH and 2 IU of laccase.H5 mutant CDH showed higher production rate, using DCIP as electron acceptor than wtCDH.Specific productivity of 16 g L À1 kU À1 from S. rolfsii in another research (Baminger et al. 2001) was still smaller than what we obtained in this study and they used 28 times higher amount of enzyme than used in this study.Another group produced LBA with specific productivity of 16 g L À1 kU À1 using CDH from S. rolfsii (Splechtna et al. 2001) where they changed ratio of CDH: laccase (1:2.5 respectfully), which was lower than specific productivity described in this study.All three variants of CDH used in this study showed higher specific production for DCIP as electron acceptor than reported by Ludwig et al. (2004).
ABTS as redox mediator for bi-enzymatic system when tmCDH was used showed similar specific productivity as reported by Ludwig et al. (2004) at 19.3 g L À1 kU À1 , as for tmCDH and 19.1 g L À1 kU À1 , while wtCDH produced more than H5CDH for given redox mediator.In contrast one group used white mound CDH in ratio 0.1: 0.36 CDH: LAC and obtained 17.7 g L À1 kU À1 which was less than we obtained in this study (Dhariwal et al. 2006).Another group used CDH from Asperigillus fumigatus with enzyme ratio of 1 IU CDH: 2.4 IU LAC, obtained specific LBA productivity of 7.14 g L À1 kU À1 , having 2.7 times less production than we reported (Yang et al. 2021).
Hydroquinone was used as electron acceptor and from experimental results we saw that tmCDH production of 29 g L À1 kU À1 as well as wtCDH production of 27 g L À1 kU À1 exceeded production by S. rolfsii where 1,4-benzoquinone was used with 21.4 g L À1 kU À1 (Ludwig et al. 2004).
Guaiacol was first reported in use for biotransformation of lactose to lactobionic acid and although production using this dye showed four times lower production it still followed trend of previous redox mediators where production was tmCDH > wtCDH > H5CDH.In one study was shown plausible mechanism of laccase enzyme with guaiacol during the enzyme activity with the end product cyclohexa-3,5-diene-1,2-dione which can be than reduced by CDH (Senthivelan et al. 2019).As was shown from previous studies with 1,4-benzoquinon, where we can see the best production of lactobionic acid, the enzyme CDH can use 1,2-benzoquinone but with lower affinity.Ferricyanide as a redox mediator showed the lowest production rate of all mediators and twice lower than reported for S. rolfsii (Ludwig et al. 2004); however, tmCDH still produced more than wtCDH and H5CDH.This mediator had lengthiest production time of all redox mediators used.We can show that redox mediators can be enumerated as follows: hydroquinone > DCIP > ABTS > guaiacol > ferricyanide with respect to the time it takes to produce 50% lactobionic acid.We showed that guaiacol can be used as redox mediator for biotransformation of lactose.Hydroquinone was excellent redox mediator with whom was highest production of lactobionic acid.

Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.

Figure 2 .
Figure 2. Conversion of lactose to lactobionic acid with various redox mediators and different CDH variant.

Table 1 .
Kinetic constants of purified CDH proteins for lactose and cellobiose with DCIP as electron acceptor, and different dyes with lactose as substrate.

Table 2 .
Kinetic constants of commercial laccase for different dyes as electron donor.
a DCIP: 2,6-dichloro-indophenol reduced was prepared by oxidizing DCIP with wtCDH, the enzyme was inactivated with incubation at 100 C for 10 min.

Table 3 .
Conversion of lactose to lactobionic acid via coupled system of different cellobiose dehydrogenase proteins and laccase with different redox mediators.