Enzymatic production of 3’-sialyllactose in milk

Valentina N Perna,Christian Dehlholm,Anne S Meyer

doi:10.1016/j.enzmictec.2021.109829

Valentina N Perna, Christian Dehlholm + Show 1 more

Open Access

https://doi.org/10.1016/j.enzmictec.2021.109829

Copy DOI

Abstract

Human milk oligosaccharides (HMOs) are lactose-based glycan molecules present in human breast milk. HMOs are essentially not present in cow’s milk and hence not naturally available in infant formulas. HMOs possess several health and developmentally beneficial properties, and the sialylated HMOs are thought to play a particularly important role for infant brain development. Enzymatic transsialylation directly in cow’s milk, involving enzyme catalyzed transfer of sialic acid from a sialic acid donor to an acceptor, is a novel route for producing sialylated HMOs for e.g. infant formulas. The transsialidase (EC 2.4.1.-) of Trypanosoma cruzi is linked to trypanosomatid pathogenicity, but certain hydrolytic sialidases (neuraminidases), EC 3.2.1.18, from non-pathogenic organisms, can actually catalyze transsialylation. Here, we report enzymatic production of the HMO compound 3’-sialyllactose directly in cow’s milk using engineeredsialidases, Tr15 and Tr16, originating from the nonpathogenic Trypanosoma rangeli. Both Tr15 and Tr16 readily catalyzed transsialylation in milk at 5 °C–40 °C using κ(kappa)-casein glycomacropeptide (cGMP) as sialyl donor substrate. Tr15 was the most efficient as this enzyme produced 1160 mg/L (1.8 mM) 3’-sialyllactose in whole milk during 10 min of reaction at 5 °C. The activation energy values, Ea, of the enzymatic transsialylation reactions were similar in milk and in buffer solutions containing cGMP and lactose. The Ea of the Tr15 catalyzed transialylation reaction in milk was 16.5 kJ/mol, which was three times lower than the Ea of Tr16 (66 kJ/mol) and of T. cruzi transsialidase (50 kJ/mol), corroborating that Tr15 was the fastest of the three enzymes and a promising candidate for potential industrial production of 3’-sialyllactose in milk. 3’sialyllactose was stable during pasteurization (30 min. at 62.5 °C) and freeze-drying.

Highlights

Human breast milk is characterized by the presence of human milk oligosaccharides (HMOs) in the range of 5− 15 g/L [1]
We demonstrate that 3’-SL production directly in cow’s milk using Tr15 and Tr16 is possible and show that it is possible to achieve 3’-SL concentrations that are comparable to those found in breast milk
The casein glycomacropeptide (cGMP) solution was concentrated to 37.1 g/L corre sponding to a sialic acid (Neu5Ac) content of 10 mM determined after 48 h desialylation using the sialidase from Arthrobacter ureafaciens (Sigma–Aldrich, Steinheim, Germany). 10 mM sialic acid concentration is the sum of the α2,3- and α2,6-bound sialic acid that are distributed 50:50 in cGMP [25]

Summary

Introduction

Human breast milk is characterized by the presence of human milk oligosaccharides (HMOs) in the range of 5− 15 g/L [1]. HMOs are lactose-based oligosaccharides that beyond lactose generally contain one or more units of N-acetylglucosamine and galactose to form repeat backbone moieties of lacto-N-biose and N-acetyllactosamine (N-ace tyllactosamine provides for further backbone extension, whilst lacto-N-biose terminates further structural elongation). The HMO backbone structures, including lactose, may be decorated with sialic acid (N-acetyl-neuraminic acid form, Neu5Ac) and/or fucose res idues [1,2]. The two simplest sialylated HMOs are 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL) These HMOs have a sialic acid moiety (Neu5Ac) linked to the galactosyl sub unit of lactose at position 3 or 6, respectively [1]. The concentration of sialylated HMOs in breast milk varies during the lactation period and depends on the length of the pregnancy. The concentration of 3’-SL in breast milk usually ranges from 90 to 350 mg/L during the first 35 lactation days [6,7], and increases to up to 840 μg/mL during 0.5–8

Methods

Results

Conclusion