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Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity

Paper ID Volume ID Publish Year Pages File Format Full-Text
34792 45048 2014 6 PDF Available
Title
Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity
Abstract

•A mutated sialidase from Trypanosoma rangeli was expressed in Pichia pastoris.•The sialidase mutant was tuned for trans-sialidase activity by optimizing reaction conditions.•3′-Sialyllactose and other sialylated HMO molecules were produced via trans-sialidase biocatalysis.•Casein glycomacropeptide was used as substrate for the trans-sialidase mutant biocatalysis.

Casein glycomacropeptide (cGMP) and lactose, which are purified (or semi-purified) components obtained from side streams from dairy industry operations, were used as substrates for enzyme catalyzed production of 3′-sialyllactose, a model case compound for human milk oligosaccharides (HMOs). The enzyme employed was a mutated sialidase, Tr6, derived from Trypanosoma rangeli, and expressed in Pichia pastoris after codon-optimization. The Tr6 contained 6 point mutations and exhibited trans-sialidase activity. The Tr6 trans-sialidase reaction conditions were tuned for maximizing Tr6 catalyzed 3′-sialyllactose production by optimizing pH, temperature, acceptor, and donor concentrations using response surface designs. At the optimum reaction conditions, the Tr6 catalyzed the transfer of sialic acid from cGMP to lactose at high efficiency without substantial hydrolysis of the 3′-sialyllactose product. The robustness of the Tr6 catalyzed reaction was verified at 5 L-scale providing a yield of 3.6 g 3′-sialyllactose at an estimated molar trans-sialylation yield of 50% on the 3′-sialyl in cGMP. Lacto-N-tetraose and lacto-N-fucopentaoses also functioned as acceptor molecules demonstrating the versatility of the Tr6 trans-sialidase for catalyzing sialyl-transfer for generating different HMOs. The data signify the applicability of enzymatic trans-sialylation on dairy side-stream components for production of human milk oligosaccharides.

Keywords
cGMP, casein glycomacropeptide; HMOs, human milk oligosaccharides; LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose; LNFP I, lacto-N-fucopentaose; LNFP V, lacto-N-fucopentaose V.trans-Sialylation; Human milk oligosaccharides; Trypanosoma rangeli; 3′-Sialy
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Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 49, Issue 2, February 2014, Pages 265–270
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us