Development and validation of a screening system for a β-galactosidase with increased specific activity produced by directed evolution

Rentschler, Eva; Schwarz, Thilo; Stressler, Timo; Fischer, Lutz

doi:10.1007/s00217-016-2709-x

Cited by 7 publications

(2 citation statements)

References 43 publications

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“…The production of the respective CE was carried out as previously reported for another enzyme. 26 The E. coli BL21 (DE3) cells containing either pET-20bB15CE, pET-20bBFCE, pET-20bDFCE, pET-20bFBCE, pET-20bRACE, or pET-20bRMCE were cultivated separately in 0.6 L of 2-yeasttryptone medium containing 16 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of NaCl, 20 g/L of glucose, and 100 μg/mL of ampicillin in a stirred tank reactor (Multifors, Infors, Bottmingen, Switzerland) for 11 h, respectively. The expression of the respective CE was induced with 0.5 mM isopropyl-β-Dthiogalactopyranoside (IPTG) after 2.5 h of cultivation at 37 °C.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Hidden Reaction: Mesophilic Cellobiose 2-Epimerases Produce Lactulose

Kuschel

Seitl

Glück

et al. 2017

J. Agric. Food Chem.

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Lactulose (4-O-β-d-galactopyranosyl-d-fructofuranose) is a prebiotic sugar derived from the milk sugar lactose (4-O-β-d-galactopyranosyl-d-glucopyranose). In our study we observed for the first time that known cellobiose 2-epimerases (CEs; EC 5.1.3.11) from mesophilic microorganisms were generally able to catalyze the isomerization reaction of lactose into lactulose. Commonly, CEs catalyze the C2-epimerization of d-glucose and d-mannose moieties at the reducing end of β-1,4-glycosidic-linked oligosaccharides. Thus, epilactose (4-O-β-d-galactopyranosyl-d-mannopyranose) is formed with lactose as substrate. So far, only four CEs, exclusively from thermophilic microorganisms, have been reported to additionally catalyze the isomerization reaction of lactose into lactulose. The specific isomerization activity of the seven CEs in this study ranged between 8.7 ± 0.1 and 1300 ± 37 pkat/mg. The results indicate that very likely all CEs are able to catalyze both the epimerization as well as the isomerization reaction, whereby the latter is performed at a comparatively much lower reaction rate.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Hidden Reaction: Mesophilic Cellobiose 2-Epimerases Produce Lactulose

Kuschel

Seitl

Glück

et al. 2017

J. Agric. Food Chem.

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“…Using classical error-prone PCR, engineered Pseudomonas fluorescens and Penicillium cyclopium lipase were produced with enhanced thermostability and alkali stability which expanded their uses as catalysts in bakery and dairy [ 16 , 17 ]. A new β-galactosidase obtained using error-prone PCR had improved specific activity for lactose hydrolysis in milk processing [ 18 ]. Liu et al [ 19 ] developed Bacillus alcalophilus alkaline protease using error-prone PCR with high cold adaptation suitable for cold-temperature food processing.…”

Section: Strategies and Challenges For The Development Of Engineered ...mentioning

confidence: 99%

Bioengineered Enzymes and Precision Fermentation in the Food Industry

Boukid¹,

Ganeshan²,

Wang³

et al. 2023

IJMS

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Enzymes have been used in the food processing industry for many years. However, the use of native enzymes is not conducive to high activity, efficiency, range of substrates, and adaptability to harsh food processing conditions. The advent of enzyme engineering approaches such as rational design, directed evolution, and semi-rational design provided much-needed impetus for tailor-made enzymes with improved or novel catalytic properties. Production of designer enzymes became further refined with the emergence of synthetic biology and gene editing techniques and a plethora of other tools such as artificial intelligence, and computational and bioinformatics analyses which have paved the way for what is referred to as precision fermentation for the production of these designer enzymes more efficiently. With all the technologies available, the bottleneck is now in the scale-up production of these enzymes. There is generally a lack of accessibility thereof of large-scale capabilities and know-how. This review is aimed at highlighting these various enzyme-engineering strategies and the associated scale-up challenges, including safety concerns surrounding genetically modified microorganisms and the use of cell-free systems to circumvent this issue. The use of solid-state fermentation (SSF) is also addressed as a potentially low-cost production system, amenable to customization and employing inexpensive feedstocks as substrate.

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Biotechnical production of trehalose through the trehalose synthase pathway: current status and future prospects

Cai

Seitl

et al. 2018

Appl Microbiol Biotechnol

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Trehalose (α-D-glucopyranosyl-(1 → 1)-α-D-glucopyranoside) is a non-reducing disaccharide composed of two glucose molecules linked by an α,α-1,1-glycosidic bond. It possesses physicochemical properties, which account for its biological roles in a variety of prokaryotic and eukaryotic organisms and invertebrates. Intensive studies of trehalose gradually uncovered its functions, and its applications in foods, cosmetics, and pharmaceuticals have increased every year. Currently, trehalose is industrially produced by the two-enzyme method, which was first developed in 1995 using maltooligosyltrehalose synthase (EC 5.4.99.15) and subsequently using maltooligosyltrehalose trehalohydrolase (EC 3.2.1.141), with starch as the substrate. This biotechnical method has lowered the price of trehalose and expanded its applications. However, when trehalose synthase (EC 5.4.99.16) was later discovered, this method for trehalose production using maltose as the substrate soon became a popular topic because of its simplicity and potential in industrial production. Since then, many trehalose synthases have been studied. This review summarizes the sources and characteristics of reported trehalose synthases, and the most recent advances on structural analysis of trehalose synthase, catalytic mechanism, molecular modification, and usage in industrial production processes.

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Development and validation of a screening system for a β-galactosidase with increased specific activity produced by directed evolution

Cited by 7 publications

References 43 publications

Hidden Reaction: Mesophilic Cellobiose 2-Epimerases Produce Lactulose

Hidden Reaction: Mesophilic Cellobiose 2-Epimerases Produce Lactulose

Bioengineered Enzymes and Precision Fermentation in the Food Industry

Biotechnical production of trehalose through the trehalose synthase pathway: current status and future prospects

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