Importance of the 5' untranslated region for recombinant enzyme production in isolated Bacillus subtilis 007.

The production of industrial enzymes requires an efficient expression system with a suitable host. This study investigated the isolated Bacillus subtilis 007 as a host for expressing three enzymes with potential application in the food industry. Firstly, testing the P and P promoters and the corresponding 5' untranslated regions revealed great differences in the production of the recently discovered β-galactosidase from Paenibacillus wnnyii.

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii.

Background: The β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low K value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes.

Secretion of the cytoplasmic and high molecular weight β-galactosidase of Paenibacillus wynnii with Bacillus subtilis.

Background: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B.

A new β-galactosidase from Paenibacillus wynnii with potential for industrial applications.

Commercial β-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [K] for lactose) and product inhibition (inhibitor constant [K] for galactose). An in silico screening of gene sequences was done and identified a putative β-galactosidase (Paenibacillus wynnii β-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P.

The behavior of cathepsin D during milk processing and its contribution to bitterness in a model fresh cheese.

The bovine endopeptidase cathepsin D was investigated regarding its temperature-dependent inactivation and ability to form bitter peptides within a spiked model fresh cheese. Cathepsin D was found to be more susceptible than other milk endogenous peptidases to temperature treatments in skim milk. Inactivation kinetics revealed decimal reduction times of 5.

Recent advances in the application of microbial diamine oxidases and other histamine-oxidizing enzymes.

The consumption of foods fraught with histamine can lead to various allergy-like symptoms if the histamine is not sufficiently degraded in the human body. The degradation occurs primarily in the small intestine, naturally catalyzed by the human diamine oxidase (DAO). An inherent or acquired deficiency in human DAO function causes the accumulation of histamine and subsequent intrusion of histamine into the bloodstream.

Toward Oral Supplementation of Diamine Oxidase for the Treatment of Histamine Intolerance.

A new diamine oxidase (DAO-1) was discovered recently in the yeast PO1f and investigated for its histamine degradation capability under simulated intestinal conditions. DAO-1 was formulated together with catalase as a sucrose-based tablet. The latter (9 × 7 mm; 400 mg) contained 690 nkat of DAO-1 activity, which was obtained from a bioreactor cultivation of a genetically modified with optimized downstream processing.

Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine.

Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA.

Production and characterization of a new diamine oxidase from Yarrowia lipolytica.

A putative diamine oxidase (DAO) from Yarrowia lipolytica PO1f (DAO-1) was homologously recombinantly integrated into the genome of Y. lipolytica PO1f using the CRISPR-Cas9 system for the subsequent DAO production in a bioreactor. Thereby, it was proven that the DAO-1 produced was indeed a functional DAO.

Secretion of a low and high molecular weight β-glycosidase by Yarrowia lipolytica.

Background: The secretory production of recombinant proteins in yeast simplifies isolation and purification but also faces possible complications due to the complexity of the secretory pathway. Therefore, correct folding, maturation and intracellular transport of the recombinant proteins are important processing steps with a higher effort needed for complex and large proteins. The aim of this study was to elucidate the secretion potential of Yarrowia lipolytica for low and high molecular weight β-glycosidases in a comparative cultivation approach.

Evaluation of porcine diamine oxidase for the conversion of histamine in food-relevant amounts.

Histamine exists in a multitude of foods and displays an emerging role within food intolerances. Our aim was to identify the activity of porcine diamine oxidase (DAO) required for the in vitro degradation of histamine amounts that are found in typical meals containing histamine (75 mg, equaled 150 mg/L). Furthermore, we investigated an actual dietary supplement that is commercially available for histamine intolerant individuals for its histamine reduction capability.

Characterization of a Recombinant Trehalose Synthase from Arthrobacter chlorophenolicus and its Unique Kinetics Indicating a Substrate Cooperativity.

Trehalose is a non-reducing disaccharide with beneficial physiological properties and commercial potential. Trehalose synthase (EC 5.4.

Combination of sequence-based and in silico screening to identify novel trehalose synthases.

A combined approach of sequence-based screening from metagenomic soil DNA and subsequent in silico screening was established to identify novel trehalose synthases (TS, EC 5.4.99.

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

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.

A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity.

Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as β-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of β-galactosidase preparations produced from K.

Hidden Reaction: Mesophilic Cellobiose 2-Epimerases Produce Lactulose.

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.

Detection, production, and application of microbial arylsulfatases.

Arylsulfatases are enzymes which catalyze the hydrolysis of arylsulfate ester bonds to release a free sulfonate. They are widespread in nature and are found in microorganisms, most animal and human tissues, and plant seeds. However, this review focuses on arylsulfatases from microbial origin and gives an overview of different assays and substrates used to determine the arylsulfatase activity.

Membrane Targeting and Insertion of the C-Tail Protein SciP.

C-tailed membrane proteins insert into the bilayer post-translationally because the hydrophobic anchor segment leaves the ribosome at the end of translation. Nevertheless, we find here evidence that the targeting of SciP to the membrane of Escherichia coli occurs co-translationally since signal elements in the N-terminal part of the SciP protein sequence are present. Two short hydrophobic sequences were identified that targeted a green fluorescent protein-SciP fusion protein to the membrane involving the signal recognition particle.

The C-terminal regions of YidC from Rhodopirellula baltica and Oceanicaulis alexandrii bind to ribosomes and partially substitute for SRP receptor function in Escherichia coli.

The marine Gram-negative bacteria Rhodopirellula baltica and Oceanicaulis alexandrii have, in contrast to Escherichia coli, membrane insertases with extended positively charged C-terminal regions similar to the YidC homologues in mitochondria and Gram-positive bacteria. We have found that chimeric forms of E. coli YidC fused to the C-terminal YidC regions from the marine bacteria mediate binding of YidC to ribosomes and therefore may have a functional role for targeting a nascent protein to the membrane.