Secretion of the cytoplasmic and high molecular weight ß-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. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. RESULTS: In this study, the cytoplasmic and 120 kDa ß-galactosidase of Paenibacillus wynnii (ß-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the ß-gal-Pw gene led to an increase in extracellular ß-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular ß-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular ß-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. CONCLUSION: For the first time, the ß-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

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

Commercial ß-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [KM] for lactose) and product inhibition (inhibitor constant [Ki] for galactose). An in silico screening of gene sequences was done and identified a putative ß-galactosidase (Paenibacillus wynnii ß-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low ß-galactosidase activities of a maximum of 150 nkat per liter of medium with o-nitrophenyl-ß-d-galactopyranoside (oNPGal) as substrate. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield ∼9,000-fold. Here, a volumetric activity of 1,350.18 ± 11.82 µkatoNPGal/Lculture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C, and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer (Novo buffer), respectively. Remarkably, the KM value of BgaPw with lactose was as low as 0.63 ± 0.045 mM in milk. It was found that the resulting products of lactose hydrolysis, namely galactose and glucose, did not inhibit the ß-galactosidase activity of BgaPw, but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known ß-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield).

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 5.6 min to 10 s in a temperature range from 60 to 80°C. High temperature and ultra-high temperature (UHT) treatments from 90 to 140°C completely inactivated cathepsin D within 5 s. A residual cathepsin D activity of around 20% was detected under pasteurization conditions (72°C for 20 s). Therefore, investigations were done to estimate the effect of residual cathepsin D activity on taste in a model fresh cheese. The UHT-treated skim milk was spiked with cathepsin D and acidified with glucono-δ-lactone to produce a model fresh cheese. A trained bitter-sensitive panel was not able to distinguish cathepsin D-spiked model fresh cheeses from the control model fresh cheeses in a triangle test. Model fresh cheese samples were also analyzed for known bitter peptides derived from casein fractions using a HPLC-tandem mass spectrometry (MS) approach. In accordance with the sensory evaluation, the MS analyses revealed that the bitter peptides investigated within the cathepsin D-spiked model fresh cheese were not found or were below the limit of detection. Even though cathepsin D may be present during the fermentation of pasteurized milk, it does not seem to be responsible for bitter peptide formation from milk proteins on its own.

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. The histamine exerts its effects acting on different histamine receptors all over the body but also directly in the intestinal lumen. The inability to degrade sufficient amounts of dietary histamine is known as the 'histamine intolerance'. It would be preferable to solve this problem initially by the production of histamine-free or -reduced foods and by the oral supplementation of exogenous DAO supporting the human DAO in the small intestine. For the latter, DAOs from mammalian, herbal and microbial sources may be applicable. Microbial DAOs seem to be the most promising choice due to their possibility of an efficient biotechnological production in suitable microbial hosts. However, their biochemical properties, such as activity and stability under process conditions and substrate selectivity, play important roles for their successful application. This review deals with the advances and challenges of DAOs and other histamine-oxidizing enzymes for their potential application as processing aids for the production of histamine-reduced foods or as orally administered adjuvants to humans who have been eating food fraught with histamine.

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

A new diamine oxidase (DAO-1) was discovered recently in the yeast Yarrowia lipolytica 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 Y. lipolytica with optimized downstream processing. The DAO-1 tablet was tested in a histamine bioconversion experiment under simulated intestinal conditions in the presence of food constituents, whereby about 30% of the histamine was degraded in 90 min. This amount might already be sufficient to help people with histamine intolerance. Furthermore, it was found that the stability of DAO-1 in a simulated intestinal fluid is influenced distinctively by the presence of a food matrix, indicating that the amount and type of food consumed affect the oral supplementation with DAO. This study showed for the first time that a microbial DAO could have the potential for the treatment of histamine intolerance by oral supplementation.

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. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine-5'-phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed-batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.

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. The cultivation yielded 2343 ± 98 nkat/Lculture with a specific DAO activity of 1301 ± 54.2 nkat/gprotein, which was a 93-fold increase of specific DAO activity compared to the native Y. lipolytica PO1f DAO-1 production. The DAO-1 showed a broad substrate selectivity with tyramine, histamine, putrescine and cadaverine being the most favored substrates. It was most active at 40 °C, pH 7.2 in Tris-HCl buffer (50 mM) (with histamine as substrate), which is comparable to human and porcine DAOs. The affinity of DAO-1 towards histamine was lower compared to mammalian DAOs (Km = 2.3 ± 0.2 mM). Nevertheless, DAO-1 degraded around 75% of the histamine used in a bioconversion experiment with a food-relevant concentration of 150 mg/L. With its broad selectivity for the most relevant biogenic amines in foods, DAO-1 from Y. lipolytica PO1f is an interesting enzyme for application in the food industry for the degradation of biogenic amines.

Secretion of a low and high molecular weight ß-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 ß-glycosidases in a comparative cultivation approach. RESULTS: A low sized ß-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized ß-galactosidase isolated from the metagenome (M1; 120 kDa) were integrated into the acid extracellular protease locus using the CRISPR-Cas9 system to investigate the size dependent secretion of heterologous proteins in Y. lipolytica PO1f. The recombinant strains were cultivated in the bioreactor for 78 h and the extra- and intracellular enzyme activities were determined. The secretion of CelB resulted in an extracellular volumetric activity of 187.5 µkatoNPGal/Lmedium, while a volumetric activity of 2.98 µkatoNPGal/Lmedium was measured during the M1 production. However, when the amount of functional intra- and extracellular enzyme was investigated, the high molecular weight M1 (85%) was secreted more efficiently than CelB (27%). Real-time PCR experiments showed a linear correlation between the transcript level and extracellular activity for CelB, while a disproportional high mRNA level was observed regarding M1. Interestingly, mass spectrometry data revealed the unexpected secretion of two endogenous intracellular glycolytic enzymes, which is reported for the first time for Y. lipolytica. CONCLUSION: The results of this study provide deeper insights into the secretion potential of Y. lipolytica. A secretion limitation for the low-size CelB was observed, while the large size M1 enzyme was produced in lower amounts but was secreted efficiently. It was shown for the first time that Y. lipolytica is a promising host for the secretion of heterologous high molecular weight proteins (> 100 kDa), although the total secreted amount has to be increased further.

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. Kinetic investigations of porcine DAO showed a substrate inhibition by histamine concentrations greater than 56 mg/L (0.5 mM). The stability of free porcine DAO was tested in a fed state simulated intestinal fluid and exhibited a half-life period of around 19 min. A total of 50 nanokatal (nkat) free porcine DAO, which equaled the amount of enzyme isolated from around 100 g pig kidney, were necessary for the in vitro reduction of around 90% of the histamine. The dietary supplement that contains a pig kidney extract did not show DAO activity. Instead, the used histamine (0.75 mg) was apparently reduced due to the adsorption of histamine onto a capsule component by 18.9 ± 2.3% within 5 hr. Although the capsule preparation retained its overall structure and shape for at least 90 min in simulated gastric fluid, the apparent histamine reduction was significantly reduced to 12.1 ± 2.3% (P ≤ 0.05). In conclusion, an alternative to the pig kidney DAO or an improved capsule preparation is needed to ensure an adequate supplementation for histamine-intolerant humans. PRACTICAL APPLICATION: Histamine intolerance is an emerging issue in our society and the intolerance-related physiological symptoms are currently not reliably treatable due to a lack of scientific investigation. A commercially available dietary supplement for histamine intolerance does not fulfil the requirements for a satisfactory histamine reduction in intolerant humans. The activity of the histamine degrading enzyme diamine oxidase, required for a satisfactory histamine degradation, is by far higher than the theoretical amount apparently given in the dietary supplement. With this knowledge, it is obvious that improved food supplements must be developed to help histamine intolerant humans.

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 5.4.99.16) catalyzes the reversible conversion between maltose and trehalose. A recombinant trehalose synthase from Arthrobacter chlorophenolicus SK 33.001 (ACTS) was cloned, expressed, and characterized. The recombinant enzyme encoded a protein of 598 amino acids with a molecular mass of 66 kDa. Gel filtration showed that ACTS is a tetramer in sodium phosphate buffer. The enzyme was metal ion independent and exhibited maximal activity in sodium phosphate buffer (pH 7.5) at 30 °C. The kinetic investigations resulted in a KM value of 120.5 ± 4.5 mM for maltose and a KM value of 343.1 ± 13.8 mM for trehalose. The catalytic efficiency (Vmax/KM) for maltose and trehalose were 0.2 and 0.15 U mg-1 mM-1, respectively. In addition, a cooperative substrate binding was found displayed by the determined Hill coefficients (nH) of 2.8 for maltose and 2.1 for trehalose as a substrate, respectively. The final trehalose yield of various maltose concentrations (50-1000 mM) was constant between 58 and 59%, implying that substrate concentration had no inhibitory influence on ACTS activity.

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 5.4.99.16). Metagenomic DNA was isolated from diverse soil samples and used as template for PCR-based screening targeted against conserved regions of trehalose synthases. This resulted in four metagenomic TS-like fragments with broad sequence diversity (41-67% identity to each other). The encoded open reading frames were used as templates for further in silico screening. Two trehalose synthases were discovered using this novel approach and their enzymatic properties were further investigated. The trehalose synthase from Micrococcus terreus MtTS exhibited a broad pH optimum between 6.5 and 7.5 with highest reaction velocity at 35 °C and a protruding stability at this temperature (t1/2 = 50 h). Characteristic of enzymes from thermophilic organisms, the trehalose synthase from Thermobaculum terrenum had a distinct temperature optimum at 50 °C, exhibiting also a prominent half time with t1/2 = 45 h at pH 6.5. Both bioconversions resulted in final trehalose levels of 60%, whereas TtTS produced reduced amounts of the byproduct glucose (10%) compared with MtTS (15%), which is favorable for trehalose production. This combined screening approach intended to circumvent the bottleneck of metagenomic enzyme mining, regarding time and cost of intensive screening procedures for industrial relevant biocatalysts such as trehalose synthases.

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 ß-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of ß-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of arylsulfatase (EC 3.1.6.1). Due to the action of arylsulfatase, an unpleasant "cowshed-like" off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.

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. 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.

Hidden Reaction: Mesophilic Cellobiose 2-Epimerases Produce Lactulose.

Lactulose (4-O-ß-d-galactopyranosyl-d-fructofuranose) is a prebiotic sugar derived from the milk sugar lactose (4-O-ß-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 ß-1,4-glycosidic-linked oligosaccharides. Thus, epilactose (4-O-ß-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.

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. Furthermore, the production of microbial arylsulfatases using wild-type organisms as well as the recombinant production using Escherichia coli and Kluyveromyces lactis as expression hosts is discussed. Finally, various potential applications of these enzymes are reviewed.

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. After targeting, the membrane insertion of SciP is catalyzed by YidC independent of the SecYEG translocase. However, when the C-terminal tail of SciP was extended to 21 aa residues, we found that SecYEG becomes involved and makes its membrane insertion more efficient.

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. Here, we show in E. coli that an extended C-terminal region of YidC can compensate for a loss of SRP-receptor function in vivo. Furthermore, the enhanced affinity of the ribosome to the chimeric YidC allows the isolation of a ribosome nascent chain complex together with the C-terminally elongated YidC chimera. This complex was visualized at 8.6 Šby cryo-electron microscopy and shows a close contact of the ribosome and a YidC monomer.