Browsing by Person "Fischer, Lutz"

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Enzymatic hydrolysis of vegetable and insect proteins using technical enzyme preparations
(2021) Großmann, Kora Kassandra; Fischer, Lutz
The present dissertation covers the usage of technical enzyme preparations (TEPs) for vegetable and insect protein hydrolysis, due to the mounting interest in alternative protein sources to cover the increasing demand for food from a growing world population. The TEPs, as defined in this study, are enzyme preparations that include side activities and are used in food processing. Today, TEPs are used by food manufacturers based on the supplier’s information that usually states the main enzyme activity and includes information on side activities only in some cases. However, knowledge about the activity profile is crucial as side activities can contribute to the properties of the protein hydrolysates produced (e.g. degree of hydrolysis [DH], liberation of amino acids) and the final food product quality. In the first study, an automated photometric analyzer (GalleryTM Plus, Thermo Fisher Scientific) was introduced for the comprehensive activity determination of TEPs. The new setup of the analyzer covered 32 synthetic and natural substrates in order to determine aminopeptidase, carboxypeptidase, dipeptidylpeptidase and endopeptidase activities distinguishably. Accordingly, the overall proteolytic activity of TEPs was quantified and detailed information about the substrate spectra and peptidase side activities was generated. Furthermore, several batches of the industrial TEP Flavourzyme1000L were measured. By determining 32 peptidase activities, batch variations were shown. As Flavourzyme1000L is standardized by its supplier Novozymes on only one activity (leucine aminopeptidase), the additional 31 new peptidase activities determined showed differences of the side activities of the batches. In addition, the study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting lupine protein hydrolysates (DH and liberation of amino acids). Due to the determination of 32 peptidase activities (so-called “activity fingerprint”), TEPs were selected specifically to increase, for example, the DH. The two TEPs P278 and DZM were selected due to their complementary peptidase activities, as an example of this study. The combination of these two TEPs resulted in an increase of the DH of 47%. Now, TEPs can be selected targeted more on the basis of their peptidase activities to, for example, increase the hydrolysis efficiency of lupine protein by combining complementary peptidase activities. In the second study, six food-grade TEPs (Flavourzyme1000L, ProteaseP “Amano”6SD, DeltazymAPS-M-FG, Promod278, ProteAX-K and PeptidaseR) were investigated regarding their influence on the hydrolysis of soy, pea and canola protein. The hydrolysates were investigated analytically concerning their DH and free amino acid profiles and sensorically concerning the taste attributes umami and bitter. By using a random forest model, the taste attributes bitter and umami were connected to specific peptidase activities (exo- and endopeptidase activities). Furthermore, out of the six selected TEPs, the usage of ProteAX-K showed high umami and low bitter taste of the vegetable protein hydrolysates (soy, pea and canola). In line with the first study, the second study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting vegetable protein hydrolysates. Based on these new insights, TEPs can be selected more specifically based on their peptidase activity profiles to direct the formation of desired taste attributes of the protein hydrolysates. In the third study, two TEPs with various peptidase activities (Flavourzyme1000L, ProteaseA “Amano”2SD) were applied for the hydrolysis of insect proteins. This study investigated the potential of cricket and mealworm protein and their hydrolysates regarding their sensory potential. The sensory profiles of the insect proteins were altered by, firstly, applying proteolytic hydrolysis and then a Maillard reaction (30 min, T = 98°C, 1% (w/v) xylose) to the hydrolysates. The initially earthy-like flavor of the insect proteins resulted in modified taste profiles described by e.g., savory-like attributes, due to both processing steps. Furthermore, 38 odor-active molecules (1 alcohol, 5 acids, 11 aldehydes, 5 ketones and 16 heterocyclic compounds) were identified by gas chromatography-olfactometry (GC-O). The identified molecules are also found in meat and edible seafood products. The third study showed that the flavoring profile of insect proteins was modified and can be developed further by the respective processing.
Food-grade Lactobacilli expression systems for recombinant enzymes
(2013) Böhmer, Nico; Fischer, Lutz
Lactobacilli are Gram-positive bacteria used throughout the food industry as traditional starters for various fermented foods. Lactobacilli would be superior for recombinant enzyme production regarding the food safety demands since most of them are Generally Recognised As Safe (GRAS) organisms. The major advantages of Lactobacilli as food-associated microorganisms used for recombinant enzyme production are their safe and sustainable use as overall safety food-grade expression systems. In the work presented, Lactobacilli were studied in detail as food-grade expression systems for recombinant enzyme production. In a first analysis, the two pSIP expression systems, pSIP403 and pSIP409, were investigated to produce a hyper-thermophilic Beta-glycosidase (CelB) from Pyrococcus furiosus in Lactobacillus plantarum NC8 and Lactobacillus casei as hosts, respectively. Both Lactobacilli harbouring the pSIP409-celB vector produced active CelB in batch bioreactor cultivations, while the specific CelB activity of the cell-free extract was about 44% higher with Lb. plantarum (1,590 ± 90 nkatpNPGal/mgprotein) than with Lb. casei (1,070 ± 66 nkatpNPGal/mgprotein). A fed-batch bioreactor cultivation of Lb. plantarum NC8 pSIP409-celB resulted in a specific CelB activity of 2,500 ± 120 nkatpNPGal/mgprotein. A basal whey medium with supplements was developed as an alternative to the cost intensive MRS medium used. About 556 ± 29 nkat pNPGal/mgprotein of CelB activity was achieved in bioreactor cultivations using this medium. It was shown that both Lactobacilli were potential expression hosts for recombinant enzyme production. An additional approach was performed to produce a metagenome-beta-galactosidase using Lb. plantarum NC8 with the pSIP expression system. Using this system, a quite low maximal galactosidase activity of only 0.18 nkatoNPGal/mgprotein was detected. A 13 times higher activity of 2.42 nkatoNPGal/mgprotein was produced after the knock out of the interfering native Kluyveromyces lactis Beta-galactosidase in the well-known food-grade K. lactis pKLAC2 expression system. Nevertheless, the best performing expression system for the recombinant production of the metagenome-derived enzyme was the Escherichia coli BL21 strain with a pET vector, resulting in the highest Beta-galactosidase of 82.01 nkatoNPGal/mgprotein. Beside the use of the pSIP expression system, a novel expression system for Lb. plantarum was developed. This system is based on the manganese starvation-inducible promoter from the specific manganese transporter of Lb. plantarum NC8 which was cloned for the first time. The expression of CelB was achieved by cultivating Lb. plantarum NC8 at low manganese concentrations with MRS medium and the pmntH2-celB expression vector. A CelB activity of 8.52 µkatoNPGal/L was produced in a bioreactor. The advantages of the novel expression system are that no addition of an external inducing agent was required, and additionally, no further introduction of regulatory genes was necessary. The new promoter meets the general demands of food-grade expression systems. The glutamic acid racemase of Lb. plantarum NC8 was cloned and characterized in this work for the first time as a possible target for a food-grade selection system for this species. Glutamic acid racemases (MurI, E.C. 5.1.1.3) catalyse the racemisation of L- and D-glutamic acid. MurIs are essential enzymes for bacterial cell wall synthesis, which requires D-glutamic acid as an indispensable building block. Therefore, these enzymes are suitable targets for antimicrobial drugs as well as for the potential design of auxotrophic selection markers. A high expression system in E. coli BL21 was constructed to produce and characterize the biochemical properties of the MurI from Lb. plantarum NC8. The recombinant, tag-free Murl was purified by an innovative affinity chromatography method using L-glutamic acid as the relevant docking group, followed by an anion exchange chromatography step (purification factor 9.2, yield 11%). This two-step purification strategy resulted in a Murl sample with a specific activity of 34.06 µkatD-Glu/mgprotein, comprising a single protein band in SDS-PAGE. The purified Murl was used for biochemical characterization to gain in-depth knowledge about this enzyme. Only D- and L-glutamic acid were recognised as substrates for the Murl with similar kcat/Km ratios of 3.6 sec-1/mM for each enantiomer. The findings in this study may contribute to further development and implementation of food-grade Lactobacilli expression systems for recombinant enzyme production. Furthermore, the results obtained may help to optimise and select hosts and expression systems for industrial enzyme production for the needs of the food industry.
Herstellung und Charakterisierung einer rekombinanten, sequenzspezifischen Protease zur Generierung bioaktiver Peptide
(2009) Hug, Thomas; Fischer, Lutz
The aim of the present thesis was the production and biochemical characterization of a sequence specific microbial protease. This enzyme should be applied in food protein hydrolyzation in order to generate bioactive peptides. To determine the substrate specificity of serine protease PRT1 from Xanthomonas campestris pv. campestris this strain was cultivated in a shaking flusk. After dialysis of the culture broth and addition of 1,10-phenantroline for metalloprotease PRT2 inactivation an enzyme activity of 0,34 nkat Caseine/mL was detected. The conversion of several chromogenic peptide substrates revealed that PRT1 does not offer a clear substrate specificity. The lysyl endopeptidase LysC from Lysobacter enzymogenes ssp. enzymogenes was obtained by cultivation of the wild type strain (ATCC 27796). In a bioreactor (1 L scale) a maximum protease activity of 0,084 nkat Tos-Gly-Pro-Lys-pNA/mL in the culture broth was detected after 45 h. The LysC gene was amplified by PCR using genomic template DNA and was cloned into the E. coli expression vector pET20b(+), leading to no detectable recombinant protease when expressed in E. coli BL21(DE3). Thus for the heterologous expression a synthetic gene construct was applied which was formerly described in literature. It contained a short N-terminal pro-sequence (MGSK) and a codon usage adapted to E. coli. The bioreactor cultivation (5 L scale) of E. coli BL21(DE3) pET20b-MGSK-LysC led to LysC inclusion bodies. The solubilization of the inclusion bodies and the following enzyme renaturation using L-arginine as an unspecific folding additive resulted in a maximum protease activity of 0,06 nkat Tos-Gly-Pro-Lys-pNA/L Culture 5h after IPTG induction. To increase the yield of recombinant protease activity the influence of the LysC propeptides on the in vitro renaturation of the protease was investigated. For this purpose both pro-peptide DNAs were sequenced, cloned and heterologously expressed in E. coli BL21(DE3). The addition of C-terminal and N-terminal propeptide to the LysC renaturation led to a maximum of 27fold (1.56 Μkat Tos-Gly-Pro-Lys-pNA/L Culture) LysC activity increasion compared to the L-arginine renaturation. As an alternative food-grade expression system the in the literature already established system of Lactobacillus plantarum NC8, L. sakei Lb790 and the E. coli-Lactobacillus shuttle vector pSIP409 was tested. However, the shaking flusk cultivations of neither L. plantarum NC8 pSIP409-MGSK-LysC nor L. sakei Lb790 pSIP409-MGSK-LysC led to detectable recombinant lysyl endopeptidase. As a possible reason therefor the different codon usage of E. coli and Lactobacillus was assumed. So expression experiments were performed using point mutated variants of the beta-galctosidase gene from Kluyveromyces lactis. It could be shown that the exchange of the serine codons tca/agt and tcc had a significant effect on the resulting enzyme activity. The exchange of three codons led to a decreation of beta-galactosidase activity of 38 %. The characterization of the recombinant lysyl endopeptidase LysC confirmed the high substrate specificity for lysine residues at P1 position. The pH and temperature optimum was 8.5 and 45°C, respectively. At 4°C and pH 9 the enzyme was stable for at least 20.5 h, whereas at 45°C only 40 % residual activity were detected after 1 h. An inhibiting effect on LysC was demonstrated for Ba2+, NH+ and PMSF. Hydrolysis of bovine caseine by LysC for generated the ACE inhibiting peptides EMPFPK, FALPQYLK, NMAINPSK and ALNEINQFYQK as well as the antioxidative VLPVPQK, which all were unambiguously identified by LC-ESI-MS/MS. Performing appropriate in vitro assays, the radical scavenging acticvity (IC50 = 4,85 Μg/mL), lipoxygenase inhibition (IC50 = 23,6 Μg/mL) and ACE inhibition (IC50 = 2,78 Μg/mL) of the caseine hydrolysate were quantified.
Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine
(2021) Swietalski, Paul; Hetzel, Frank; Klaiber, Iris; Pross, Eva; Seitl, Ines; Fischer, Lutz
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.