Lutz Fischer

Enzymatic deamidation using protein-glutamine glutaminases (PGs) is a promising method for improving the solubility, foaming properties, and emulsifying properties of plant proteins. This study compared the deamidation of various plant proteins using PGs from Chryseobacterium proteolyticum (PGC), Bacteroides helcogenes (PGB), and Flavobacterium sp. (PGF) and examined the effects on the foaming and emulsifying properties of sunflower and oat proteins. PGB produced the largest increases in protein solubility, though PGF usually gave the highest maximal degree of deamidation (DD(max)). For oat protein, all PGs reduced maximum foam volume (V_F(max)), but PGC and PGB doubled foam half-time (t_{1/2 foam}). For sunflower protein, PGB reduced V_F(max), while PGC increased t_{1/2 foam} by up to 17.5-fold. Thus, different PGs affect techno-functional properties distinctly, reflecting different substrate specificities. Improvements in solubility, foaming, and emulsifying properties are not dependent on achieving the highest DD(max). Expanding the range of available PGs would allow for better matching to plant substrates and enhance industrial applicability.

Background: Cellobiose 2-epimerases (CEs) are promising enzymes that catalyze the conversion of lactose to the bioactive disaccharides epilactose and lactulose. Low-temperature (≤8 °C) lactose bioconversion is essential in dairy applications to prevent microbial contamination. Unfortunately, most CEs are not particularly active at these low temperatures. Therefore, this work aims to discover and characterize novel cold-active CEs. Results: Two novel mesophilic CEs from Teredinibacter haidensis ( Th CE) and Cellvibrio japonicus ( Cj CE) were characterized, which showed both epi- and isomerization activity additionally at low temperatures. Th CE showed a maximum epimerization activity (595.4 ± 4.4 nkat/mg protein) at pH 7.5 and 35 °C, while Cj CE achieved maximum epimerization activity (887.4 ± 3.9 nkat/mg protein) at pH 9.5 and 40 °C. In addition, Cj CE maintained an >80% activity across a wide pH (6–9.5) and temperature range (30–45 °C). Both CEs exhibited significant activity and stability at 8 °C. Specifically, Th CE and Cj CE retained around 20 and 40% of their epimerization activity at 8 °C and showed half-lives of 96.3 and 72.2 days, respectively. Moreover, Cj CE was also found to be capable of catalyzing the isomerization reaction at 8 °C. The isomerization activity of Th CE and Cj CE increased ~370-fold (70.8 ± 2.7 nkat/mg protein) and ~230-fold (66.1 ± 0.3 nkat/mg protein), respectively, at lactose concentrations up to 600 mM (35 °C for Th CE and 40 °C for Cj CE). This demonstrates how important it is to conduct investigations of CEs for lactose isomerization to lactulose up to the solubility limit of lactose. Conclusions: This study identified two new CEs that exhibit interesting catalytic properties for possible application in the generation of disaccharides with prebiotic properties from lactose at low temperatures.

The recombinant production of the protein-glutaminase (PG) from Bacteroides helcogenes (PGB) was investigated in the three Bacillus subtilis strains: 168, RIK1285 and the isolated strain 007. B. subtilis 007 produced the highest PG activity (7.6 ± 0.7 µkat L _{Culture supernatant} ⁻¹ ) in shake flask cultivations and, thus, was used for further investigations. As a wild-type strain, B. subtilis 007 exhibited high extracellular proteolytic activity and formed large amounts of foam in a bioreactor cultivation. While the proteolytic activity would be favorable for the extracellular cleavage of PGB’s propeptide, foaming is undesirable and should be prevented. Therefore, the influence of the two foam-demolishing substances anti-foaming agent 204 and rapeseed oil on the PGB production was investigated in bioreactor cultivations. A PGB activity of 11.8 ± 1.1 µkat L _{Culture supernatant} ⁻¹ was obtained using anti-foaming agent 204, while rapeseed oil led to a lower maximal PGB activity of 7.6 ± 0.2 µkat L _{Culture supernatant} ⁻¹ . PGB was partially purified from the culture supernatant by fractionated ammonium sulphate precipitation followed by hydrophobic interaction chromatography. A final yield of 14.8% was obtained, while the specific PGB activity increased from 6.7 to 28.6 nkat mg ⁻¹ . Alternatively, PGB was purified from culture supernatant by cross-flow filtration and subsequent heating step (1 h, 60 °C) to inactivate the extracellular peptidases. Thereby, the total proteolytic activity was decreased to below 4% while the specific PGB activity increased to 32.1 ± 2.0 nkat mg ⁻¹ . The PGB preparation obtained was applied in gluten deamidation experiments which showed that PGB deamidated a 1% (w/v) gluten suspension by 95 ± 2%.

Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/L _culture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/L _culture , and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/L _culture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics. Key points: • Site-specific PGB integration enabled by genome sequencing of B. subtilis 007 . • Antibiotic-free and secretory PGB production with an optimized B. subtilis host . • Increased PGB production reaching 9.5 µkat/L _culture .

Background: The monosaccharide D-tagatose is a promising alternative to sucrose because of its similar sweetness and lower glycemic index. A novel L-arabinose isomerase (L-AI) from Lentilactobacillus parakefiri DSM 10551 (L-AI-Lp) has been biochemically characterized and used to isomerize D-galactose to D-tagatose in skim milk ultrafiltration permeate at pH 4.5 and 6.5. However, like most L-AIs described in the literature, this enzyme has only been produced recombinantly in Escherichia coli . This study aimed to systematically investigate the intracellular recombinant production of L-AI-Lp in Bacillus subtilis , which has qualified for a presumption of safety (QPS) designation from the European Food Safety Authority. Results: The influence of four promoters on L-AI-Lp production in B. subtilis 007 was investigated in shake flask cultivations. Among these, the P _AprE promoter yielded the highest volumetric L-AI activity of 69.2 ± 7.4 µkat _{Gal, 65 °C} /L _Culture . The production yield was further increased to 147.7 ± 1.0 µkat _{Gal, 65 °C} /L _Culture by using the nonsporulating, surfactin-deficient strain B. subtilis 007 ∆ sfp ∆ sigF , which was constructed by deleting sigF and sfp in B. subtilis 007. Furthermore, the influence of pH and dissolved oxygen (DO) on bioreactor cultivations of B. subtilis 007 ∆ sfp ∆ sigF was analyzed. In bioreactor cultivations, the highest L-AI activity of 88.6 ± 2.4 µkat _{Gal, 65 °C} /L _Culture was measured under unregulated pH and low oxygen conditions (DO ≤ 5%), representing a 3.2-fold increase compared with previous recombinant production in E . coli . The L-AI-Lp was subsequently partially purified by heat treatment and precipitation methods, resulting in a 7.8-fold increase in specific activity to 128.2 nkat _{Gal, 65 °C} /mg and a yield of 84%. Conclusions: The L-AI-Lp was recombinantly produced for the first time in a microbial species with QPS status using the nonsporulating and surfactin-deficient strain B. subtilis 007 ∆ sfp ∆ sigF . The L-AI-Lp was subsequently partially purified via nonchromatographic methods, providing a basis for a low-cost downstream process. These results represent an important step toward potential industrial application of L-AI-Lp and highlight the potential of B. subtilis 007 ∆ sfp ∆ sigF as an expression host for the recombinant production of L-AIs compared with previously used hosts from the order Lactobacillales .

The diamine oxidase from Glutamicibacter halophytocola (DAO-GH) was recombinantly produced in K. phaffii using the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter for methanol-free production. Firstly, K. phaffii clones were generated for intracellular and secretory DAO-GH production that still possessed antibiotic resistance due to the cloning procedure. For intracellular production, a maximum intracellular DAO activity of 15,404 nkat/L _culture was achieved in fed-batch bioreactor cultivations, while for secretory production, the highest extracellular DAO activity of 6,078 nkat/L _culture was achieved using the αMF signal peptide without its EAEA sequence. The intracellularly produced DAO-GH was partially purified in several purification steps with a yield of 80%, a purification factor of about 10 and specific DAO activity of 16.7 nkat/mg _protein . The secretory DAO-GH production resulted in a specific DAO activity of 15.4 nkat/mg _protein already in the cell-free culture supernatant at the end of cultivation without further purification steps. The food industry aims to avoid the use of antimicrobial resistance in enzyme production, therefore, a new cassette plasmid with self-excisable antibiotic resistance markers was constructed for secretory DAO-GH production. The antibiotic-resistance-free K. phaffii clone generated with this plasmid achieved a maximum extracellular DAO activity of 4,770 nkat/L _culture in a fed-batch bioreactor cultivation. The DAO-GH obtained in this cultivation was spray-dried, resulting in a storable powder with 23 nkat/g _powder DAO activity and a water activity value of 0.12. This study demonstrated the secretion of recombinant DAO in a microbial host such as K. phaffii for the first time and provides a strategy for generating antibiotic-resistance-free K. phaffii clones.

A novel diamine oxidase (DAO) was discovered in the bacterium Glutamicibacter halophytocola (DAO-GH). The gene of DAO-GH was integrated into the genome of the yeast Komagataella phaffii and recombinantly produced under control of the methanol-inducible AOX1 promoter in a bioreactor cultivation. A high DAO activity of 70.2 ± 5.2 µkat/L_culture (5.25 ± 0.22 µkat/g_protein) was yielded after 90 h of cultivation. The DAO-GH was partially purified by the polyethyleneimine precipitation of nucleic acids, fractionated ammonium sulfate precipitation and hydrophobic interaction chromatography, resulting in a specific DAO activity of 19.7 µkat/g_Protein. The DAO-GH was then biochemically investigated regarding its potential for histamine and tyramine degradation in fermented foods and the human small intestine. Interestingly, the DAO-GH showed activity even at a low pH of 5 and low temperature of 6 °C. Both histamine and tyramine were effectively degraded and DAO-GH showed especially very high affinity towards tyramine (K_m of 0.009 mM). The DAO-GH was shown to be capable of degrading around 20% of the initially applied histamine in tuna paste (pH 5.6) at 5 °C within 24 h and completely degraded the histamine in a simulated intestinal fluid within 1.5 h in bioconversion experiments. The DAO-GH was spray-dried for the production of a storable enzyme preparation. Only around 17% of activity were lost in this process and the DAO-GH remained stable at room temperature for at least 3 months. The discovery of this DAO with its very advantageous biochemical properties allows the preparation of histamine-reduced or -free fermented foods by a simple enzymatic treatment or the treatment of histamine intolerance symptoms as a dietary supplement or medicine.

A novel diamine oxidase (DAO) was discovered in the bacterium Glutamicibacter halophytocola (DAO-GH). The gene of DAO-GH was integrated into the genome of the yeast Komagataella phaffii and recombinantly produced under control of the methanol-inducible AOX1-promoter in a bioreactor cultivation. A high DAO activity of 70.2 ± 5.2 µkat/Lculture (5.25 ± 0.22 µkat/gprotein) was yielded after 90 h of cultivation. The DAO-GH was partially purified by the polyethyleneimine precipitation of nucleic acids, fractionated ammonium sulfate precipitation and hydrophobic interaction chromatography, resulting in a specific DAO activity of 19.7 µkat/gProtein. The DAO-GH was then biochemically investigated regarding its potential for histamine and tyramine degradation in fermented foods and the human small intestine. Interestingly, the DAO-GH showed activity even at a low pH of 5 and low temperature of 6 °C. Both histamine and tyramine were effectively degraded and DAO-GH showed especially very high affinity towards tyramine (Km of 0.009 mM). The DAO-GH was shown to be capable of degrading around 20 % of the initially applied histamine in tuna paste (pH 5.6) at 5 °C within 24 h and completely degraded the histamine in a simulated intestinal fluid within 1.5 h in bioconversion experiments. The DAO-GH was spray-dried for the production of a storable enzyme preparation. Only around 17 % of activity were lost in this process and the DAO-GH remained stable at room temperature for several months. The discovery of this DAO with its very advantageous biochemical properties allows the preparation of histamine-reduced or -free fermented foods by a simple enzymatic treatment or the treatment of histamine intolerance symptoms as a dietary supplement or medicine.

A new horseradish peroxidase (HRP)-based assay for the determination of aqueous ammonia was developed. This new assay utilizes the concentration-dependent, linearly increasing activation effect of aqueous ammonia on the enzyme HRP at alkaline pH values. The special feature of this assay is that the analyte, ammonia, is not directly involved in the reaction catalyzed by the enzyme HRP. Instead, the concentration of the analyte is determined by measuring the increase of the activity of the HRP in the absence and presence of the sample. Accordingly, all relevant assay parameters and concentrations of the components were first carefully evaluated to enable the determination of low aqueous ammonia concentrations while simultaneously achieving a broad linear range. The optimized conditions in the newly developed method for the determination of ammonia were pH 10.0, 4.8 nkat mL⁻¹ HRP, 2.3 mmol L⁻¹ o-dianisidine, and 400 µM hydrogen peroxide. The limit of detection and the limit of quantification of the new method were 0.24 mmol L⁻¹ and 0.36 mmol L⁻¹, respectively. One possible application for the newly developed ammonia determination assay was the determination of protein-glutamine glutaminase (PG) activities. This assay was employed as a two-step assay, starting with the PG reaction conducted at the optimal pH value for the PG used. After this step, the pH was increased to 10, and the ammonia released was measured in a second reaction. The results obtained with this method showed less than 10% variation compared to two established methods.