Mutation, food-grade expression, and characterization of a lactonase for zearalenone degradation.

Zearalenone (ZEN) is a mycotoxin that causes serious threats to human health. People are exposed to ZEN contamination externally and internally through many ways, while environmental-friendly strategies for efficient elimination of ZEN are urgently needed worldwide. Previous studies revealed that the lactonase Zhd101 from Clonostachys rosea can hydrolyze ZEN to low toxicity compounds. In this work, the enzyme Zhd101 was conducted with combinational mutations to enhance its application properties. The optimal mutant (V153H-V158F), named Zhd101.1, was selected and introduced into the food-grade recombinant yeast strain Kluyveromyces lactis GG799(pKLAC1-Zhd101.1), followed by induced expression and secretion into the supernatant. The enzymatic properties of this mutant were extensively examined, revealing a 1.1-fold increase in specific activity, as well as improved thermostability and pH stability, compared to the wild-type enzyme. The ZEN degradation tests and the reaction parameters optimization were carried out in both solutions and the ZEN-contaminated corns, using the fermentation supernatants of the food-grade yeast strain. Results showed that the degradation rates for ZEN by fermentation supernatants reached 96.9% under optimal reaction conditions and 74.6% in corn samples, respectively. These new results are a useful reference to zearalenone biodegradation technologies and indicated that the mutant enzyme Zhd101.1 has potential to be used in food and feed industries. KEY POINTS: • Mutated lactonase showed 1.1-fold activity, better pH stability than the wild type. • The strain K. lactis GG799(pKLAC1-Zhd101.1) and the mutant Zhd101.1 are food-grade. • ZEN degradation rates by supernatants reached 96.9% in solution and 74.6% in corns.

A Novel Strategy for Whole-Cell Biotransformation Enabling Simultaneous l-Phenyllactic Acid Production and Coenzyme Regeneration.

l-Phenyllactic acid (l-PLA) is a small molecular organic acid that exhibits a powerful capacity for inhibition against foodborne pathogens. In this work, we developed a new cost-effective and environmentally friendly process for the biosynthesis of l-PLA. This strategy designed a novel whole-cell biotransformation system employing two heterologous enzymes, namely, phenylalanine dehydrogenase (PheDH) and l-hydroxyisocaproate dehydrogenase (l-HicDH). The novelty of this strategy lies in the first-time utilization of these two enzymes, which not only enables cascade catalysis for the production of l-PLA but also facilitates the regeneration of the coenzymes (NAD+/NADH) using only two enzymes rather than introducing more heterologous enzymes to the system. Consequently, this strategy can effectively simplify the biosynthesis process of l-PLA and minimize production costs. The initial l-PLA yield using this process achieved 2.53 ± 0.07 g/L. Furthermore, through meticulous optimization of the parameters for inducible enzyme expression and l-PLA biosynthesis, the l-PLA yield was successfully increased to 4.68 ± 0.04 g/L with a yield rate of 64.54 ± 0.29%. Moreover, this novel strategy is versatile in the biosynthesis of other organic acids, which can be achieved by easily modulating the combinations of substrates and enzymes.

Preparation of recombinant Kluyveromyces lactis agents for simultaneous degradation of two mycotoxins.

Aflatoxin B1 (AFB1) and zearalenone (ZEN) are widely distributed in corns, peanuts, and other cereals, causing serious threat to food safety and human health. As shown by our previous studies, the recombinant yeast strain Kluyveromyces lactis GG799(pKLAC1-ZPF1) had the ability of degrading AFB1 and ZEN simultaneously. In this work, the agent preparation process was optimized for K. lactis GG799(pKLAC1-ZPF1), and the storage conditions of the prepared yeast agents were investigated, for obtaining the products with high storage activities and potent mycotoxin degradation efficiency. The optimal preparation process was as follows: centrifugation at 6000 rpm for 15 min for collection of the yeast cells, spray drying with the ratio of protective compounds to yeast cells at 3:1 (w/w) and then stored at - 20 °C. Simultaneous degradation tests of AFB1 and ZEN were performed using the supernatants of reactivated yeast agents after three months of storage, and the degradation ratios for AFB1 and ZEN in reaction system 1 (70.0 mmol/L malonic buffer, pH 4.5, with 1.0 mmol/L MnSO4, 0.1 mmol/L H2O2, 5.0 µg/mL AFB1 and ZEN, respectively) were 48.2 ± 3.2% and 34.8 ± 2.8%, while that for ZEN in reaction system 2 (50.0 mmol/L Tris-HCl, pH 7.5, with 5.0 µg/mL AFB1 and ZEN, respectively) was 30.1 ± 2.7%. Besides, the supernatants of reactivated yeast agents degraded more than 80% of AFB1 and 55% of ZEN in contaminated peanuts after twice treatments. Results of this work suggested that the optimized process for K. lactis GG799(pKLAC1-ZPF1) was with high value for industrial applications.

Research Advances of d-allulose: An Overview of Physiological Functions, Enzymatic Biotransformation Technologies, and Production Processes.

d-allulose has a significant application value as a sugar substitute, not only as a food ingredient and dietary supplement, but also with various physiological functions, such as improving insulin resistance, anti-obesity, and regulating glucolipid metabolism. Over the decades, the physiological functions of d-allulose and the corresponding mechanisms have been studied deeply, and this product has been applied to various foods to enhance food quality and prolong shelf life. In recent years, biotransformation technologies for the production of d-allulose using enzymatic approaches have gained more attention. However, there are few comprehensive reviews on this topic. This review focuses on the recent research advances of d-allulose, including (1) the physiological functions of d-allulose; (2) the major enzyme families used for the biotransformation of d-allulose and their microbial origins; (3) phylogenetic and structural characterization of d-allulose 3-epimerases, and the directed evolution methods for the enzymes; (4) heterologous expression of d-allulose ketose 3-epimerases and biotransformation techniques for d-allulose; and (5) production processes for biotransformation of d-allulose based on the characterized enzymes. Furthermore, the future trends on biosynthesis and applications of d-allulose in food and health industries are discussed and evaluated in this review.

Simultaneous degradation of two mycotoxins enabled by a fusion enzyme in food-grade recombinant Kluyveromyces lactis.

Aflatoxin B1 (AFB1) and zearalenone (ZEN) are two predominant mycotoxins ubiquitously found in corn, peanuts, and other grains, which pose a great threat to human health. Therefore, safe and effective methods for detoxification of these mycotoxins are urgently needed. To achieve simultaneous degradation of multiple mycotoxins, a fusion enzyme ZPF1 was constructed by linking zearalenone hydrolase and manganese peroxidase with a linker peptide GGGGS. This fusion enzyme was secretory expressed successfully in the newly constructed food-grade recombinant strain Kluyveromyces lactis GG799(pKLAC1-ZPF1), and was investigated with the mycotoxins degradation efficiency in two reaction systems. Results showed that both AFB1 and ZEN can be degraded by ZPF1 in reaction system 1 (70.0 mmol/L malonic buffer with 1.0 mmol/L MnSO4, 0.1 mmol/L H2O2, 5.0 µg/mL AFB1 and ZEN, respectively) with the ratios of 46.46% and 38.76%, respectively. In reaction system 2 (50.0 mmol/L Tris-HCl, with 5.0 µg/mL AFB1 and ZEN, respectively), AFB1 cannot be degraded while ZEN can be degraded with the ratio of 35.38%. To improve the degradation efficiency of these mycotoxins, optimization of the induction and degradation conditions were fulfilled subsequently. The degradation ratios of AFB1 and ZEN by ZPF1 in reaction system 1 reached 64.11% ± 2.93% and 46.21% ± 3.17%, respectively. While in reaction system 2, ZEN was degraded by ZPF1 at a ratio of 41.45% ± 3.34%. The increases of degradation ratios for AFB1 and ZEN in reaction system 1 were 17.65% and 7.45%, respectively, while that for ZEN in reaction system 2 was 6.07%, compared with the unoptimized results.

Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea).

In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.