Abstract:Mycotoxins are a group of secondary metabolites produced by fungi during their growth. Mycotoxins not only seriously affect the yield of food crops, but also pose big threat to human health. There are various physical treatments for mycotoxins including cleaning, peeling, exposure, irradiation, ion pulse and so on, however, these physical methods are difficult to ensure complete removal of toxins. Also, the loss of nutrients in crops would be possibly caused during the physical treatment. Chemical methods can also eliminate mycotoxins to some extent, but the introduction of chemical reagents and the potential indirect contamination can limit the further application in practical field. With a continuous development of biotechnology, microbial enzymes have shown great advantages in the degradation of mycotoxins due to the mild reaction conditions, high degradation efficiency and low toxicity of degradation products. The discovery, identification and application of mycotoxin-degrading enzymes which have been reported so far were thoroughly reviewed in this manuscript. It is believed that with the deepening of research, the mycotoxin-degrading enzymes are expected to be further developed and employed in the feed and food industries.

Abstract:As a linear polysaccharide, alginate has different physiological and biochemical characteristics according to its structure and composition. It holds great application value and potential in food, medicine and cosmetics. These characteristics of alginate are mainly controlled by the action of alginate-modifying enzymes such as alginate lyase, mannuronan C5-epimerase, alginate acetylase and alginate deacetylase. This review mainly introduced the synthesis of alginate-modifying enzymes and the mechanism of alginate modification, and summarized the source, classification, structure, mode of action and research progress of several alginate modifying enzymes. The emphasis was laid on the research progress of alginate lyase and mannuronan C5-epimerase. And we also prospected the future development of related research, providing reference for the further development and application of alginate and its related modifying enzymes.

Abstract:Xylan is the most abundant heterogeneous polysaccharide in plant cell walls, which has great potential to synthesize high-value bio-based products. In the food industry, efficient enzymatic conversion of xylan can produce functional food polysaccharides such as xylo-oligosaccharides, xylitol and ferulic acid. Compared with the multi-enzyme synergistic catalysis, multifunctional enzymes combining multiple xylanolytic activities in one single peptide have significant advantages in reducing enzyme costs, improving catalytic efficiency and simplifying the enzymatic process, and therefore have broad application prospects. This paper firstly introduced the structural composition of xylan from different plant sources and the enzymatic systems for the degradation of main and side chains, then systematically reviewed the research progress of multifunctional xylanolytic enzyme with respect to structure, function and mechanism of action and their potential applications in the food industry, and finally prospected the research direction of multifunctional xylanolytic enzymes, laying a foundation for their precise design and industrial application.

Abstract:Isomaltulose is a new multifunctional sweetener and an internationally recognized safe sucrose substitute. Isomaltulose has wide application prospects in the medicine and food industries. Sucrose isomerase (SIase) is the most effective biological enzyme reagent for the production of isomaltulose by biological transformation. This review briefly presents the physiological characteristics of isomaltulose as well as the existing problems in its production. The source, three-dimensional structure, catalysis mechanism, rational design of high efficient enzymes and heterologous efficient expression of SIase were discussed in detail. In addition, the wide application of cell and enzyme immobilization in the production of isomaltulose was then introduced. Finally, the application of SIase in isomaltulose production was prospected.

Abstract:Isomaltooligosaccharides (IMO) are oligosaccharides mainly composed of α-1,6 glycosidic bonds linked glucoses, which have physiological characteristics of hypoglycemic index and promoting probiotics proliferation. The current commercial IMO production has low yield and high isomaltose content in the product, while the probiotic function of IMO is mainly attributed to the high polymerization fractions like disaccharides and above. In this study, α-glucosidase from Aspergillus niger and cyclodextrin glucosyltransferase (CGTase) from Bacillus stearothermophilus NO2 were combined for synthesis of IMO with improved yield and polymerization (named IMO^H). In this study, the effects of reaction pH, temperature, time, substrate type and enzyme addition on the yield were investigated by single-factor experiments, and the effects of compound enzyme amount and reaction time were subsequently explored. The results showed that the yield of IMOH reached 64.27%, and the high polymerization content of trisaccharides and above was 71.57% under pH 5.5 at 50 ℃, using 300 g/L maltodextrin with DE values ranges from 15 to 20 as the substrate, and with α-glucosidase of 5 U/g substrate (calculated by substrate mass) and CGTase of 20 U/g substrate (calculated by substrate mass). The structural analysis of IMO^H products showed that the ratio of α-1,6 bond ratio was 70.67%, and the average molecular weight of products was 706, which was higher than that of commercial IMO50. The in vitro fermentation experiments of intestinal microorganisms suggested that compared with commercial IMO50, the IMOH had a better proliferative effect on Bifidobacterium adolescentis, and could further inhibit the growth of opportunistic bacteria Escherichia coli in intestinal tract, showing a certain in vitro probiotic effect. This study proposed a new process of the IMO synthesis by the dual-enzyme compounds, providing a new idea for the industrial synthesis of IMO with high yield and high degree of polymerization.

Abstract:Laccase is a copper-containing polyphenol oxidase, which can catalyze the oxidation of various phenolic compounds and aromatic amines. It has wide application prospects in food, paper, energy, macromolecular compound synthesis and other fields. In recent years, more and more attention has been focused on the development and utilization of laccase. In this study, during the fermentation via Trametes versicolor in flasks, the morphology of mycelium was induced and regulated by adding farnesol, and the yield of laccase was increased. Besides, the activity of laccase fermented from Trametes versicolor and the mass concentration of protein reached (161.37±1.58) U/L and (29.19±1.30) mg/L, respectively, which was more than ten times higher than those of the control group. In addition, the optimum reaction temperature was 40 ℃, the optimum pH was 4.0, and the laccase had good thermal stability in the range of 25~55 ℃. Furthermore, based on the study of enzymatic reaction kinetics, it was found that laccase had a certain decolorization effect on evans blue, Congo red, and eriochrome black T dyes, and the decolorization effect of evans blue was the strongest, exhibiting good application potential.

Abstract:A β-glucosidase gene SmBgl3A, which might play a key role in cellobiose degradation and utilization, was screened from Sporocytophaga sp. CX11 through the analysis and comparison of expression level. The gene contains 2 283 bp and encodes 760 amino acids. The encoded protein of SmBgl3A has 46.76% homology with the β-glucosidase BoGH3B from Bacteroides ovatus. The gene SmBgl3A was heterologously expressed in Escherichia coli. The electrophoretically pure recombinant protein SmBgl3A was achieved by the purification of crude enzymes using Ni-NTA affinity chromatography, and the relative molecular weight of recombinant protein was consistent with the theoretical value (81.2×10⁴). Characterization of enzymatic properties showed that the optimum temperature and pH of SmBgl3A were 35 ℃ and 6.5, respectively. When the pH value was 5.0~7.0 and the temperature was below 40 ℃, SmBgl3A was stable. In addition, SmBgl3A showed good NaCl tolerance. The optimal substrate for SmBgl3A was pNPG, and the specific enzyme activity with pNPG as substrate was 14.74 U/mg. K_m and k_cat of SmBgl3A towards pNPG were 10.88 mmol/L and 46.72 s^-1, respectively. SmBgl3A is a novel enzyme with excellent enzymatic properties, further expanding the kinds of β-glucosidase and laying a foundation for the enzyme application in cellulose degradation, food industry and other fields.

Abstract:Naringinase can hydrolyze bitter substances, such as naringin in citrus fruits, which is applied in juice debittering or preparation of pharmaceutical intermediates, for example, prunin. In this study, calcium alginate/carboxymethyl chitosan/silica gel composite microspheres, which were environmental friendly and accessible with abundant resources, were prepared by dropping method and used as carriers for naringinase immobilization. The optimized specific enzyme activity, enzyme loading rate and enzyme activity recovery of immobilized naringinase could reach 203.33 U/g, 36.80% and 62.15%, respectively, under the following conditions: silica gel/carboxymethyl chitosan mass ratio of 1.0∶1.5, crosslinking time of 2.0 h, immobilization temperature of 25 ℃, coupling time of 4 h, and enzyme dosage of 465.6 U/mL. Meanwhile, compared with the free naringinase, this immobilized naringinase exhibited better stability to pH and thermal changes, and showed the feasibility of operation and storage. Besides, the application of naringinase immobilized on calcium alginate/silica gel/carboxymethyl chitosan composite microspheres would further expand the development of naringinase in food, medicine and other fields.

Abstract:α-L-Rhamnosidase can specifically hydrolyze α-L-rhamnose on glycosides and has been widely used in food, medicine and other industrial fields. This research aimed to obtain high-temperature-tolerant α-L-rhamnosidase, and to improve the efficiency of industrial production and reduce the cost of application. In this study, the α-L-rhamnosidase gene screened from the thermophilic bacterium Sulfolobus islandicus was cloned and heterogeneously expressed in Escherichia coli. In addition, the enzymatic properties of recombinant SisRha was determined, and the enzymatic preparation process of isoquercetin was subsequently optimized. The results showed that the recombinant SisRha was most active at 90 ℃with excellent thermal stability, and the enzyme activity was hardly lost after incubation at 80 ℃ for 120 min. The optimum reaction pH of the enzyme was 5.5, and the enzyme was quite stable in the pH range of pH 4.5~7.0. The K_m， V_max and k_cat values of the recombinant enzyme SisRha against pNPR as the artificial substrate were (0.15±0.03) mmol/L, (6.26±0.51) U/mg and (10.48±0.86) s^-1, respectively. The specific enzymatic activities of SisRha on the substrates pNPR, rutin, epimedin C, naringin, hesperidin and icariin were 25.06, 11.89, 6.74, 3.14, 2.77 ,1.68 U/mg, respectively. The optimum process of isoquercetin conversed from rutin by the recombinant SisRha was as follows: 2 mmol/L rutin could be almost converted with a molar conversion rate of 98.56% using 0.4 U/mL enzyme dosage of the recombinant SisRha under 85 ℃ at pH 5.0 for 1 hour incubation. This study enriched the current resources of α-L-rhamnosidase, established a theoretical foundation for the study of α-L-rhamnosidase derived from thermophilic bacteria, and provided a method of converting rutin to isoquercetin at high temperature.