Abstract:Strong-flavor Baijiu is a typical representative of traditional Chinese distilled alcoholic beverages, with a long history and unique flavor. Flavor analysis showed that fatty acid ethyl esters have a key influence on the sensory properties of strong-flavor Baijiu. Esterification reaction catalyzed by ester synthases from microorganisms is an important way to synthesize fatty acid ethyl esters using alcohols and acids as the substrates. However, the mechanism of enzymatic catalysis is not clear until now, and leads to the batch instability for synthesizing esters in strong-flavor Baijiu during fermentation process. The classical enzyme catalyzed esterification of acids and alcohols is under organic phase system, while the water content of fermented grains is 53% ~58% during Baijiu fermentation. It is generally recognized as a water phase system. Identifying enzymes for synthesizing fatty acid ethyl esters and revealing the catalytic mechanism can provide a theoretical basis for improving the quality of strong-flavor Baijiu. Baijiu flavor esters and microbial metabolism were summarized in this review. The structure and catalytic mechanism of microbial enzymes from Baijiu were also discussed to provide a scientific basis for clarifying the synthetic mechanism of ethyl esters in strong-flavor Baijiu.

Abstract:Carrageenan is a kind of linear sulphated polysaccharide extracted from the cell wall of marine red algae, which is formed by the alternating connection of 1,3-β-D-galactose and 1,4-α-D -galactose as the basic skeleton. Studies have shown that carrageenan and its molecular modified derivatives have many biological activities such as anti-tumor, anti-virus, anti-coagulation, enhancing cellular immunity and humoral immunity. Carrageenase is a glycoside hydrolase that degrades carrageenan by breaking theβ-1,4-glycosidic bond. Carrageenan sulfase, also known as carrageenan sulfase, is an enzyme that acts on the sulfate group of oligo-carrageenan to free inorganic sulfuric acid. These two enzymes have a synergistic effect on the degradation of carrageenan polysaccharides. However, due to the complex structure of carrageenan polysaccharides, the degradation and molecular modification of carrageenan have not been explored in the past. Nowadays, with the introduction of technology and the various biological activities of carrageenan polysaccharides, the molecular modification of carrageenan polysaccharides has attracted the continuous attention of relevant researchers. In this paper, the molecular modification of carrageenan polysaccharides in recent years is reviewed, with emphasis on the research progress of carrageenan and sulfatase. The changes of physiological activity after modification were also described.

Abstract:The development and application of functional starch are one of the hot research areas in food and medicine industries. Functional starch draws more and more attention from customs. It is an efficient method for the synthesis of functional starch by enzymatic catalysis. Due to its green environmental protection, safety and health characteristics, it has received extensive attention from scholars in many fields. In this paper, the structural regulation principle of artificial synthesis of amylose and highly branched starch using sucrose is summarized, and the research progress of amylosucrase and glycogen branching enzyme in the synthesis of functional starch is reviewed. The research trends and application prospects of starch in the field of functional food and medicine is illustrated, providing a reference for further development of functional starch.

Abstract:The β-galactosidase (AoBgal35A) from Aspergillus oryzae was modified by directed evolution to improve its hydrolysis efficiency of lactose. A mutation library of AoBgal35A was constructed by error-prone PCR. The mutants with high hydrolysis efficiency of lactose were screened by high-throughput screening and efficiently expressed in Pichia pastoris to prepare lactose-free milk. A positive mutant (mAoBgal35A) was selected and its enzyme activity reached 4 760 U/mL after high cell density fermentation. Purified mAoBgal35A showed the highest activity at pH 5.0 and 55 ℃. The optimum pH and temperature of mAoBgal35A were increased by 0.5 and decreased by 5 ℃ compared with the wild-type AoBgal35A, respectively. The mAoBgal35A could effectively hydrolyze the lactose in milk in 72 h at room temperature with the enzyme dosage of 2 U/mL, and the content of lactose in milk was 0.38 g/dL, which was within the standard range of lactose-free milk. However, for the wild-type enzyme, the content of lactose in milk was 0.6 g/dL with the enzyme dosage of 20 U/mL under the same conditions. The results showed that directed evolution significantly improved the hydrolysis efficiency of AoBgal35A, and the positive mutant mAoBgal35A showed great application potential in lactose-free milk.

Abstract:γ-Gminobutyric acid is an important biologically active factor, which is synthesized though the decarboxylation of L-glutamic acid by glutamate decarboxylase (GAD). The author firstly cloned and expressed the glutamate decarboxylase-encoding gene from Saccharomyces cerevisiae in E. coli. The specific activity of the recombinant ScGAD purified by affinity chromatography reached a maximum value of 66.55 U/mg. Further enzymatic property analysis results indicated that ScGAD exhibited an optimum reaction temperature of 60 ℃, an optimum reaction pH of 4.0, excellent stability in the range of 30~50 ℃ and pH 4.0~9.0, and the value of 14.28 mmol/L for kinetic constant Km indicating ScGAD of a good affinity to L-glutamic acid. Finally, through the investigation of the optimal conditions for GABA preparation by whole-cell catalysis using ScGAD, the highest generation efficiency of GABA was achieved at the conditions of 60 ℃ and pH 4.0. On this basis, 100 mmol/L of the substrate (sodium L-glutamate) could be converted to 35.9 g/(g·h) of GABA through whole-cell catalysis. This study provides a basis for efficient production of GABA.

Abstract:Cold-adapted maltopentaose-forming amylases have relatively high catalytic activities at low temperatures and enable an efficient and specific conversion of starch to functional maltopentaose at room temperature, resulting in a broad applications in food, medical and pharmaceutical fields. In this study, SdG5A and SdG5A-CD from Saccharophagus degradans were expressed in Bacillus subtilis. Further, the cold adaptation of the recombinant enzymes was characterized and compared. The results showed that recombinant SdG5A retained 27.8% of its maximal activity at 0 ℃ and could produce 48.6% of maltopentaose at room temperature, indicating a strong cold adaptation of SdG5A. In contrast, SdG5A-CD, lacking the linker and starch-binding domain (SBD), did not show any cold adaptation. To investigate the mechanisms that might underlie the cold adaptation of SdG5A, the structure was predicted by RoseTTAFold and the structural flexibility was analyzed through molecular dynamics simulation. An extremely high flexibility in the C-terminal linker-SBD region was observed and its root-mean-square fluctuation at 0 ℃ was consistent with that at 45 ℃, indicating that the highly flexible linker-SBD region acted as a critical component for the cold adaptation of SdG5A.

Abstract:As an extension of multi-enzyme immobilization strategy, enzyme-cell co-immobilization has become a new hot topic in enzyme engineering research because of its ability to enhance the catalytic performance of the system by hierarchically encapsulating different biomolecules of immobilized enzymes and cells. In order to use this new technology to enhance the synthesis of butyric acid, zinc zeolitic imidazolate framework-8@cellulase-Clostridium tyrobutyricum(ZIF-8@cellulase-Ct) co-immobilization system was prepared by constructing ZIF-8 separated with cellulase and Ct based on biomimetic mineralization method. The system was characterized by SEM, FT-IR and XRD techniques and analyzed by enzymatic and fermentation experiments, which showed that the system had better simultaneous saccharification and fermentation performance. 56.02% of corn cobs were hydrolyzed within at 40 ℃ and pH 4.5, and the glucose conversion rate was about 85.61%. Placed under different pH and oxygen concentrations, cells with ZIF-8@cellulase coating were found to have higher and more stable specific growth rate, cell viability and lower relative reactive oxygen levels, and the final concentration of ZIF-8-SSF butyric acid yield reached 6.74 g/L in 32 h, which was 12.52% higher than that of non-immobilized cells.

Abstract:A gene (sbnag2550) encoding N-acetyl-glucosaminidase (NAGase) was cloned from Streptomyces bacillaris and expressed in Escherichia coli BL21(DE3). The enzymatic properties of SbNag2550 were studied using the purified enzyme obtained by nickel ion affinity chromatography. The optimal temperature of SbNag2550 was 50 ℃, and it could show more than 90% activity at temperature ranging from 45 ℃ to 60 ℃. The enzyme also showed excellent thermostability, retaining nearly 90% activity after incubating at 55 ℃ for 60 h. SbNag2550 was used to catalyze the reverse hydrolysis reaction of N-acetyl-glucosamine (NAG) and glycerol, realizing the synthesis of glyceryl N-acetyl-glucosamine (GNAG). Under optimal conditions, the conversion rate reached 28.20% and 34.82% at 24 h and 72 h, respectively. GNAG was firstly synthesized by enzymatic catalysis in this study, which laid a foundation for its functional research and application development.

Abstract:The thermal stability of enzyme is an important target of industrial enzyme property modification. As an exogenous additive widely used to improve enzyme stability, the mechanism of sorbitol affecting enzyme thermal stability is still unclear. Therefore, the authors focused onα-L-rhamnosidase (r-Rha1) derived from Aspergillus niger and investigated the mechanism of sorbitol on the thermal stability of α-L-rhamnosidase by thermal inactivation kinetics, circular dichroism and spectroscopic analysis of fluorescence mapping. Adding sorbitol improved the thermal stability of α-L-rhamnosidase at 60~70 ℃， and 1.2 mol/L sorbitol increased the half-life of α-L-rhamnosidase 29-fold, 25-fold and 10-fold at 60, 65 ℃ and 70 ℃, respectively. The results of circular dichroism and fluorescence mapping plus indicated that sorbitol improved the thermal stability of α-L-rhamnosidase by enhancing its surface hydrophobic activity and structural rigidity. The effect of sorbitol on the thermal stability of α-L-rhamnosidase was investigated to further prove the general applicability of sorbitol in improving the thermal stability of the enzyme, while providing a reference for the subsequent industrial application of α-L-rhamnosidase.

Abstract:Sucrose isomerase (SIase) plays an important role in the enzymatic preparation of isomaltulose. In this study, a SIase gene from Klebsiella sp. LX3 was expressed in E. coli by using the molecular chaperone co-expression system, and then purified and characterized. The SIase gene was cloned into pET-24b to yield the construct pET-24b-SIase, which was introduced into E. coli BL21(DE3) for expression. Unfortunately, the enzyme was expressed mainly as inclusion bodies. In order to enhance the soluble form of the enzyme, SIase was coexpressed with a molecular chaperone. Four plasmids (pKJE7, pGro7, pG-Tf2, pTf16), each carrying a molecular chaperone gene, were separately introduced into E. coli BL21(DE3) carrying pET-24b-SIase and the expression of SIase was evaluated. The plasmid pGro7 was the most effective at improving the expression of recombinant SIase as a soluble enzyme, yielding a specific activity of 14.8 U/mL for the crude cell extract compared with a mere 3.5 U/mL for the crude cell extract derived from cells harbouring only pET-24b-SIase. The expressed SIase was purified by affinity chromatography using Ni-NTA. The purified enzyme exhibited the maximum activity at 40 ℃ and pH 6.0. Its K_m for sucrose was(179.10±20.65) mmol/L and the k_cat/K_m was (5.44±0.72) L/(mmol·s). The results of the product specificity study showed that the proportion of isomaltulose and trehalulose in the products decreased and the proportion of monosaccharides increased with increasing reaction temperature. The results of this study showed that the level of soluble SIase expressed in E. coli could be enhanced by coexpressing the enzyme with a molecular chaperone, suggesting a potential strategy for the large-scale production of SIase and its applications.