Starch is composed of glucose units linked by α-1, 4-glucoside bond and α-1, 6-glucoside bond. It is the main component of foods and the primary raw material for starch processing industry. Pullulanase can effectively hydrolyze the α-1, 6-glucoside bond in starch molecules. Combined with other starch processing enzymes, it can effectively improve the starch utilization rate. Therefore, it has been widely used in the starch processing industry. This paper summarized the screening of pullulanase-producing strain and its encoding genes. In addition, the effects of expression elements and fermentation conditions on the production of pullulanase were summarized. Moreover, the progress in crystal structure elucidation and molecular modification of pullulanase was discussed. Lastly, future perspectives on pullulanase research were proposed.
Glycoside Hydrolases/genetics* ; Starch/metabolism*

Enzyme separation, purification, immobilization, and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis. Thus, the development of novel methods for enzyme purification, immobilization, and improvement of their catalytic performance and storage are of great significance. Herein, ferritin was fused with the lichenase gene to achieve the purpose. The results showed that the fused gene was highly expressed in the cells of host strains, and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo. Through low-speed centrifugation, the purity of the enzymes was up to > 90%, and the activity recovery was 61.1%. The activity of the enzymes after storage for 608 h was higher than the initial activity. After being used for 10 cycles, it still maintained 50.0% of the original activity. The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm. The specific activity of them was 12.09 times that of the free lichenase, while the catalytic efficiency was 7.11 times and the half-life at 50 ℃ was improved 11.09 folds. The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo, which can simplify the preparation of the target enzymes, improve their catalysis performance, and facilitate their storage.
Biocatalysis ; Enzymes, Immobilized/metabolism* ; Ferritins/metabolism* ; Glycoside Hydrolases/metabolism*

Water solubility, stability, and bioavailability, can be substantially improved after glycosylation. Glycosylation of bioactive compounds catalyzed by glycoside hydrolases (GHs) and glycosyltransferases (GTs) has become a research hotspot. Thanks to their rich sources and use of cheap glycosyl donors, GHs are advantageous in terms of scaled catalysis compared to GTs. Among GHs, sucrose phosphorylase has attracted extensive attentions in chemical engineering due to its prominent glycosylation activity as well as its acceptor promiscuity. This paper reviews the structure, catalytic characteristics, and directional redesign of sucrose phosphorylase. Meanwhile, glycosylation of diverse chemicals with sucrose phosphorylase and its coupling applications with other biocatalysts are summarized. Future research directions were also discussed based on the current research progress combined with our working experience.
Glucosyltransferases/metabolism* ; Glycoside Hydrolases/metabolism* ; Glycosylation ; Glycosyltransferases/genetics*

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range. Furthermore, the enzymatic properties of α-L-rhamnosidases of different origins differ in terms of the optimal temperature, the thermal stability, and the substrate specificity, which determine the different applications of these enzymes. In this connection, it is crucial to elucidate the similarities and differences in the catalytic mechanism and substrate specificity of α-L-rhamnosidase of different origins through analyzing its enzymatic properties. Moreover, it is important to explore and understand the effects of aglycon and metal cations on enzyme activity and the competitive inhibition of L-rhamnose and glucose on enzymes. These knowledge can help discover α-L-rhamnosidase of industrial significance and promote its industrial application.
Glycoside Hydrolases/metabolism* ; Hydrogen-Ion Concentration ; Rhamnose ; Substrate Specificity ; Temperature

Consolidated bioprocessing technology can be used for Kluyveromyces marxianus YX01 to produce ethanol from Jerusalem artichoke, which is one of the potential processes to produce biofuel from non-cereal crops. In this study, we combined the aeration rate with the substrate concentration to conduct cross-over experiments for K. marxinaus YX01, and studied ethanol fermentation and the influence of inulin enzyme activity. The substrate concentration had a little repressive effect on ethanol productivity. When substrate concentration reached 250 g/L under anaerobic conditions, ethanol concentration was 84.8 g/L, and ethanol yield was reduced from 86.4% (50 g/L substrate concentration) to 84.7% of the theoretical value. Aeration rate could accelerate K. marxinaus YX01 ethanol fermentation, but reduced ethanol yield. When substrate concentration reached 250 g/L under aeration at 1.0 vvm, ethanol yield was reduced from 84.7% under anaerobic conditions to 73.3% of the theoretical value. With increased concentration of the carbon source and reduced aeration rate, the inulinase of K. marxinaus YX01 reduced and the concentration of glycerol increased, however, the acetic acid increased with the increased concentration of the carbon source and aeration rate. When substrate concentration reached 250 g/L under anaerobic conditions, inulinase activity was only 6.59 U/mL; when substrate concentration reached 50 g/L under aeration at 1.0 vvm, inulinase activity was 21.54 U/mL.
Ethanol ; metabolism ; Fermentation ; Glycoside Hydrolases ; metabolism ; Helianthus ; metabolism ; Inulin ; metabolism ; Kluyveromyces ; classification ; metabolism ; Substrate Specificity

Jerusalem artichoke tubers with inulin as major component are potential feedstock for fuel ethanol production, and Kluyveromyces cicerisporus Y179 expressing high level of inulinase is suitable for ethanol production with this feedstock by simultaneous saccharification and fermentation approach. In this article, the impact of inoculum, aeration and temperature on ethanol production by the yeast was studied. The experimental results illustrated that inoculum with different levels and seed collected at different cultivation times had negligible effect, while anaerobic conditions enhanced ethanol production, and more ethanol was produced by the yeast at 30 degrees C than at 37 degrees C or 42 degrees C. The medium using Jerusalem artichoke tuber meal as sole component with 22% (W/V) total sugars was inoculated with 36 h-precultured seed at 10% (V/V), and the batch fermentation was conducted in a 5 L fermentor at 30 degrees C with a stirring speed of 300 r/min under anaerobic conditions. After 144 h, 12.3% (V/V) ethanol was produced and the yield of ethanol from sugars was 86.9% of its theoretical one, with 93.6% sugars consumed. These results indicate that K. cicerisporus Y179 is a promising candidate for industrial ethanol production using Jerusalem artichoke tuber feedstock.
Ethanol ; metabolism ; Fermentation ; Glycoside Hydrolases ; metabolism ; Helianthus ; chemistry ; Industrial Microbiology ; methods ; Kluyveromyces ; metabolism

A novel chitinolytic and chitosanolytic bacterium, Sphingomonas sp. CJ-5, has been isolated and characterized. It secretes both chitinase and chitosanase into surrounding medium in response to chitin or chitosan induction. To characterize the enzymes, both chitinase and chitosanase were purified by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-Sepharose Fast Flow. SDS-PAGE analysis demonstrated molecular masses of chitinase and chitosanase were 230 kDa and 45 kDa respectively. The optimum hydrolysis conditions for chitinase were about pH 7.0 and 36 degrees C, and these for chitosanase were pH 6.5 and 56 degrees C, respectively. Both enzymes were quite stable up to 45 degrees C for one hour at pH 5~8. These results show that CJ-5 may have potential for industrial application particularly in recycling of chitin wastes.
Chitinases ; metabolism ; Enzyme Stability ; Fermentation ; Glycoside Hydrolases ; metabolism ; Hydrogen-Ion Concentration ; Sphingomonas ; enzymology

Research on novel pullulanase has major significance on the domestic industrialization of pullulanase and the breakdown of foreign monopoly. A thermophilic bacteria LM 18-11 producing thermostable pullulanase was isolated from Lunma hot springs of Yunnan province. It was identified as Anoxybacillus sp. by 16S rDNA phylogenetic analysis. Full-length pullulanase gene was cloned from Anoxybacillus sp. LM18-11. The optimum temperature of the pullulanase was between 55 and 60 degrees C with a half-life as long as 48 h at 60 degrees C; and its optimum pH was between 5.6 and 6.4. V(max) and K(m) of the pullulanase was measured as 750 U/mg and 1.47 mg/mL, which is the highest specific activity reported so far. The pullulanase crystals structure showed a typical alpha-amylase family structure. The N-terminal has a special substrate binding domain. Activity and substrate binding were decreased when the domain was deleted, the V(max) and K(m) were 324 U/mg and 1.95 mg/mL, respectively. The pullulanase was highly heterologous expressed in Bacillus subtilis by P43 promoter. The extracellular enzyme activity was 42 U/mL, which increased more than 40 times compared to the initial strain. This pullulanase has good application prospects.
Anoxybacillus ; classification ; enzymology ; China ; Glycoside Hydrolases ; metabolism ; Hydrogen-Ion Concentration ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Temperature

We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT.
Bacillus ; enzymology ; Glycoside Hydrolases ; metabolism ; Molecular Weight ; Protein Conformation ; Sequence Deletion ; Substrate Specificity ; Temperature

Northern corn leaf blight, caused by the fungus Exserohirum turcicum Pass. (Leonard and Suggs), is one of the major diseases in most corn-growing areas of the world. Research on gene tagging of E. turcicum has been limited due to the lack of an efficient transformation system. Since E. turcicum produces and accumulates melamin in cell walls during vegetative growth, it is difficult to efficiently isolate its protoplast. To isolate the protoplast of this pathogen with a high frequency, the effects of cell wall degradation enzymes, including beta-1,3-glucanase (Fungase, Funcelase, Novozyme and Glucanex) and beta-glucuronidase (Driselase, Uskizyme and Kitalase), enzyme concentrations, combinations, strains and medium on the isolation frequency were tested. The isolation frequencies were high enough for transformation when the combinations of (Kitalase + Glucanex + Driselase), (Kitalase + Glucanex) or (Kitalase + Uskizyme) were used. Moreover, the isolation frequencies of protoplast were significantly affected by the cultural morphologies of strain and the growth stage of mycelia. Among the plasmids tested, only plasmid pAN71 is efficient for transformation of E. turcicum. This result will provide some useful information for gene tagging of E. turcicum and other species in Exserohirum.
Ascomycota ; cytology ; metabolism ; Cell Wall ; metabolism ; Fungal Proteins ; metabolism ; Glycoside Hydrolases ; metabolism ; Protoplasts ; cytology ; metabolism ; Transformation, Genetic ; genetics