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*

Glycosidases are widely used in food and pharmaceutical industries due to its ability to hydrolyze the glycosidic bonds of various sugar-containing compounds including glycosides, oligosaccharides and polysaccharides to generate derivatives with important physiological and pharmacological activity. While glycosidases often need to be used under high temperature to improve reaction efficiency and reduce contamination, most glycosidases are mesophilic enzymes with low activity under industrial production conditions. It is therefore critical to improve the thermo-stability of glycosidases. This review summarizes the recent advances achieved in engineering the thermo-stability of glycosidases using strategies such as directed evolution, rational design and semi-rational design. We also compared the pros and cons of various techniques and discussed the future prospects in this area.
Glycoside Hydrolases/genetics* ; Oligosaccharides ; Polysaccharides ; Protein Engineering

Glycoside compounds are widely used in medicine, food, surfactant, and cosmetics. The glycosidase-catalyzed synthesis of glycoside can be operated at mild reaction conditions with low material cost. The glycosidase-catalyzed processes include reverse hydrolysis and transglycosylation, appropriately reducing the water activity in both processes may effectively improve the catalytic efficiency of glucosidase. However, glucosidase is prone to be deactivated at low water activity. Thus, glucosidase was immobilized to maintain its activity in the low water activity environment, and even in neat organic solvent system. This article summarizes the advances in glycosidase immobilization in the past 30 years, including single or comprehensive immobilization techniques, and immobilization techniques combined with genetic engineering, with the aim to provide a reference for the synthesis of glycosides using immobilized glycosidases.
Catalysis ; Enzymes, Immobilized ; Glycoside Hydrolases/genetics* ; Glycosides/biosynthesis* ; Hydrolysis

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*

To improve the inulinase application in biotechnology, the characteristic of inulinase from Kluyveromyces marxianus YX01 was investigated. The inu gene of K. marxianus YX01 was transformed into Pichiapastoris GS115 host cells with molecular biology techniques. Then we achieved the heterologous expression of exo-inulinase whose molecular mass was about 86.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, six His-tag was added to the inulinase and a two-step method was applied in the purification of inulinase, including concentration via dialysis by polyethylene glycol 20 000 and metal Ni-NTA Agarose affinity adsorption. The purification factor of purified protein was 3.6 and the recovery rate of enzyme activity was 33.1%. We characterized the purified inulinase. The optimum temperature was 60 degrees C and pH was 4.62. When inulin and sucrose were used as substrates, the K(m) and V(max) values were 80.53 g/L vs 4.49 g/(L x min) and 183.10 g/L vs 20.20 g/(L x min), respectively. In addition, metal ions including Mn2+, Ca2+, Cu2+, Zn2+ and Fe2+ exhibited different degrees of inhibition on the enzyme activity, and Cu2+, Zn2+ and Fe2+ exhibited the most significant inhibition. Our findings might lay a good foundation for industrial application of inulinase.
Glycoside Hydrolases ; chemistry ; genetics ; Industrial Microbiology ; Inulin ; Kluyveromyces ; enzymology ; genetics ; Pichia ; Sucrose ; Temperature

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

Chitosanases represent a class of glycoside hydrolases with high catalytic activity on chitosan but nearly no activity on chitin. Chitosanases can convert high molecular weight chitosan into functional chitooligosaccharides with low molecular weight. In recent years, remarkable progress has been made in the research on chitosanases. This review summarizes and discusses its biochemical properties, crystal structures, catalytic mechanisms, and protein engineering, highlighting the preparation of pure chitooligosaccharides by enzymatic hydrolysis. This review may advance the understandings on the mechanism of chitosanases and promote its industrial applications.
Chitosan/chemistry* ; Chitin ; Glycoside Hydrolases/genetics* ; Protein Engineering ; Oligosaccharides/chemistry* ; Hydrolysis

Exploring excellent new pullulanase genes, and enriching pullulanase theory are of great importance to realize the industrialization of pullulanase. Three genes, pulA, pulB and pulC, encoding pullulanases, were cloned from Bacillus cereus GXBC-3 by bioinformatics analyzing the open reading frame in Bacillus cereus, annotated as putative I and II pullulanases in the GenBank database. Characteristics of these recombinant enzymes were inducible intracellular expressed in Escherichia coli, the results showed PulA was typical II pullulanase. Recombinant PulA could hydrolyze alpha-1,4- and alpha-1,6-glycosidic bonds. Its specific activity was 32.89 U/mg with an optimum temperature of 40 degrees C and optimum pH 6.5 using pullulan as substrate. And for soluble starch substrate, its specific activity was 25.71 U/mg with an optimum temperature of 50 degrees C and optimum pH 7.0. PulB and PulC were I pullulanases and only hydrolyzed alpha-1,6-glycosidic bond. The specific activities, optimum temperature and optimum pH of PulB and PulC for pullulan substrate were 228.54 U/mg, 45 degrees C, 7.0 and 229.65 U/mg, 45 degrees C, 6.5, respectively.
Bacillus cereus ; enzymology ; genetics ; Cloning, Molecular ; Escherichia coli ; Glucans ; metabolism ; Glycoside Hydrolases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

Molecular engineering of cellulases can improve enzymatic activity and efficiency. Recently, the Carbohydrate-Active enZYmes Database (CAZy), including glycoside hydrolase (GH) families, has been established with the development of Omics and structural measurement technologies. Molecular engineering based on GH families can obviously decrease the probing space of target sequences and structures, and increase the odds of experimental success. Besides, the study of cellulase active-site architecture paves the way toward the explanation of catalytic mechanism. This review focuses on the main GH families and the latest progresses in molecular engineering of catalytic domain. Based on the combination of analysis of a large amount of data in the same GH family and their conservative active-site architecture information, rational design will be an important direction for molecular engineering and promote the rapid development of the conversion of biomass.
Catalytic Domain ; genetics ; Cellulase ; chemistry ; genetics ; Directed Molecular Evolution ; methods ; Evolution, Molecular ; Glycoside Hydrolases ; chemistry ; genetics ; Protein Engineering ; methods

The xylanolytic enzymes found in Thermotoga maritima showed extremely high thermostability and considerable potential in industrial application. Yet expression level of the genes encoding these enzymes was very low. The alpha-glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The alpha-glucuronidase was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL with plasmid pET-28a(+), and made up about 20% of the total proteins present in the intracellular soluble fraction. The results proved the assumption that rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of aguA gene from hyperthermophilic bacterium T. maritima in E. coli. Purification procedure included two steps, heat treatment and immobilized metal affinity chromatography, and over 13.5mg of pure enzyme was obrained from 1L of induced culture. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 5.1 fold, and a yield of 55.1%. The optimum activity of recombinant alpha-glucuronidase was found at pH 6.0 and 85 degrees C, the enzyme retained 70% of its activity after 1 h of incubation at 85 degrees C. The induction conditions for expression of recombinant strain BL21-CodonPlus(DE3)-RIL/pET-28a-aguA were studied on induction time and duration by IPTG. The results showed that the activity of thermostable alpha-glucuronidase reach the maximum in 5-hour after inducted at the exponential phase (OD600 of 0.7 - 0.8).
Escherichia coli ; genetics ; Glycoside Hydrolases ; genetics ; isolation & purification ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Thermotoga maritima ; enzymology