Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.     

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.     

Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme

A chitinase gene (pCHI52) encoding the 52-kDa chitinase was isolated from a Serratia marcescens KCTC2172 cosmid library. This chitinase gene consists of 2526 bp with an open reading frame that encodes 485 amino acids. Escherichia coli harboring the pCHI52 gene secreted not only a 52-kDa but also a 35-kDa chitinase into the culture supernatant. We purified both 52-kDa and 35-kDa chitinases using a chitin affinity column and Sephacryl-S-300 gel filtration chromatography. We determined that the 17 N-terminal amino acid sequences of the 52-kDa and the 35-kDa chitinase are identical. Furthermore, a protease obtained from S. marcescens KCTC2172 cleaved the 52-kDa chitinase into the 35-kDa protein with chitinase activity. These results suggest that the 35-kDa chitinase derives from the 52-kDa chitinase by post-translational proteolytic modification. The optimal reaction temperature of 45°C and the optimal pH of 5.5 were identical for both enzymes. The specific activities of the 52-kDa and 35-kDa chitinases on natural swollen chitin were 67 μmol min⁻¹ mg⁻¹ and 60 μmol min⁻¹ mg⁻¹, respectively.     

Activation of old cuticle chitin as a substrate for chitinase in the molt of Manduca

During the molt, chitin in the old cuticle of $\text{Manduca}$ is digested by chitinase taken up from molting fluid, but the chitin in intact (= premolt) cuticle is not accessible to chitinase. As a prerequisite of digestion, old cuticle chitin is rendered competent to serve as chitinase substrate in a reaction attributable to trypsin-like proteolytic activity of molting fluid.     

Structure of human chitotriosidase. Implications for specific inhibitor design and function of mammalian chitinase-like lectins

Chitin hydrolases have been identified in a variety of organisms ranging from bacteria to eukaryotes. They have been proposed to be possible targets for the design of novel chemotherapeutics against human pathogens such as fungi and protozoan parasites as mammals were not thought to possess chitin-processing enzymes. Recently, a human chitotriosidase was described as a marker for Gaucher disease with plasma levels of the enzyme elevated up to 2 orders of magnitude. The chitotriosidase was shown to be active against colloidal chitin and is inhibited by the family 18 chitinase inhibitor allosamidin. Here, the crystal structure of the human chitotriosidase and complexes with a chitooligosaccharide and allosamidin are described. The structures reveal an elongated active site cleft, compatible with the binding of long chitin polymers, and explain the inactivation of the enzyme through an inherited genetic deficiency. Comparison with YM1 and HCgp-39 shows how the chitinase has evolved into these mammalian lectins by the mutation of key residues in the active site, tuning the substrate binding specificity. The soaking experiments with allosamidin and chitooligosaccharides give insight into ligand binding properties and allow the evaluation of differential binding and design of species-selective chitinase inhibitors.     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.     

Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation.

Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharide-degrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (A1, A2, B1, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pI 4.7 (A1); Mr 69,000, pI 4.5 (A2); Mr 38,000, pI 6.6 (B1); Mr 38,000, pI 5.9 (B2); Mr 39,000, pI 8.5 (C); and Mr 52,000, pI 5.2 (D). Among these chitinases, A1 and A2 had the highest colloidal-chitin-hydrolyzing activities. Chitinase A1 showed a strong affinity to insoluble substrate chitin. Purified chitinase A1 released predominantly chitobiose [(GlcNAc)2] and a trace amount of N-acetylglucosamine (GlcNAc) from colloidal chitin. N-terminal amino acid sequence analysis of chitinases A1 and A2 indicated that chitinase A2 was generated from chitinase A1, presumably by proteolytic removal of a C-terminal portion of chitinase A1. Since chitinase A2 did not have the ability to bind to chitin, the importance of the C-terminal region of chitinase A1 to the strong affinity of chitinase A1 to substrate chitin was suggested. Strong affinity of the chitinase seemed to be required for complete degradation of insoluble substrate chitin. From these results, it was concluded that chitinase A1 is the key enzyme in the chitinase system of this bacterium.     

The Role of Chitinase in the Antifungal Activity of Bacillussp. 739

Investigation of the crude extracellular chitinase of Bacillussp. 739, an antagonist of phytopathogenic fungi, discerned a relationship between the chitinase and antifungal activities of this bacterium. Purified chitinase lost its ability to inhibit the growth of micromycetes. The antagonistic (antifungal) activity of crude chitinase was found to be located in a low-molecular-weight fraction of the enzyme, which does not possess chitinase activity. Both crude and purified chitinase were able to lyse the cell walls of intact mycelium. Accordingly, it may be inferred that the antagonistic activity of Bacillussp. 739 against micromycetes is largely determined by low-molecular-weight nonenzymatic substances, whereas the role of chitinase is to utilize chitin, which is ubiquitously present in soil.     

Molecular detection of bacterial and streptomycete chitinases in the environment

Sets of PCR primers were designed to amplify bacterial chitinases at different levels of specificity. The bacterial chitinase group primers were successful in targeting enzymes classified within the group A glycosyl hydrolases of family 18. The widespread occurrence of group A bacterial chitinases in actinomycetes was demonstrated. Streptomycete chitinase specific primers were designed and a collection of type strains of species changed in the genes Streptomyces were screened and shown to have at least one and usually multiple chitinase genes. The presence of the gene for the chitin binding protein was also demonstrated within the streptomycete type strains. These data indicate that streptomycetes are well equipped to degrade chitin. The detection of group A chitinases in total community DNA is described and a sandy soil shown to contain more than 10 different genes using DGGE to indicate genetic diversity.     

The role of chitinase in antifungal activity of Bacillus sp. 739]

Investigation of the crude extracellular chitinase of Bacillus sp. 739, an antagonist of phytopathogenic fungi, discerned a relationship between the chitinase and antifungal activities of this bacterium. Purified chitinase lost its ability to inhibit the growth of micromycetes. The antagonistic (antifungal) activity of crude chitinase was found to be located in a low-molecular-weight fraction of the enzyme, which does not possess chitinase activity. Both crude and purified chitinase were able to lyse the cell walls of intact mycelium. Accordingly, it may be inferred that the antagonistic activity of Bacillus sp. 739 against micromycetes is largely determined by low-molecular-weight nonenzymatic substances whereas the role of chitinase is to utilize chitin, which is ubiquitously present in soil.     

Distribution of Chitinases in the Entomopathogen <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine in Protein Secretion

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.     

Biochemistry of insect differentiation: A system for studying the mechanism of chitinase activity in vitro

Results obtained with an in vitro system for the study of chitinase are described. The system involves soluble enzyme protein(s) and an insoluble substrate preparation. With insect molting fluid chitinase, it shows properties that parallel those observed during in vivo breakdown of cuticle during the molt. For example, molting fluid chitinase activity not previously exposed to chitin is stronly and specifically adsorbed to the substrate, in contrast to other enzymatic activities including hexosaminidase (chitobiase) present in molting fluid. This leads to partial purification of molting fluid chitinase activity reflected in increased specific activity of chitinase associated with the insoluble chitin substrate; we have previously reported increase of specific chitinase activity of (deproteinized) cuticle resulting from its incubation with molting fluid (M. L. Bade and A. Stinson, 1978, Biochem. Biophys. Res. Commun.84, 381–388). Soluble end product is generated rapidly and linearly with time by the in vitro system; the end product is assumed to be N-acetylglucosamine since the specific radioactivity of this compound is unchanged during the 10 min required for assay. Molting fluid chitinase activity may involve a number of polypeptides ranging in molecular weight from 145,000 to less than 20,000 daltons. The system described gives results consistent with a processive mechanism for molting fluid chitinase, i.e., data are given demonstrating that molting fluid chitinase continues to act on the same chitin particle(s) with which it initially associates rather than diffusing freely from substrate particle to substrate particle, and the product of its action appears to be a monosaccharide rather than a mixture of oligosaccharides. Processive behavior for chitinase would be predicted from the known structure, and the in vivo measured rate of breakdown, of cuticle chitin during the molt; the preliminary nature of this conclusion, based on what is so far known about the structure of the substrate used in the in vitro system, is briefly discussed.     

Chitinolytic activities in the gut of Aedes aegypti (Diptera:Culicidae) larvae and their role in digestion of chitin-rich structures

Mosquito larvae are believed to be capable of digesting chitin, an insoluble polysaccharide of N-acetylglucosamine, for their nutritional benefit. Studies based on physiological and biochemical assays were conducted in order to detect the presence of chitinase activities in the gut of the detritus-feeding Aedes aegypti larvae. Larvae placed for 24 h in suspensions of chitin azure were able to digest the ingested chitin. Semi-denaturing PAGE using glycol chitin and two fluorogenic substrate analogues showed the presence of two distinct chitinase activities: an endochitinase that catalyzed the hydrolysis of chitin and an endochitinase that cleaved the short substrates [4MU(GlcNAc)(3)] and [4MU(GlcNAc)(2)] that hydrolyzed the chitobioside [4MU(GlcNAc)(2)]. The endochitinase had an extremely broad pH-activity against glycol chitin and chitin azure, pH ranging from 4.0 to 10.0. When the substrate [4MU(GlcNAc)(3)] was used, two activities were observed at pH ranges 4.0-6.0 and 8.0-10.0. Chitinase activity against [4MU(GlcNAc)(3)] was detected throughout the gut with the highest specific activity in the hindgut. The pH of the gut contents was determined by observing color changes in gut after feeding the larvae with color indicator dyes. It was observed a correlation between the pH observed in the gut of feeding larvae (pH 10-6.0) and the optimum pH for gut chitinase activities. In this work, we report that gut chitinases may be involved in the digestion of chitin-containing structures and also in the partial degradation of the chitinous peritrophic matrix in the hindgut.     

Comparative Proteome Analysis Reveals that Cuticular Proteins Analogous to Peritrophin‐Motif Proteins are Involved in the Regeneration of Chitin Layer in the Silk Gland of Bombyx mori at the Molting Stage

The silk gland of silkworm produces silk proteins during larval development. Many studies have long focused on the silk gland of the fifth instar larvae, but few have investigated this gland at other larval stages. In the present study, the silk gland proteomes of the fourth instar and fourth molt are analyzed using liquid chromatography–tandem mass spectrometry. In total, 2654 proteins are identified from the silk gland. A high abundance of ribosomal proteins and RR‐motif chitin‐binding proteins is identified during day 2 of the fourth instar (IV‐2) larval developmental stage, and the expression of cuticular proteins analogous to peritrophin (CPAP)‐motif chitin‐binding proteins is higher during the fourth molt (IV‐M). In all, nine enzymes are found to be involved in the chitin regeneration pathway in the silk gland. Among them, two chitinase and two chitin deacetylases are identified as CPAP‐motif proteins. Furthermore, the expression of CPAP3‐G, the most abundant CPAP‐motif cuticular protein in the silk gland during the IV‐M stage, is investigated using western blot and immunofluorescence analyses; CPAP3‐G shows a reverse changing trend with chitin in the silk gland. The findings of this study suggest that CPAP‐motif chitin‐binding proteins are involved in the degradation of the chitin layer in the silk gland. The data have been deposited to the ProteomeXchange with identifier PXD008677.     

Studies on the Chitinolytie Enzymes of Black-koji Mold

In an attempt to separate the enzyme system participating in the decomposition of glycol chitin to constituent aminosugar, the purification of chitinase of Aspergillus niger was carried out by detemining both liquefying and saccharifying activities. Using fractionation with ammonium sulfate and column chromatography by hydroxylapatite, the chitinase system of the mold was separated into different enzyme fractions, which were required for the complete hydrolysis of glycol chitin. It was found that one of these enzymes caused a rapid decrease in viscosity of glycol chitin solution, another enzyme possessed N-acetyl-β-glucosaminidase activity upon N, N′-diacetylchitobiose and β-methyl-N-acetylglucosaminide, and that glycol chitin was decomposed to constituent aminosugar by a successive action of the two different enzymes.     

细菌几丁质酶结构、功能及分子设计的研究进展

几丁质是仅次于纤维素的第二大天然多糖，由N-乙酰-D-氨基葡萄糖聚合而成，具有重要的应用价值。自然界中几丁质可被细菌高效降解。细菌可分泌多种几丁质降解酶类，主要分布在GH18家族和GH19家族中。细菌中几丁质降解酶基因存在明显的基因扩增及多结构域组合现象，不同家族、不同作用模式的几丁质酶系协同作用打破复杂的抗降解屏障，完成结晶几丁质的高效降解。因此，深入分析细菌几丁质酶结构与功能，对几丁质高效降解与高值转化应用具有重要意义。本文介绍了细菌几丁质酶的分类、结构特点与催化作用机制；总结了不同细菌胞外几丁质降解酶系的协同降解模式；针对几丁质酶家族分子改造的研究进展，展望了以结构生物信息学及大数据深度学习为基础的蛋白质工程设计策略在今后改造中的作用，为几丁质酶的设计与理性改造提供新的视角与思路。     

Chitinolytic enzymes and the bacterial microflora in the digestive tract of cod, Gadus morhua

The levels of chitinolytic enzymes and chitinolytic bacteria in the digestive tract of feeding and fasting cod were compared. Enzyme activities within a given tissue were of a similar order irrespective of the presence of chitinolytic bacteria and/or chitin. Cod chitinase and chitobiase are therefore endogenous and constitutive enzymes. Fasting cod had similar numbers of bacteria within the gastro-intestinal compartments as feeding fish. Representative bacterial isolates from fasting fish were characterized.