Chitinase A Research Articles

Development of a Chitin-Based Purification System Utilizing Chitin-Binding Domain and Tobacco Etch Virus Protease Cleavage for Efficient Recombinant Protein Recovery.

This study aims to develop an efficient chitin-based purification system, leveraging a novel design where the target proteins, superfolding green fluorescent protein (sfGFP) and Thermus antranikianii trehalose synthase (TaTS), fused with a chitin-binding domain (ChBD) from Bacillus circulans WL-12 chitinase A1 and a tobacco etch virus protease (TEVp) cleavage site. This configuration allows for the effective immobilization of the target proteins on chitin beads, facilitating the removal of endogenous proteins. A mutant TEVp, H-TEVS219V-ChBD, fused with the His-tag and ChBD, is employed to cleave the target proteins from the chitin beads specifically. Subsequently, fresh chitin beads are added for adsorption to remove H-TEVS219V-ChBD in the solution, thereby significantly improving the purity of the target protein. Our results confirm that this system can efficiently and specifically purify and recover sfGFP and TaTS, achieving electrophoretic-grade purity exceeding 90%. This system holds significant potential for industrial production and other applications.

Callus-specific CRISPR/Cas9 system to increase heritable gene mutations in maize.

A callus-specific CRISPR/Cas9 (CSC) system with Cas9 gene driven by the promoters of ZmCTA1 and ZmPLTP reduces somatic mutations and improves the production of heritable mutations in maize. The CRISPR/Cas9 system, due to its editing accuracy, provides an excellent tool for crop genetic breeding. Nevertheless, the traditional design utilizing CRISPR/Cas9 with ubiquitous expression leads to an abundance of somatic mutations, thereby complicating the detection of heritable mutations. We constructed a callus-specific CRISPR/Cas9 (CSC) system using callus-specific promoters of maize Chitinase A1 and Phospholipid transferase protein (pZmCTA1 and pZmPLTP) to drive Cas9 expression, and the target gene chosen for this study was the bZIP transcription factor Opaque2 (O2). The CRISPR/Cas9 system driven by the maize Ubiquitin promoter (pZmUbi) was employed as a comparative control. Editing efficiency analysis based on high-throughput tracking of mutations (Hi-TOM) showed that the CSC systems generated more target gene mutations than the ubiquitously expressed CRISPR/Cas9 (UC) system in calli. Transgenic plants were generated for the CSC and UC systems. We found that the CSC systems generated fewer target gene mutations than the UC system in the T0 seedlings but reduced the influence of somatic mutations. Nearly 100% of mutations in the T1 generation generated by the CSC systems were derived from the T0 plants. Only 6.3-16.7% of T1 mutations generated by the UC system were from the T0 generation. Our results demonstrated that the CSC system consistently produced more stable, heritable mutants in the subsequent generation, suggesting its potential application across various crops to facilitate the genetic breeding of desired mutations.

Recycling of hyoscyamine 6β-hydroxylase for the in vitro production of anisodamine and scopolamine.

The tropane alkaloids hyoscyamine, anisodamine, and scopolamine are extensively used medicines. In particular, scopolamine has the greatest value in the market. Hence, strategies to enhance its production have been explored as an alternative to traditional field-plant cultivation. In this work, we developed biocatalytic strategies for the transformation of hyoscyamine into its products utilizing a recombinant Hyoscyamine 6β-hydroxylase (H6H) fusion protein to the chitin-binding domain of the chitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis was carried out in batch, and recycling of H6H constructions was performed via affinity-immobilization, glutaraldehyde crosslinking, and adsorption-desorption of the enzyme to different chitin matrices. ChBD-H6H utilized as free enzyme achieved complete conversion of hyoscyamine in 3- and 22-h bioprocesses. Chitin particles demonstrated to be the most convenient support for ChBD-H6H immobilization and recycling. Affinity-immobilized ChBD-H6H operated in a three-cycle bioprocess (3h/cycle, 30°C) yielded in the first and third reaction cycle 49.8% and 22.2% of anisodamine and 0.7% and 0.3% of scopolamine, respectively. However, glutaraldehyde crosslinking decreased enzymatic activity in a broad range of concentrations. Instead, the adsorption-desorption approach equaled the maximal conversion of the free enzyme in the first cycle and retained higher enzymatic activity than the carrier-bound strategy along the consecutive cycles. The adsorption-desorption strategy permitted the reutilization of the enzyme in a simple and economical manner while exploiting the maximal conversion activity displayed by the free enzyme. This approach is valid since other enzymes present in the E. coli lysate do not interfere with the reaction. KEY POINTS: • A biocatalytic system for anisodamine and scopolamine production was developed. • Affinity-immobilized ChBD-H6H in ChP retained catalytic activity. • Enzyme-recycling by adsorption-desorption strategies improves product yields.

Plant Chitinase Mutants as the Catalysts for Chitooligosaccharide Synthesis Using the Sugar Oxazoline Derivatives.

Sugar oxazolines, (GlcNAc)n-oxa (n = 2, 3, 4, and 5), were synthesized from a mixture of chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, and 5), and utilized for synthesis of (GlcNAc)7 with higher elicitor activity using plant chitinase mutants as the catalysts. From isothermal titration calorimetry, the binding affinity of (GlcNAc)2-oxa toward an inactive mutant obtained from Arabidopsis thaliana GH18 chitinase was found to be higher than those of the other (GlcNAc)n-oxa (n = 3, 4, and 5). To synthesize (GlcNAc)7, the donor/acceptor substrates with different size combinations, (GlcNAc)2-oxa/(GlcNAc)5 (1), (GlcNAc)3-oxa/(GlcNAc)4 (2), (GlcNAc)4-oxa/(GlcNAc)3 (3), and (GlcNAc)5-oxa/(GlcNAc)2 (4), were incubated with hypertransglycosylating mutants of GH18 chitinases from A. thaliana and Cycas revoluta. The synthetic activities of these plant chitinase mutants were lower than that of a mutant of Bacillus circulans chitinase A1. Nevertheless, in the plant chitinase mutants, the synthetic efficiency of combination (1) was higher than those of the other combinations (2), (3), and (4), suggesting that the synthetic reaction is mostly dominated by the binding affinities of (GlcNAc)n-oxa. In contrast, the Bacillus enzyme mutant with a different subsite arrangement synthesized (GlcNAc)7 from combination (1) in the lowest efficiency. Donor/acceptor-size dependency of the enzymatic synthesis appeared to be strongly related to the subsite arrangement of the enzyme used as the catalyst. The A. thaliana chitinase mutant was found to be useful when combination (1) is employed for the substrates.

High-Efficiency Secretion and Directed Evolution of Chitinase BcChiA1 in Bacillus subtilis for the Conversion of Chitinaceous Wastes Into Chitooligosaccharides.

Limitations of enzyme production and activity pose a challenge for efficient degradation of chitinaceous wastes. To solve this problem, we engineered a system for high-yielding extracellular secretion of chitinase A1 from Bacillus circulans (BcChiA1) in B. subtilis. Furthermore, an innovative chitinase high-throughput screening method based on colloidal chitin stained with Remazol Brilliant Blue R (CC-RBB) was established and used to identify three mutants with improved chitinase activity: Y10A/R301A/E327A (Mu1), Y10A/D81A/E327A (Mu2), and F38A/K88A/R301A (Mu3). Their highest specific activity reached 1004.83 ± 0.87 U/mg, representing a 16.89-fold increase in activity compared to native BcChiA1. Additionally, we found that there is a synergistic effect between BcChiA1 and a lytic polysaccharide monooxygenase from Bacillus atrophaeus (BatLPMO10), which increased the chitin processing efficiency by 50% after combining the two enzymes. The yield of chitooligosaccharide (COS) production using the mutant Mu1 and BatLPMO10 reached 2885.25 ± 2.22 mg/L. Taken together, the results indicated that the CC-RBB high-throughput screening method is a useful tool for chitinase screening, and evolution of BcChiA1 in collaboration with BatLPMO10 has tremendous application potential in the biological treatment of chitinaceous wastes for COS production.

Solid-state NMR C-13 and N-15 chemical shifts of the chitin binding domain of chitinase A1 from Bacillus circulans WL-12

Solid-state NMR C-13 and N-15 chemical shifts of the chitin binding domain of chitinase A1 from Bacillus circulans WL-12

A novel chitin-binding mode of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12 revealed by solid-state NMR.

Chitin-binding domain of chitinase A1 (ChBDChiA1 ) is characteristic because it binds only to insoluble crystalline chitin. While binding sites of major carbohydrate-binding modules carry multiple aromatic rings aligned on a surface, lethal mutations for ChBDChiA1 were reported only at W687, a location completely different from the site mentioned above, in spite of their similar main-chain folds. Here, the structural mechanism underlying its crystalline chitin binding was uncovered by solid-state NMR. Based on 13 C- and 15 N-signal assignment of microcrystalline ChBDChiA1 , the chemical shift perturbation on chitin binding was carefully examined. The perturbation was greatest at W687 and nonaromatic residues surrounding it, revealing their direct involvement in chitin binding. These residues and Q679 should provide a novel chitin-binding platform parallel to the W687 ring.

Trehalose production via merged secretion, purification, and immobilization of trehalose synthase in Bacillus subtilis

Abstract Here, we established a one-step reaction method using trehalose synthase to produce trehalose from maltose. Bacillus subtilis was used as an expression host, and the various B. subtilis signal peptides and chitin-binding domains (ChBD) from Bacillus circulans WL-12 chitinase A1 were fused with the N- and C-termini, respectively, of trehalose synthase from the Picrophilus torridus DSM 9790 (PTTS) gene. Results showed that trehalose synthase was secreted by the YwbN signal peptide of B. subtilis and bound specifically to the chitin beads. The conversion yield of the immobilized enzyme was up to 69.07%, which was higher than that of the free enzyme (65.40%). Furthermore, the immobilized enzyme also retained 50% of its residual activity after the 21st repeated use. The chitin beads maintained 55% adsorption capacity after being reused 53 times. The results indicate the potential usefulness of the developed approach for trehalose production.

A chitin film containing basic fibroblast growth factor with a chitin-binding domain as wound dressings

Basic fibroblast growth factor (bFGF) can stimulate wound healing. However, consistent delivery of bFGF has many disadvantages. To decrease their instability and diffusible, we introduced chitin-binding domain (ChtBD) into bFGF. Two expression plasimids were constructed. The first one (named bFGF) contained bFGF (154 amino acids), the second (named ChtBD-bFGF) contained bFGF and the ChtBD (54 amino acids). ChtBD was derived from chitinase A1 (ChiA1) of Bacillus circulans WL-12. The recombinant protein ChtBD-bFGF had the same biological activity as bFGF in promoting fibroblast proliferation. Chitin powder was dissolved in 11wt% NaOH and 4wt% urea aqueous solution via the freezing/thawing method. A chitin solution was spread on a glass plate and coagulated with anhydrous alcohol. The chitin binding ability of ChtBD-bFGF was 11.4-fold higher (up to 286μg/cm2) than bFGF in vitro. The immunofluorescence data indicated that the ChtBD-bFGF@chitin film promoted cell adhesion and proliferation. The ChtBD-bFGF@chitin film and bFGF@chitin films were implanted subcutaneously. Histological analysis showed that ChtBD-bFGF promoted vascularization at the implanted site more effectively than bFGF. These results suggest that the ChtBD-bFGF@chitin film is a stabile delivery vehicle for accelerating wound healing.

The role of exochitinase type A1 in the fungistatic activity of the rhizosphere bacterium Paenibacillus sp. M4

The aim of the study was to detect the activity and characterize potentially fungistatic chitinases synthesized by rhizosphere bacteria identified as Paenibacillus sp. M4. Maximum chitinolytic activity was achieved on the fifth day of culturing bacteria in a growth medium with 1% colloidal chitin. Analysis of a zymogram uncovered the presence of four activity bands in the crude bacterial extract. The used three-stage protein purification procedure resulted in a single band of chitinase activity on the zymogram. The purified enzyme exhibited maximum activity at pH 6.5 and temperature 45oC, and thermal stability at 40oC for 4 h. In terms of substrate specificity, it is an exochitinase (chitobiose). The amino acid sequence obtained after mass spectrometry showed similarity to chitinase A1 synthesized by Bacillus circulans. The M4 isolate demonstrated the highest growth inhibiting activity against plant pathogens belonging to the genera Fusarium, Rhizoctonia and Alternaria. Fungistatic activity, although to a somewhat lesser degree, was also demonstrated by purified chitinase. The obtained results confirm the participation of the studied exochitinase in antagonism towards pathogenic molds. However, the lower fungistatic effectiveness of the chitinases points to the synergistic action of different metabolites in biocontrol by these bacteria.

Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

The β-glucosidase Tm-bglA gene from hyperthermophile Thermotoga maritima was cloned into expression vectors pET-28a, producing two proteins of 55 kDa and 52 kDa in cell, which could be referred to Tm-BglA with and without 23 amino acids. The results showed that fusion of 23 amino acids to Tm-BglA improved its soluble expression and product tolerance, resulting over 60% yield of recombinant Tm-BglA secreted into the growth medium in Escherichia coli JM109 (DE3). Tm-BglA with 23 amino acids had higher k cat and K M values for p-nitrophenyl-β-D-glycopyranoside and much stronger product inhibition than Tm-BglA without 23 amino acids. Subsequently, the Tm-BglA was immobilized on chitin efficiently by genetically fusing the chitin-binding domain of chitinase A1 from Thermoanaerobacterium thermosaccharolyticum DSM571. The immobilized Tm-BglA had higher optimal temperature (95°C) and was more thermostable at the range from 75 to 100°C than its free form. This immobilized protein exhibited high stability and the conversion yield exceeding 90%. The high-level soluble expression, combined simultaneous purification and immobilization of the enzyme on chitin offers a novel approach for the low cost production of β-glucosidase to produce lactose-free fresh dairy products.

Molecular Properties Prediction, Docking Studies, and Antimicrobial Screening of 1,3,4-Thiadiazole and s-Triazole Derivatives

A series of 1,3,4-thiadiazoles and s-triazoles were subjected to Molinspiration, ALOGPS 2.1, and Osiris programs to predict their molecular properties that are important for drug candidates. Subsequently, all of them were docked into the active sites of enzymes namely glucosamine-6-phosphate synthase (GlcN-6-P), VIM-2 metallo-β- lactamase (VIM-2), chitinase A1 (ChiA1), and sterol 14 alpha-demethylase (CYP51) that were considered in antimicrobial studies of thiadiazole and s-triazole derivatives. Since all compounds fulfilled the criteria for good membrane permeability, oral bioavailability, low toxicity and the potential inhibitory activities towards VIM-2, ChiA1, and CYP51, most of them were synthesized and their antimicrobial activity has been tested.

Involvement of Gln679, in addition to Trp687, in chitin-binding activity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III domains, and a C-terminal chitin-binding domain (ChBD). The ChBD of ChiA1 (ChBDChiA1) belongs to carbohydrate-binding module (CBM) family 12 and specifically binds to insoluble or crystalline chitin. It has been suggested that tryptophan-687 (Trp687) is involved in the chitin-binding activity of this ChBD. Site-directed mutagenesis was used to identify additional amino acid residues required for chitin-binding activity of this domain. Furthermore, a total of 14 amino acid residues in ChBDChiA1 were carefully selected, and it was found that mutation of Gln679, which is not well-conserved in CBM family 12, significantly decreased the binding activity to colloidal chitin. A nuclear magnetic resonance study demonstrated that neither the Q679A nor the W687A mutation altered the overall structure of ChBDChiA1. Therefore, Gln679 was identified as a new residue that is involved in the chitin-binding activity of ChBDChiA1 in addition to Trp687. However, the mechanism of chitin binding by ChBD is still unknown.

Characterization of a Novel Thermostable Chitin-Binding Domain and Its Application in Immobilization of a Multifunctional Hemicellulase

A novel thermostable chitin-binding domain (Tt-ChBD) of chitinase A1 from Thermoanaerobacterium thermosaccharolyticum DSM571 was cloned, characterized, and compared for its binding activity with another mesophilic chitin-binding domain (Bc-ChBD). Recombinant protein with Tt-ChBD exhibits stronger affinity to chitin than those with Bc-ChBD at temperatures from 65 °C to at least 75 °C, but not to other polysaccharides including xylan, chitosan, cellulose, and agarose. For repeated production of xylose from arabinoxylan-containing feedstocks, a best-characterized trifunctional chimeric enzyme Xar-L1-Xyn (XX) constructed in our previous work was attempted to be immobilized on chitin efficiently by genetically fusing Tt-ChBD to the N-terminal region of XX (named CXX) and the C-terminal region of XX (named XXC), respectively. The fusing position of Tt-CBD affected the affinity-binding activity to chitin. Recombinant XX, XXC, and CXX were purified to homogeneity and characterized. According to the xylosidase activities, the optimum temperature and pH profiles of the CXX and XXC both in free and immobilized form were the same as those of XX. However, the thermal and pH stabilities of the immobilized XXC and CXX were both greatly improved in the range from 70 to 90 °C and pH 4.2-8.2. The immobilized multifunctional hemicellulase exhibited high stability to producing xylose for at least 19 or 30 times in continuous operation with the achievement of 60% or 80% conversion yield at temperatures up to 65 °C. These results indicate the usefulness of Tt-ChBD as an affinity tag for the simultaneous purification and immobilization of the enzyme on chitin and the great potential applications for thermophilic enzyme immobilization at higher temperatures.

Immobilization of nisin producer Lactococcus lactis strains to chitin with surface-displayed chitin-binding domain

In this study, nisin producer Lactococcus lactis strains displaying cell surface chitin-binding domain (ChBD) and capable of immobilizing to chitin flakes were constructed. To obtain ChBD-based cell immobilization, Usp45 signal sequence with ChBD of chitinase A1 enzyme from Bacillus circulans was fused with different lengths of PrtP (153, 344, and 800 aa) or AcmA (242 aa) anchors derived from L. lactis. According to the whole cell ELISA analysis, ChBD was successfully expressed on the surface of L. lactis cells. Scanning electron microscope observations supported the conclusion of the binding analysis that L. lactis cells expressing the ChBD with long PrtP anchor (800 aa) did bind to chitin surfaces more efficiently than cells with the other ChBD anchors. The attained binding affinity of nisin producers for chitin flakes retained them in the fermentation during medium changes and enabled storage for sequential productions. Initial nisin production was stably maintained with many cycles. These results demonstrate that an efficient immobilization of L. lactis cells to chitin is possible for industrial scale repeated cycle or continuous nisin fermentation.

Engineering chitinases for the synthesis of chitin oligosaccharides: Catalytic amino acid mutations convert the GH-18 family glycoside hydrolases into transglycosylases

Abstract Two family GH-18 chitinases mutated on the key catalytic amino acids were evaluated as “glycosynthases” for the coupling of oxazoline activated donor to chitin oligosaccharide (COs) acceptors obtained from microbial fermentation. Bacillus circulans WL-12 chitinase A1 ( Bc ChiA1) and Trichoderma harzanium chitinase 42 ( Th Chit42) were individually mutated on the three conserved carboxylic acids, all suggested to have a role in catalysis: the general acid/base glutamate and the two aspartates, defined as the putative stabilizer and its assistant. The mutants D200A and D202A of Bc ChiA1, together with D170N and to a lesser extent D170A of Th Chit42 proved to be active for chitinbiose oxazoline polymerization, and also for coupling reaction between Gal(β1 → 4) chitinbiose oxazoline and chitinpentaose at neutral pH. These mutants have additionally retained the ability to catalyze transglycosylation reaction on natural COs, whereas their hydrolytic activity is abolished. Such mutants can be considered as chitin transglycosylases, verifying also the fact that the two conserved aspartic acids, the stabilizer and its assistant, are not a prerequisite for the formation of oxazolinium intermediate by acetamido anchimeric assistance, i.e. for the substrate-assisted catalysis mechanism of family GH-18 chitinases.

Expression of modified oxidase of D-aminoacids of Trigonopsis variabilis in methylotrophic yeasts Pichia pastoris

Effective recombinant strains Pichia pastoris that produce functionally active hybrid of Trigonopsis variabilis D-aminoacids bond with chitin-connecting domain of chitinase A1 of Bacillus circulans (DAOcbd) were obtained. The dependence of DAOcbd production levels from production of the number of copies of "expression cassette" integrated in the AOX1 locus of recombinant strains was studied. It was indicated that synthesized DAOcbd may be easily purified and immobilized on chitin sorbents and possessed high specific activity. Produced strains and methods of their cultivation and DAOcbd extraction may be used for development of technologies of obtaining of biocatalyzers in technological processes of obtaining of 7-aminocephalosporane acid.

Role of Chitin Binding Domain of Chitinase A ofStreptomyces cyaneusSP-27 in Protoplast Formation fromSchizophyllum commune

Chitinase A (CHIA) of Streptomyces cyaneus SP-27 forms protoplasts from Schizophyllum commune mycelia when it is combined with alpha-1,3-glucanase of Bacillus circulans KA-304. An N-terminal chitin-binding domain truncated mutant (CatCHIA), which was expressed in Escherichia coli Rosetta-gami B (DE 3), lost most of its colloidal chitin- and powder chitin-binding activity. The colloidal chitin-hydrolyzing, the powder chitin-hydrolyzing, and the protoplast-forming activities of CatCHIA were lower than those of CHIA, suggesting that the chitin-binding domain contributes to the hydrolysis of chitin in the cell-wall of S. commune.

Facile Immobilization of EvolvedAgrobacterium radiobacterCarbamoylase with High Thermal and Oxidative Stability

Optically pure amino acids have been widely used as intermediates in the synthesis of antibiotics, antifungal agents, pesticides, and sweeteners. Of particular importance, d- p-hydroxyphenylglycine (D-HPG) can be produced from d, l-hydroxyphenly hydantoin (D,L-HPH) in a two-step reaction mediated by d-hydantoinase and N-carbamoyl- d-amino acid amidohydrolase (or carbamoylase). To make this production more industrially appealing, the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 cloned in an Escherichia coli strain was intensively mutated to improve the thermal stability of carbamoylase by three rounds of DNA shuffling. After an extensive screening of the mutant library, the mutant E. coli strain M303 was obtained to produce variant carbamoylase, CBL303, with three critical mutated residues, including V40A, G75S, and V237A. Further characterization showed that in comparison with the wild-type counterpart the evolved carbamoylase exhibited more than 20-fold tolerance to heat and, in addition, hydrogen peroxide as a result of the synergistic effect caused by the three mutations. Moreover, with the fusion of the chitin-binding domain (ChBD) of Chitinase A1, the evolved carbamoylase CBL303 was specifically adsorbed on chitin beads. Subsequent analysis indicated that the linkage between the enzyme and the affinity matrix was substantially stable. The half-life of the immobilized carbamolyase CBL303 could reach 210 h at 45 degrees C, whereas its free form had that of 17 h. In particular, when applied to D-HPG production, the immobilized enzyme could be recycled 16 times with the achievement of 100% conversion yield. Along with the previous illustration of d-hydantoinase immobilization, the success achieved by immobilization of the evolved carbamoylase in this work apparently offers a promising way for the efficient production of D-HPG from D,L-HPH.

Purification and immobilization of recombinant variants of Brevundimonas diminuta glutaryl-7-aminocephalosporanic acid acylase expressed in Escherichia coli cells

Modified chitin-binding domain (ChBD) from Bacillus circulans chitinase A1 with W42F mutation in chitin-binding site was genetically fused to different positions within α-subunit of glutaryl-7-aminocephalosporanic acid acylase (GLA) gene. Hybrid proteins were efficiently expressed in E. coli cells as soluble, enzymatically active and correctly processed holoenzymes. ChBD-GLA fusions were easily affinity purified on chitin column by changing the salt concentration of binding and elution buffer. The developed one-step affinity purification procedure is thus a promising approach for scaled-up isolation of GLA variants for preparation of industrial biocatalysts as well as for structure-functional studies.