Processive Chitinase Research Articles

Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation have been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

Product inhibition slow down the moving velocity of processive chitinase and sliding-intermediate state blocks re-binding of product

Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 μM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 μM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 μM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

Chemical-State-Dependent Free Energy Profile from Single-Molecule Trajectories of Biomolecular Motors: Application to Processive Chitinase

The mechanism of biomolecular motors has been elucidated using single-molecule experiments for visualizing motor motion. However, it remains elusive how changes in the chemical state during the catalytic cycle of motors lead to unidirectional motions. In this study, we use single-molecule trajectories to estimate an underlying diffusion model with chemical-state-dependent free energy profile. To consider nonequilibrium trajectories driven by the chemical energy consumed by biomolecular motors, we develop a novel framework based on a hidden Markov model, wherein switching among multiple energy profiles occurs reflecting the chemical state changes in motors. The method is tested using simulation trajectories and applied to single-molecule trajectories of processive chitinase, a linear motor that is driven by the hydrolysis energy of a single chitin chain. The chemical-state-dependent free energy profile underlying the burnt-bridge Brownian ratchet mechanism of processive chitinase is determined. The novel framework allows us to connect the chemical state changes to the unidirectional motion of biomolecular motors.

A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high-level expression in Pichia pastoris.

Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60°C and over a broad pH range between 5.0 and 9.0, and was stable at 50°C and over the pH range 4.0-9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of β-N-acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

Combined Approach to Engineer a Highly Active Mutant of Processive Chitinase Hydrolyzing Crystalline Chitin.

Serratia marcescens chitinase A (SmChiA) processively hydrolyzes recalcitrant biomass crystalline chitin under mild conditions. Here, we combined multiple sequence alignment, site-saturation mutagenesis, and automated protein purification and activity measurement with liquid-handling robot to reduce the number of mutation trials and shorten the screening time for hydrolytic activity improvement of SmChiA. The amino acid residues, which are not conserved in the alignment and are close to the aromatic residues along the substrate-binding sites in the crystal structure, were selected for site-saturation mutagenesis. Using the previously identified highly active F232W/F396W mutant as a template, we identified the F232W/F396W/S538V mutant, which shows further improved hydrolytic activity just by trying eight different sites. Importantly, valine was not found in the multiple sequence alignment at Ser538 site of SmChiA. Our combined approach allows engineering of highly active enzyme mutants, which cannot be identified only by the introduction of predominant amino acid residues in the multiple sequence alignment.

Chemical-State-Dependent Free Energy Profile from Single-Molecule Trajectories of Biomolecular Motors: Application to Processive Chitinase.

The mechanism of biomolecular motors has been elucidated using single-molecule experiments for visualizing motor motion. However, it remains elusive that how changes in the chemical state during the catalytic cycle of motors lead to unidirectional motions. In this study, we use single-molecule trajectories to estimate an underlying diffusion model with chemical-state-dependent free energy profile. To consider nonequilibrium trajectories driven by the chemical energy consumed by biomolecular motors, we develop a novel framework based on a hidden Markov model, wherein switching among multiple energy profiles occurs reflecting the chemical state changes in motors. The method is tested using simulation trajectories and applied to single-molecule trajectories of processive chitinase, a linear motor that is driven by the hydrolysis energy of a single chitin chain. The chemical-state-dependent free energy profile underlying the burnt-bridge Brownian ratchet mechanism of processive chitinase is determined. The novel framework allows us to connect the chemical state changes to the unidirectional motion of biomolecular motors.

Expression and biochemical characterization of a novel chitinase ChiT-7 from the metagenome in the soil of a mangrove tidal flat in China

Chitinases play an important role in the process of chitin bioavailability. In this study, we cloned a new chitinase gene and characterized its recombinant protein. The new 1251 bp gene of chitinase (ChiT-7) was cloned from the metagenome of the mangrove tidal flat soil in the city of Zhangzhou in Fujian Province (China) by genome walking. The gene encoded a mature protein with 381 amino acids, which manifested certain sequence similarity (59% identity) to characterized GH18 chitinases. The mature protein of ChiT-7 was successfully expressed in E. coli BL21 (DE3). After purification, the specific activity of the recombinant enzyme was 0.63 U/mg at the optimal pH of 6.0 and the optimal temperature of 45 °C. The rChiT-7 was active over a wide pH range, and the residual enzyme activity reached 80% or higher at 30 °C–50 °C. rChiT-7 hydrolyzed colloidal chitin with (GlcNAc)2 and GlcNAc as the main final products. Structural analysis of ChiT-7 indicated that ChiT-7 could be a processive chitinase. rChiT-7 manifested characteristics analogous to those of fungi and actinomycetes and exhibited sequence homology.

Processive Chitinase is Burnt-Bridge Brownian Motor Operated by Fast Catalysis after Peeling Rail from Crystalline Chitin

Processive Chitinase is Burnt-Bridge Brownian Motor Operated by Fast Catalysis after Peeling Rail from Crystalline Chitin

Treatment of recalcitrant crystalline polysaccharides with lytic polysaccharide monooxygenase relieves the need for glycoside hydrolase processivity

Processive glycoside hydrolases associate with recalcitrant polysaccharides such as cellulose and chitin and repeatedly cleave glycosidic linkages without fully dissociating from the crystalline surface. The processive mechanism is efficient in the degradation of insoluble substrates, but comes at the cost of reduced enzyme speed. We show that less processive chitinase variants with reduced ability to degrade crystalline chitin, regain much of this ability when combined with a lytic polysaccharide monooxygenase (LPMO). When combined with an LPMO, several less processive chitinase mutants showed equal or even increased activity on chitin compared to the wild-type. Thus, LPMOs affect the need for processivity in polysaccharide degrading enzyme cocktails, which implies that the composition of such cocktails may need reconsideration.

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin

Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Here, we analyse the mechanism underlying unidirectional movement of Serratia marcescens chitinase A (SmChiA) using high-precision single-molecule imaging, X-ray crystallography, and all-atom molecular dynamics simulation. SmChiA shows fast unidirectional movement of ~50 nm s−1 with 1 nm forward and backward steps, consistent with the length of reaction product chitobiose. Analysis of the kinetic isotope effect reveals fast substrate-assisted catalysis with time constant of ~3 ms. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the product-binding site of SmChiA. Our results demonstrate that SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.

Human Chitotriosidase Is an Endo-Processive Enzyme.

Human chitotriosidase (HCHT) is involved in immune response to chitin-containing pathogens in humans. The enzyme is able to degrade chitooligosaccharides as well as crystalline chitin. The catalytic domain of HCHT is connected to the carbohydrate binding module (CBM) through a flexible hinge region. In humans, two active isoforms of HCHT are found–the full length enzyme and its truncated version lacking CBM and the hinge region. The active site architecture of HCHT is reminiscent to that of the reducing-end exo-acting processive chitinase ChiA from bacterium Serratia marcescens (SmChiA). However, the presence of flexible hinge region and occurrence of two active isoforms are reminiscent to that of non-processive endo-chitinase from S. marcescens, SmChiC. Although the studies on soluble chitin derivatives suggest the endo-character of HCHT, the mode of action of the enzyme on crystalline chitin is not known. Here, we made a thorough characterization of HCHT in terms of the mode of action, processivity, binding, and rate constants for the catalysis and dissociation using α-chitin as substrate. HCHT efficiently released the end-label from reducing-end labelled chitin and had also high probability (95%) of endo-mode initiation of processive run. These results qualify HCHT as an endo-processive enzyme. Processivity and the rate constant of dissociation of HCHT were found to be in-between those, characteristic to processive exo-enzymes, like SmChiA and randomly acting non-processive endo-enzymes, like SmChiC. Apart from increasing the affinity for chitin, CBM had no major effect on kinetic properties of HCHT.

A broad pH range and processive chitinase from a metagenome library.

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Corynebacterium glutamicum possesses β-N-acetylglucosaminidase

BackgroundIn Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae, which includes mycobacteria, cell elongation and peptidoglycan biosynthesis is mainly due to polar growth. C. glutamicum lacks an uptake system for the peptidoglycan constituent N-acetylglucosamine (GlcNAc), but is able to catabolize GlcNAc-6-phosphate. Due to its importance in white biotechnology and in order to ensure more sustainable processes based on non-food renewables and to reduce feedstock costs, C. glutamicum strains have previously been engineered to produce amino acids from GlcNAc. GlcNAc also is a constituent of chitin, but it is unknown if C. glutamicum possesses chitinolytic enzymes.ResultsChitin was shown here not to be growth substrate for C. glutamicum. However, its genome encodes a putative N-acetylglucosaminidase. The nagA2 gene product was active as β-N-acetylglucosaminidase with 0.27 mM 4-nitrophenyl N,N’-diacetyl-β-D-chitobioside as substrate supporting half-maximal activity. NagA2 was secreted into the culture medium when overproduced with TAT and Sec dependent signal peptides, while it remained cytoplasmic when overproduced without signal peptide. Heterologous expression of exochitinase gene chiB from Serratia marcescens resulted in chitinolytic activity and ChiB secretion was enhanced when a signal peptide from C. glutamicum was used. Colloidal chitin did not support growth of a strain secreting exochitinase ChiB and β-N-acetylglucosaminidase NagA2.ConclusionsC. glutamicum possesses β-N-acetylglucosaminidase. In the wild type, β-N-acetylglucosaminidase activity was too low to be detected. However, overproduction of the enzyme fused to TAT or Sec signal peptides led to secretion of active β-N-acetylglucosaminidase. The finding that concomitant secretion of endogenous NagA2 and exochitinase ChiB from S. marcescens did not entail growth with colloidal chitin as sole or combined carbon source, may indicate the requirement for higher or additional enzyme activities such as processive chitinase or endochitinase activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0795-3) contains supplementary material, which is available to authorized users.

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.