In vivo and in silico toxicity assessment of four common liquid crystal monomers to Daphnia magna: Novel endocrine disrupting chemicals in crustaceans?

Liquid crystal monomers (LCMs) are widely used in liquid crystal displays (LCDs) and are proposed to be a new generation of environmentally persistent, bioaccumulative and toxic (PBT) substances that are increasingly detected in rivers and seas. However, there is a lack of in vivo data that characterize adverse responses and toxic mechanisms of LCMs on aquatic organisms. The aim of this study was to comprehensively investigate the effect of four typical LCMs on the lethality, growth, molting, and reproductive capacity of Daphnia magna (D. magna), a highly studied aquatic species in environmental toxicology. Whole body and enzymatic biomarkers (i.e., body length, chitobiase, acetylcholinesterase, antioxidant defense) were measured to assess the toxicity of LCMs. The 48 h mortality rate and observations of disrupted thorax development and inhibition of ecdysis indicate that D. magna are sensitive to LCMs exposure. Oxidative stress, impaired neurotransmission, and disruptions in molting were observed in short-term biomarker tests using LCMs. A 21 day exposure of D. magna to LCMs resulted in reduced growth, reproduction, and population intrinsic growth rate. In addition, chitobiase and 20-hydroxyecdysone, enzymes important for the molting process, were altered at 7, 14 and 21 d. This is hypothesized to be related to endocrine imbalance resulting from LCM exposure. Based on molecular docking simulations, there is evidence that LCMs bind directly to ecdysteroid receptors; this may explain the observed endocrine disrupting effects of LCMs. These data support the hypothesis that LCMs are endocrine disrupting chemicals in aquatic species, impacting the process of molting. This may subsequently lead to lower reproduction and unbalanced population dynamics.

A novel endo-type chitinase possessing chitobiase activity derived from the chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T.

One of the chitinases (ChiG) derived from the chitinolytic bacterium Chitiniphilus shinanonensis SAY3T exhibited chitobiase activity cleaving dimers of N-acetyl-D-glucosamine (GlcNAc) into monomers, which is not detected in typical endo-type chitinases. Analysis of the reaction products for GlcNAc hexamers revealed that all the five internal glycosidic bonds were cleaved at the initial stage. The overall reaction catalyzed by chitobiases toward GlcNAc dimers was similar to that catalyzed by N-acetyl-D-glucosaminidases (NAGs). SAY3 possesses two NAGs (ChiI and ChiT) that are thought to be important in chitin catabolism. Unexpectedly, a triple gene-disrupted mutant (ΔchiIΔchiTΔchiG) was still able to grow on synthetic medium containing GlcNAc dimers or powdered chitin, similar to the wild-type SAY3, although it exhibited only 3% of total cellular NAG activity compared to the wild-type. This indicates the presence of unidentified enzyme(s) capable of supporting normal bacterial growth on the chitin medium by NAG activity compensation.

Effects of diflubenzuron on shrimp (Neocaridina palmata) in freshwater systems dominated by submerged plant (Ceratophyllum demersum).

Diflubenzuron (DFB) is a benzoylbenzourea insect growth regulator widely used in agriculture, horticulture, and vector control. Therefore, it can easily pollute water bodies and cause harm to aquatic life and ecosystems. To evaluate the impact of DFB on atyid shrimp Neocaridina palmate, the insecticide was applied, at 0, 0.74, 2.222, 6.667, 20, and 60 µg L-1, to indoor systems dominated by submerged plant Ceratophyllum demersum. The highest no observed effect concentration and the lowest observed effect concentration was determined to be 0.167 and 0.536 µg L-1, respectively, as it was counted with either activity or immune-reactive content of chitobiase. Subcellular indices were more sensitive, with a lowest observed effect concentration below 0.107 µg L-1. Principal response curves (PRC) and principal component analysis (PCA) showed that DFB reduced the biomass of C. demersum and the content of chlorophyll-a and phycocyanin in the media. The biomass of periphyton were promoted at the high concentrations. According to the PRC and PCA, DFB reduced the bacterial population related to photoautotrophy, sulphur reduction, and sulphur oxidation and it promoted those related to photoheterotrophy, nitrate reduction, nitrate denitrification, and nitrogen fixation. Besides, DFB reduced fungi related to denitrification. PRC and PCA showed that DFB had a negative impact on pH and dissolved oxygen levels and a positive impact on NH4-N, NO2-N, PO4-P, and conductivity, suggesting the deterioration in quality of water. This study provided useful information for understanding the ecotoxicological effects of DFB at population and community levels.

Effects of tebufenpyrad on freshwater systems dominated by Neocaridina palmata, Physa fontinalis, and Ceratophyllum demersum.

Tebufenpyrad are widely used for control leaf mites in orchard and may enter freshwater systems through runoff, spray drift, and so on. Few papers have reported the side effect of the pesticide on population dynamics of aquatic taxa such as shrimps, gastropods, macrophytes, phytoplankton, and bacteria. Here, we tested the effect of a single application of tebufenpyrad on Neocaridina palmata, Physa fontinalis, Ceratophyllum demersum, Simocephalus vetulus, Dolerocypris sinensis, and so on, by indoor systems. The TWA (Time-weighted average)-based highest no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for Neocaridina palmata, which were counted by the wet weight, were 0.67 and 2.33 µg/L, respectively, and the dose-related effect lasted 21 d. According to our study, chitobiase could be used to quantify the effects of the pesticide on shrimp despite the interference from P. fontinalis, which was finally corrected by employing of antibodies. The NOEC and LOEC were thus determined to be 1.41 and ≤ 5.64 µg/L, respectively, which were higher than the values that was counted by the wet weight. Principal component analysis (PCA) and principal response curve (PRC) investigation showed that the pesticide suppressed population of C. demersum, and phytoplankton, while the Physa fontinalis, S. vetulus, and D. sinensis were stimulated by the pesticide. Illumina MiSeq was used to determine the alteration in bacterial community within the systems. The results of PRC and PCA analyses showed that tebufenpyrad induced flora of nitrate reducing, nitrate denitrifying, thiosulfate oxidation, ureolysis, and methanol oxidation, while it suppressed flora of cellulolysis. Tebufenpyrad was found to have a negative effect on water quality indicators such as pH, DO, NO3-, NO2-, and SO42-, and a positive effect on PO43-, NH4+, and EC. This suggested that the tebufenpyrad led to water quality deterioration.

Direct and indirect effects of fenoxycarb on freshwater systems dominated by Neocaridina palmata (Decapoda: Atyidae) and macrophyte Ceratophyllum demersum.

Few studies have been conducted with regard to the effects of insecticides on population dynamics of shrimps and associated groups such as macrophytes, phytoplankton, microorganisms etc. In the present study, effects of a single application of fenoxycarb were tested using indoor freshwater systems dominated by Neocaridina palmata and Ceratophyllum demersum (Dicotyledons: Ceratophyllales). The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for the N. palmata, as scaled by liberated chitobiase, were 6.48 µg/L and 27.76 µg/L, and the dose-related effect lasted for 14 days. Results of principal components analysis (PCA) and that of principal response curves (PRC) method showed that the biomass of C. demersum and concentrations of chlorophyll-a were suppressed, while the concentrations of phycocyanin were promoted. Illumina high-throughput sequencing was adopted to determine the diversity of bacteria and fungi in the media. Result of PCA and PRC showed that the fenoxycarb promoted photosynthetic bacteria (e.g. Cyanobacteria and Rhodobacterales) and suppressed groups involved in nitrogen and sulfur the transformation (e.g. Flavobacterium, hgcI_clade, Cystobasidium, Rhodotorula and Rhizobiales). Promotion in pathogen such as Pseudomonas and Cercozoa and suppression in beneficial taxa such as Novosphingobium and Rhodotorula were also sighted. Result of study suggested a water quality deterioration due to fenoxycarb applications.

Effect of chronic UVR exposure on zooplankton molting and growth.

Molting is a crucial physiological process in arthropods development, growth, and adult reproduction, where the chitinolytic enzyme chitobiase (CB) and the apoptosis process (caspase-3 activity) play crucial roles. Both molecular endpoints have been observed to be affected by different toxics that may be present in aquatic environments. However, the role of ultraviolet radiation (UVR) in the molting process remains with poor evidence and the possible effect of the previous exposure on F1 generation is unknown. Here, we conducted laboratory experiments with chronic UVR exposure to test the effect on the molting process of Daphnia commutata. Our results showed a clear negative effect of the UVR that affected the molting process with a reduction in individual growth. This trend was also observed in CB and caspase-3 activities. Our results also suggest that the UV dose received by the mother and eggs has an additive effect with the dose received by the offspring. These results imply that the cumulative impact of small UVR doses (2 h per day, daily dose: 2520 J m-2 of 340 nm) on mothers and eggs (which cannot be discriminated in our experiments) can have an additive or synergistic effect along with the generations through a potential increase in lethal effect. Finally, the observed desynchronization in the molting process by UVR will affect the fitness of individuals and population dynamics.

Roundup® disrupts chitinolytic enzyme activity and ecdysteroid concentration in Macrobrachium potiuna.

The endocrine system of crustaceans regulates the molt cycle with ecdysteroid hormones, mainly the 20-hydroxyecdysone (20-HE). Moreover, the molt process requires the action of chitinolytic enzymes (e.g., chitinase, chitobiase) to break down the old cuticle. However, endocrine disrupting compounds (EDC) are capable of altering their normal functioning. Glyphosate-based herbicides (GBH), such as Roundup®, the most widely used herbicides, are found in freshwater environments and have been considered EDC for many aquatic organisms. Therefore, this study examined the effects of environmentally relevant GBH concentrations (0.0065, 0.065, and 0.28 mg L-1) on the 20-HE concentration and chitobiase activity in the decapod prawn Macrobrachium potiuna exposed for 14 days. Additionally, lipid peroxidation, a biomarker of membrane lipid degradation, was evaluated in hepatopancreas to assess cellular damage. Results showed that GBH decreased the 20-HE concentration in females at the two highest concentrations tested, while an increase was observed in males exposed to the highest GBH concentration. In addition, GBH also decreased chitobiase activity in males (all concentrations) and females (the two highest concentrations). Finally, GBH caused increased lipid peroxidation in males, indicating cellular damage in the hepatopancreas. In conclusion, this work suggests that GBH is an EDC for crustaceans by disrupting molting, which could lead to altered reproduction and thus population dynamics. Graphical abstract Decrease in the 20-HE concentration and chitobiase activity in muscle of males and females of the freshwater prawn Macrobrachium potiuna exposed to the herbicide Roundup® for 14 days.

A transglycosylating chitinase from Chitiniphilus shinanonensis (CsChiL) hydrolyzes chitin in a processive manner.

Chitin, mostly extracted from shrimp waste, is the second most abundant biopolysaccharide, next only to cellulose. Enzymatic conversion of chitin into useful bioactive molecules such as chitooligosaccharides (COS) has potential biotechnological applications. The current study describes the characterization of a single modular GH18 chitinase from Chitiniphilus shinanonensis (CsChiL). CsChiL was optimally active at 50 °C in sodium citrate buffer, pH 6.0 and active over a broad pH range (6-10). In addition to hydrolysis, CsChiL displayed chitobiase and transglycosylation activities on COS with degree of polymerization (DP) 2 and 4-6, respectively. CsChiL hydrolyzed chitin polymers (α, ß, and colloidal chitin) in a processive manner. Molecular dynamics simulations and residue-wise binding energy contributions provided structural insights and molecular basis of inherent transglycosylation activity by CsChiL. Overall, CsChiL could be useful in generation of COS from the chitin obtained from shrimp waste with potential applications in agriculture and food industries.

Acute and Chronic Toxicity of Carbamazepine on the Release of Chitobiase, Molting, and Reproduction in Daphnia similis.

As one of the most frequently detected pharmaceutical compounds in aquatic environments, carbamazepine (CBZ) has recently been shown to cause acute and chronic toxicity in a variety of non-target aquatic organisms. However, little is known about the ecotoxicological effects it has on the molting and reproduction of crustaceans. The aim of the present work was to evaluate the acute and chronic toxic responses to CBZ in the crustacean Daphnia similis. After acute exposure (4 days), CBZ did not cause lethal toxicity at the tested concentrations. However, CBZ did inhibit the molting and release of chitobiase at concentrations higher than 6.25 µg/L, with 96 h EC50 (median effective concentration) values of 864.38 and 306.17 µg/L, respectively. The results of chronic exposure showed that the mean number of molts, size of the first brood, mean number of offspring per brood, mean number of broods per female, and total offspring per female decreased significantly with increasing CBZ concentrations. Significant effects of CBZ on the molting or fecundity in D. similis were observed even at concentrations as low as 0.03 µg/L. In conclusion, CBZ can cause inhibition of molting, delayed reproduction, and reduced fecundity in D. similis. CBZ toxicity to D. similis depends on the timing and duration of the exposure. Moreover, our results indicated that CBZ would act as an endocrine disrupter in D. similis, as with vertebrates (e.g., fish).

Racemic, R-, and S-tebuconazole altered chitinase and chitobiase activity of Daphnia magna.

Tebuconazole is a chiral trizole fungicide and widely used in many crops for controlling disease. Tebuconazole is potential toxic to some aquatic organisms but relative information of its isomers is scarce. To detect the endocrine disrupting effects and difference of rac-, R-, and S-tebuconazole, the chitinase activity in Daphnia magna and chitobiase activity in each test medium were used as biomonitors after a 14-day exposure. Results showed that chitinase activity was significantly reduced by rac-, R-, and S-tebuconazole. The chitobiase activity in the test medium was reduced by rac- and R-tebuconazole before day 10, and only one peak was observed at day 10 or day 12 compared with two obvious peaks in the control group (days 6 and 12). S-tebuconazole delayed and reduced the reproduction of D. magna, but did not delay the first chitobiase activity peak, whereas the second peak could not be characterized as the exposure concentration and time increased. Compared with chitinase activity, chitobiase activity can still be used as a rudimentary model for identifying molt-interfering xenobiotics, and further studies should focus on the analysis of correlations between these parameters.

Advances in Biochemical Indices of Zooplankton Production.

Several new approaches for measuring zooplankton growth and production rates have been developed since the publication of the ICES (International Council for the Exploration of the Sea) Zooplankton Methodology Manual (Harris et al., 2000). In this review, we summarize the advances in biochemical methods made in recent years. Our approach explores the rationale behind each method, the design of calibration experiments, the advantages and limitations of each method and their suitability as proxies for in situ rates of zooplankton community growth and production. We also provide detailed protocols for the existing methods and information relevant to scientists wanting to apply, calibrate or develop these biochemical indices for zooplankton production.

A new chitinase-D from a plant growth promoting Serratia marcescens GPS5 for enzymatic conversion of chitin.

The current study describes heterologous expression and biochemical characterization of single-modular chitinase-D from Serratia marcescens (SmChiD) with unprecedented catalytic properties which include chitobiase and transglycosylation (TG) activities besides hydrolytic activity. Without accessory domains, SmChiD, hydrolyzed insoluble polymeric chitin substrates like colloidal, α- and ß-chitin. Activity studies on CHOS with degree of polymerization (DP) 2-6 as substrate revealed that SmChiD hydrolyzed DP2 with a chitobiase activity and showed TG activity on CHOS with DP3-6, producing longer chain CHOS. But, the TG products were further hydrolyzed to shorter chain CHOS with DP1-2 products. SmChiD with its unique catalytic properties, could be a potential enzyme for the production of long chain CHOS and also for the preparation of efficient enzyme cocktails for chitin degradation.

Effects of chlordecone on 20-hydroxyecdysone concentration and chitobiase activity in a decapod crustacean, Macrobrachium rosenbergii.

Chlordecone (CLD) is an organochlorine insecticide abundant in aquatic environment of the French West Indies. However, few studies have investigated its impact on freshwater invertebrates. Whereas CLD is suspected of inducing endocrine disruption, this work aimed to study the effects of environmentally relevant concentrations of CLD on the 20-hydroxyecdysone (20-HE) hormone concentration and on the chitobiase activity, both having key roles in the molting process of crustaceans. In addition, the bioaccumulation of CLD was measured in the muscle tissue of Macrobrachium rosenbergii to underline potential dose-response relationship. The results have shown that CLD was bioaccumulated in exposed organisms according to a trend to a dose-response relationship. Moreover, it was observed that CLD decreased the 20-HE concentration in exposed prawns when compared to control, whatever the duration of exposure, as well as it inhibited the chitobiase activity after 30days of exposure. The present study indicates that CLD could interfere with molting process of M. rosenbergii by disturbing the 20-HE concentration and the activity of chitobiase, suggesting consequences at the long term on the shrimp development. This study also confirmed that CLD could be an endocrine disruptor in decapod crustaceans, as it was already observed in vertebrates.

Enhancing the Yield of Active Recombinant Chitobiase by Physico-Chemical and In Vitro Refolding Studies.

Chitobiase (CHB) is an important enzyme for the production of N-acetyl-D-glucosamine from the chitin biopolymer in the series of chitinolytic enzymes. Majority of over-expressed CHB (58%) in E. coli expression system led to formation of inclusion bodies. The production and soluble yield of active CHB was enhanced by co-expression with GroEL/ES chaperonin, optimizing culture conditions and solubilization followed by refolding of remaining inactive chitobiase present in the form of inclusion bodies. The growth of recombinant E. coli produced 42% CHB in soluble form and the rest (~58%) as inclusion bodies. The percentage of active CHB was enhanced to 71% by co-expression with GroEL/ES chaperonin system and optimizing culture conditions (37 °C, 200 rpm, IPTG--0.5 mM, L-arabinose--13.2 mM). Of the remaining inactive CHB present in inclusion bodies, 37% could be recovered in active form using pulsatile dilution method involving denaturants (2 M urea, pH 12.5) and protein refolding studies (1.0 M L-arginine, 5% glycerol). Using combinatorial approach, 80% of the total CHB expressed, could be recovered from cells grown in one litre of LB medium is a step forward in replacing hazardous chemical technology by biotechnological process for the production of NAG from chitinous waste.

Regulation of the chitin degradation and utilization system by the ChiX small RNA in Serratia marcescens 2170.

Serratia marcescens 2170 produces three different types of chitinases and chitin-binding protein CBP21. We found that transposon insertion into the 5' untranslated region (5' UTR) of chiPQ-ctb led to defective chitinase and CBP21 production. ChiX small RNA possessed the complementary sequence of the 5' UTRs of the chiPQ-ctb and chiR and repressed the expression of chiP and chiR. ChiX was detected in a medium containing glucose, glycerol, GlcNAc, and (GlcNAc)2, but the expression of both chiP and chiR was only observed in a medium containing (GlcNAc)2. ∆chiX mutant produced chitinases, CBP21, and chitobiase without induction. chiP transcripts were more abundant than those of chiR or chiX in a medium containing (GlcNAc)2. These results suggest that the constitutively expressed ChiX binds to the highly abundant chiP 5' UTR, thereby leading to the induction of chiR mRNA translation and the subsequent expression of chitinases and CBP21.

N-Acetyl-ß-D-glucosaminidase activity in feral Carcinus maenas exposed to cadmium.

Cadmium is a priority hazardous substance, persistent in the aquatic environment, with the capacity to interfere with crustacean moulting. Moulting is a vital process dictating crustacean growth, reproduction and metamorphosis. However, for many organisms, moult disruption is difficult to evaluate in the short term, what limits its inclusion in monitoring programmes. N-acetyl-ß-D-glucosaminidase (NAGase) is an enzyme acting in the final steps of the endocrine-regulated moulting cascade, allowing for the cast off of the old exoskeleton, with potential interest as a biomarker of moult disruption. This study investigated responses to waterborne cadmium of NAGase activity of Carcinus maenas originating from estuaries with different histories of anthropogenic contamination: a low impacted and a moderately polluted one. Crabs from both sites were individually exposed for seven days to cadmium concentrations ranging from 1.3 to 2000 µg/L. At the end of the assays, NAGase activity was assessed in the epidermis and digestive gland. Detoxification, antioxidant, energy production, and oxidative stress biomarkers implicated in cadmium metabolism and tolerance were also assessed to better understand differential NAGase responses: activity of glutathione S-transferases (GST), glutathione peroxidase (GPx) glutathione reductase (GR), levels of total glutathiones (TG), lipid peroxidation (LPO), lactate dehydrogenase (LDH), and NADP(+)-dependent isocitrate dehydrogenase (IDH). Animals from the moderately polluted estuary had lower NAGase activity both in the epidermis and digestive gland than in the low impacted site. NAGase activity in the epidermis and digestive gland of C. maenas from both estuaries was sensitive to cadmium exposure suggesting its usefulness for inclusion in monitoring programmes. However, in the digestive gland NAGase inhibition was found in crabs from the less impacted site but not in those from the moderately contaminated one. Altered glutathione levels were observed in cadmium-treated crabs from the contaminated site possibly conferring enhanced tolerance to these animals through its chelator action. Investigation of enhanced tolerance should thus be accounted for in monitoring programmes employing NAGase as biomarker to avoid data misinterpretation.

Construction and basic characterization of deletion mutants of the genes involved in chitin utilization by Serratia marcescens 2170.

In order to elucidate the roles of ChiP, ChiQ, and ChiX in chitin utilization by Serratia marcescens 2170, the construction of single-gene deletion mutants of the chiP, chiQ, and chiX genes was attempted by allelic exchange mutagenesis. ΔchiP formed smaller clearing zones and ΔchiX formed larger ones than wild-type 2170 on an agar plate containing colloidal chitin. ΔchiP grew slowly on the lower concentration of (GlcNAc)2, and there was essentially no growth on chitin oligosaccharides larger than (GlcNAc)3. The gene product of chiP was detected in the outer membrane fraction, consistently with the hypothesis that chiP encodes outer membrane chitoporin. Deletion of chiQ decreased and that of chiX increased the growth rates on chitin oligosaccharides. These observations strongly suggest that all three genes are involved in chitin utilization and that the deletion mutants obtained in this study might prove useful tools to clarify the details of the chitin utilization system of this bacterium.

Effects of two PBDE congeners on the moulting enzymes of the freshwater amphipod Gammarus pulex.

Polybrominated diphenyl ethers (PBDEs) are abundant in aquatic environment. However, only few studies have investigated their impacts on freshwater invertebrates. This work aimed to study the effects of BDE-47 and BDE-99 congeners on the chitobiase and chitinolytic enzymes activities of the freshwater amphipod Gammarus pulex, according to gender, PBDE concentration and time of exposure. In addition, the bioaccumulation of BDE-47 and BDE-99 were measured. Results revealed that females have bioaccumulated more PBDE than males, and BDE-99 was more accumulated than BDE-47. PBDE exposures for 96 h have caused chitobiase and chitinolytic enzymes inhibition. This study not only indicate the importance of taking into account various confounding factors (gender, congeners, concentration) to understand PBDE effects, but underline also disruptions of molting enzymes activities. These disturbances suggest effects on the gammarid development and reproduction, and consequently effects on the gammarid population, and on a larger scale, a dysfunction of the ecosystem.

Toxicity assessments with Daphnia magna of Guadipyr, a new neonicotinoid insecticide and studies of its effect on acetylcholinesterase (AChE), glutathione S-transferase (GST), catalase (CAT) and chitobiase activities.

Guadipyr is a novel neonicotinoid insecticide, developed by the China Agricultural University. This work investigated its aquatic toxicity on Daphnia magna. The acute immobilization test showed that guadipyr was slightly toxic to daphnids, with a 48 h EC50 of 13.01 mg/L. In addition, guadipyr significantly enhanced the acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity (per gram of protein), but had no obvious impact on catalase (CAT) activity within 48 h. The 21 d chronic exposure of D. magna to guadipyr induced a significant decrease in body growth and reproduction; both share the same lowest observed effect concentration (LOEC) at 0.10 mg/L. In the 14 d chronic test, a significant increase in chitobiase activity in test media was observed at day 8 (days to the first breeding), while a significant decrease was observed from days 10 to 14, which might be due to the endocrine imbalance resulting from guadipyr stress. These results demonstrated that guadipyr can induce notable negative ecotoxicological impacts on the aquatic system in long-term exposure at a sub-lethal dose. Further research in environmental behaviors is needed to regulate guadipyr use in the future.

Production of N-acetylglucosamine using recombinant chitinolytic enzymes.

The pharmaceutically important compound N-acetylglucosamine (NAG), is used in various therapeutic formulations, skin care products and dietary supplements. Currently, NAG is being produced by an environment-unfriendly chemical process using chitin, a polysaccharide present in abundance in the exoskeleton of crustaceans, as a substrate. In the present study, we report the potential of an eco-friendly biological process for the production of NAG using recombinant bacterial enzymes, chitinase (CHI) and chitobiase (CHB). The treatment of chitin with recombinant CHI alone produced 8% NAG and 72% chitobiose, a homodimer of NAG. However, supplementation of the reaction mixture with another recombinant enzyme, CHB, resulted in approximately six fold increase in NAG production. The product, NAG, was confirmed by HPLC, TLC and ESI-MS studies. Conditions are being optimized for increased production of NAG from chitin.