Extraction of chitin from edible crab shells of Callinectes sapidus and comparison with market purchased chitin

Chitin and its derived products have immense economic value due to their vital role in various biological activities as well as biomedical and industrial application. Insects, microorganism and crustaceans are the main supply of chitin but the crustaceans shell like shrimp, krill, lobsters and crabs are the main commercial sources. Chitin content of an individual varies depending on the structures possessing the polymer and the species. In this study edible crabs’ shells (Callinectes sapidus) were demineralized and deproteinized resulting in 13.8% (dry weight) chitin recovery from chitin wastes. FTIR and XRD analyses of the experimental crude as well as purified chitins revealed that both were much comparable to the commercially purchased controls. The acid pretreatment ceded 54g of colloidal chitin that resulted in 1080% of the crude chitin. The colloidal chitin was exploited for isolation of eighty five chitinolytic bacterial isolates from different sources. Zone of clearance was displayed by the thirty five isolates (41.17%) succeeding their growth at pH 7 on colloidal chitin agar medium. Maximum chitinolytic activity i.e. 301.55 U/ml was exhibited by isolate JF70 when cultivated in extracted chitin containing both carbon and nitrogen. The study showed wastes of blue crabs can be utilized for extraction of chitin and isolation of chitinolytic bacteria that can be used to degrade chitin waste, resolve environmental pollution as well as industrial purpose.

Heterologous Expression of a Thermostable Chitinase from Myxococcus xanthus and Its Application for High Yield Production of Glucosamine from Shrimp Shell

Glucosamine (GlcN) is a widely used food supplement. Hence, enormous attention has been concerned with enzymatic production of GlcN owing to its advantage over a chemical approach. In this study, a previously unstudied chitinase gene (MxChi) in the genome of Myxococcus xanthus was cloned, expressed in recombinant soluble form and purified to homogeneity. TLC-, UPLC-, and microplate-reader- based activity tests confirmed MxChi hydrolyzes colloidal chitin to chitobiose as sole product. The optimal catalytic pH and temperature of MxChi was identified as 7.0 and 55 °C, respectively. MxChi exhibited 80% activity after 72 h incubation at 37 °C. The site-directed mutagenesis revealed that the amino acids D323A, D325A, and E327A of MxChi were in the DXDXE catalytic motif of GH18. When coupled with β-N-acetylhexosaminidase (SnHex) and deacetylase (CmCBDA), the enzyme allowed one-pot extraction of GlcN from colloidal chitin and shrimp shell. The optimal condition was 37 °C, pH 8.0, and 1/3/16.5 (MxChi/SnHex/CmCBDA), conducted by orthogonal design for the enzymatic cascades. Under this condition, the yield of GlcN was 26.33 mg from 400 mg shrimp shell. Facile recombinant in E. coli, robust thermostability and pure product herein makes newly discovered chitinase a valuable candidate for the green recycling of chitin rich waste.

A Broad-Specificity Chitinase from Penicillium oxalicum k10 Exhibits Antifungal Activity and Biodegradation Properties of Chitin

Penicillium oxalicum k10 isolated from soil revealed the hydrolyzing ability of shrimp chitin and antifungal activity against Sclerotinia sclerotiorum. The k10 chitinase was produced from a powder chitin-containing medium and purified by ammonium sulfate precipitation and column chromatography. The purified chitinase showed maximal activity toward colloidal chitin at pH 5 and 40 °C. The enzymatic activity was enhanced by potassium and zinc, and it was inhibited by silver, iron, and copper. The chitinase could convert colloidal chitin to N-acetylglucosamine (GlcNAc), (GlcNAc)2, and (GlcNAc)3, showing that this enzyme had endocleavage and exocleavage activities. In addition, the chitinase prevented the mycelial growth of the phytopathogenic fungi S. sclerotiorum and Mucor circinelloides. These results indicate that k10 is a potential candidate for producing chitinase that could be useful for generating chitooligosaccharides from chitinous waste and functions as a fungicide.

A Novel GH Family 20 β-N-acetylhexosaminidase With Both Chitosanase and Chitinase Activity From Aspergillus oryzae

Aminooligosaccharides possess various biological activities and can exploit wide applications in food, pharmaceutical and cosmetic industries. Commercial aminooligosaccharides are often prepared by the hydrolysis of chitin and chitosan. In this study, a novel GH family 20 β-N-acetylhexosaminidases gene named AoNagase was cloned from Aspergillus oryzae and expressed in Pichia pastoris. The purified AoNagase had maximal activity at pH 5.5 and 65°C. It exhibited good pH stability in the range of pH 6.0–7.5 and at temperatures below 50°C. AoNagase was capable of hydrolyzing not only colloidal chitosan (508.26 U/mg) but also chitin (29.78 U/mg). The kinetic parameters (Km and Vmax) of AoNagase were 1.51 mM, 1106.02 U/mg for chitosan and 0.41 mM, 40.31 U/mg for colloidal chitin. To our knowledge, AoNagase is the first GH family 20 β-N-acetylhexosaminidase capable of hydrolyzing both chitosan and chitin. AoNagase is an endo-type β-N-acetylhexosaminidases and can potentially be used for the manufacturing of aminooligosaccharides.

The effect of chitin on the biological activity of Bacillus subtilis strains

The aim of the work was to assess the effect of various forms of chitin and chitosan during submerged cultivation of Bacillus subtilis strains, which form the basis of the laboratory sample Vitaplan, CL, on the synthesis of chitinase, as well as on the antagonistic activity and inducing effect of B. subtilis strains in the pathosystems of wheat - Cochliobolus sativus and Puccinia recondita f. sp. tritici. The inclusion of chitin in the form of dry powder or chitin and chitosan in the form of a colloidal suspension into the medium for deep cultivation of bacteria showed that only colloidal chitin increased the antagonistic activity of B. subtilis strains against test cultures of Alternaria solani and Clavibacter michiganensis. The ability of B. subtilis strains to synthesize extracellular chitinase when cultivated in a medium containing colloidal chitin was established. A higher fungistatic effect of the laboratory sample Vitaplan CL + colloidal chitin against Cohliobolus sativus was revealed as compared to the original sample. It was shown that the laboratory sample Vitaplan, CL + colloidal chitin increases the resistance of wheat to dark brown spot and brown rust 1.5–2.0 times more effectively as compared to Vitaplan, CL. As a result of the research, a laboratory sample of Vitaplan, CL + colloidal chitin was obtained with increased antagonistic and inducing activity as compared to Vitaplan, CL.

Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002

Background Chitinases are enzymes which degrade β-1,4-glycosidid linkages in chitin. The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. However, chitinases are currently produced with low efficiency and poor tolerance, limiting the industrial utility. Therefore, identifying chitinases with higher enzymatic activity and tolerance is of great importance. Methods Primers were designed using the genomic database of Paenibacillus chitinolyticus NBRC 15660. An exochitinase (CHI) was cloned into the recombinant plasmid pET-22b (+) to form pET-22b (+)-CHI, which was transformed into Escherichia coli TOP10 to construct a genomic library. Transformation was confirmed by colony-polymerase chain reaction and electrophoresis. The target sequence was verified by sequencing. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Recombinant protein was purified by Ni-NTA affinity chromatography and enzymatic analysis was carried out. Results The exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). Purification yielded a 13.36-fold enrichment and recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg−1. The optimum pH and temperature for degradation of colloidal chitin were 5.0 and 45 °C, respectively. The enzyme showed high stability, retaining >70% activity at pH 4.0–10.0 and 25–45 °C (maximum of 90 min). The activity of CHI strongly increased with the addition of Ca2+, Mn2+, Tween 80 and urea. Conversely, Cu2+, Fe3+, acetic acid, isoamyl alcohol, sodium dodecyl sulfate and β-mercaptoethanol significantly inhibited enzyme activity. The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc)2 as products. Conclusions This is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste.

Optimization of Agitation Rate in Bioreactor Increases Chitinase Activity of Serratia marcescens PT6

Serratia marcescens PT6 is a Gram-negative bacteria isolated from shrimp pond sediment that capable of producing chitinase. This study aimed to observe the effect of agitation rate on growth and chitinase activity of S. marcescens PT-6 in a bioreactor. The production of chitinase was done in 1.5 l bioreactor using colloidal chitin broth at the condition of pH 7, the temperature of 30°C, aeration of 0.04 vvm, and variation of agitation rate (200, 350, 500 rpm). Bacterial growth was measured by colonies counting in agar medium, while chitinase activity was measured by means of colorimetric every day for four days incubation. The results of ANOVA analysis show that the agitation rate had no effect on bacterial growth, but a significant effect (P<0.05) was observed on chitinase activity. The highest growth and chitinase activity were obtained at 200 rpm, with the highest chitinase activity of 0.006 ± 0.001 U/ml was at day-2. This study implies that the optimized agitation rate in the bioreactor increased the chitinase activity produced by S. marcescens PT-6.

Optimization of Colloidal Chitin and Inoculum Concentration in Chitinase Production by Streptomyces sp. PB2 Using Response Surface Methodology

Chitinase is an enzyme capable of catalyzing the hydrolysis of chitin. Bacteria is one of the sources for chitinase and the modification process of production are continously developed. Streptomyces sp. is one of Actinomycetes group that shows high activity in hydrolyzing chitin. This study aimed to find the optimal conditions of Streptomyces sp. PB2 for producing chitinase at various colloidal chitin (0.5%, 1%, 1.5%) and inoculum (0.5%, 1%, 1.5%) concentration in the chitin broth medium using Respons Surface Methodology (RSM). The examined parameters included Total Plate Count (CFU/ml) and chitinase activity (U/ml). Chitinase activity was statistically analyzed by MiniTab 17 to obtain a mathematical model, then were validated. The mathematical model of chitinase activity was Y = -0.000075 + 0.00056 K + 0.00067 I, with the optimum colloidal chitin concentration (K) of 1.5% and inoculum concentration (I) of 1.5%. The highest chitinase activity of 0.0019 U/ml in day-2 fermentation. The validation test showed that the mathematical model had a low accuracy with the SSE value of 0.5306. This study shows that colloidal chitin and inoculum concentration are important factors to be optimized. However, further model is needed to be developed for a better estimation of process.

Increasing Chitinase Activity of Serratia marcescens PT-6 through Optimization of Medium Composition

Chitin hydrolysate is one of the value added product derived from shrimp shell waste. Production of chitin hydrolysate using biological process offers an environmental friendly method compared to chemical process. Serratia marcescens PT-6, a gram negative chitinolytic bacterium isolated from shrimp pond sediment, shows good activity in hydrolyzing chitin. This study aimed to improve the chitinase activity of S. marcescens PT-6 culture by optimizing the component of chitin-containing medium (additional nitrogen source, additional carbon source, and colloidal chitin). The optimization of chitinase by S. marcescens PT-6 culture was done using one variable at a time method. The sequence of the research were to optimize 1) the type of additional carbon source (glucose, lactose, sucrose, and starch), 2) the type of additional nitrogen source (yeast extract, peptone, ammonium sulphate, and ammonium chloride), 3) the concentration of colloidal chitin (0.5; 1; 1.5; 2; and 2.5%), and 4) the concentration of the additional carbon and nitrogen source. The culture of S. marcescens PT-6 was incubated in colloidal chitin medium at 30 oC and chitinase activity from culture supernatant was analyzed. The results showed that starch gave the highest chitinase activity compare to other carbon source, meanwhile yeast extract was chosen as the best nitrogen source among others. The combination of 1.5% colloidal chitin with 0.5% starch and 0.1% yeast extract in medium increased the chitinase activity of S. marcescens PT-6 to 0.021 U/ml. These results indicated that an appropriate medium composition could increase the chitinase activity produced by S. marcescens PT-6 culture.