Isolation and Molecular Characterisation of Alkaline Protease Producing Bacillus thuringiensis

Proteases are of particular interest because of their action on insoluble keratin substrates and generally on a broad range of protein substrates. Proteases are one of the most important groups of industrial enzymes used in detergent, protein, brewing, meat, photographic, leather, dairy, pharmaceutical and food industry. In the present study, the organism isolated from the protein rich soil sample was identified by biochemical and molecular characterisation as Bacillus thuringiensis and further optimum conditions for alkaline protease synthesis were determined. The growth conditions for B. thuringiensis was optimised by inoculating into yeast extract casein medium at different pH and incubating at different temperatures. The maximum protease production occurred at pH 8 and at 37 °C. B. thuringiensis showed proteolytic activity at various culture conditions. Optimum conditions for the protease activity were found to be 47 °C and pH 8. In the later stage, the blood removing action of crude and partially purified protease was found to be effective within 25 min in the presence of commercial detergents indicating the possible use of this enzyme in detergent industry. Enzyme also showed good activity against hair substrate keratin and can be used for dehairing.     

Cloning,purification and biochemical characterisation of a GH35 beta-1,3/beta-1,6-galactosidase from the mucin-degrading gut bacterium <Emphasis Type="Italic">Akkermansia muciniphila</Emphasis>

A putative GH35 β-galactosidase gene from the mucin-degrading bacterium Akkermansia muciniphila was successfully cloned and further investigated. The recombinant enzyme with the molecular mass of 74 kDa was purified to homogeneity and biochemically characterised. The optimum temperature of the enzyme was 42 °C, and the optimum pH was determined to be pH 3.5. The addition of sodium dodecyl sulphate (SDS) reduced the enzyme’s activity significantly. The addition of Mg²⁺-ions decreased the activity of the β-galactosidase, whereas other metal ions or EDTA showed no inhibitory effect. The enzyme catalysed the hydrolysis of β1,3- and β1,6- linked galactose residues from various substrates, whereas only negligible amounts of β1,4-galactose were hydrolysed. The present study describes the first functional characterisation of a β-galactosidase from this human gut symbiont.     

Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase)

N-Acyl homoserine lactone (AHL) quorum-sensing signals are the vital elements of bacterial quorum-sensing systems, which regulate diverse biological functions, including virulence. The AHL-lactonase, a quorumquenching enzyme encoded by aiiA from Bacillus sp., inactivates AHLs by hydrolyzing the lactone bond to produce corresponding N-acyl homoserines. To characterize the enzyme, the recombinant AHL-lactonase and its four variants were purified. Kinetic and substrate specificity analysis showed that AHL-lactonase had no or little residue activity to non-acyl lactones and noncyclic esters, but displayed strong enzyme activity toward all tested AHLs, varying in length and nature of the substitution at the C3 position of the acyl chain. The data also indicate that the amide group and the ketone at the C1 position of the acyl chain of AHLs could be important structural features in enzyme-substrate interaction. Surprisingly, although carrying a (104)HX- HXDH(109) short sequence identical to the zinc-binding motif of several groups of metallohydrolytic enzymes, AHL-lactonase does not contain or require zinc or other metal ions for enzyme activity. Except for the amino acid residue His-104, which was shown previously to not be required for catalysis, kinetic study and conformational analysis using circular dichroism spectrometry showed that substitution of the other key residues in the motif (His-106, Asp-108, and His-109), as well as His-169 with serine, respectively, caused conformational changes and significant loss of enzyme activity. We conclude that AHL-lactonase is a highly specific enzyme and that the (106)HXDH(109) approximately H(169) of AHL-lactonase represents a novel catalytic motif, which does not rely on zinc or other metal ions for activity.     

Production and activity of extracellular lipase from Luteibacter sp.

Microbial lipases are widely used in industrial applications due to their versatility, and the characterization of new lipase-producing microorganisms could provide new sources of these enzymes, with different specificities and better activities. In this context, we have improved lipase production by Luteibacter sp. by using basal medium supplemented with 2 % olive oil, a pH of 6 and a growth temperature of 37 °C. The enzyme extraction process with the addition of 0.25 % Tween 80 increased lipase activity. Implementation of these modifications increased lipase activity by approximately 430 %. The lipase activities produced in the culture supernatant (LCS) and extracted with Tween 80 (LCST80) were characterized. Both extracts hydrolyzed ρ-nitrophenyl (ρNP) esters with different acyl chain lengths, with a preference for short acyl lengths, and had optimum activity at 45 °C. The LCS was stable at acidic and alkaline pH, but LCST80 was only stable at alkaline pH. Methanol, SDS, Triton X-100, EDTA, and EGTA did not affect lipase activity, while divalent cations (Ca²⁺, Zn²⁺, Mg²⁺) - with the exception of Co²⁺— increased lipase activity. Both extracts showed transesterification activity on ρNP ester substrates, and both were able to hydrolyze different natural lipids. The characterization of lipase produced by Luteibacter sp. introduces this recently described genus as a new source of lipases with great biotechnological potential.     

Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes

Laccase is a blue copper oxidase with multiple copper ions and widely distributed in higher plant and fungi. To date, numerous fungal laccases have been reported by many researchers. In present work, a new laccase gene, named CcLCC5I, from Coprinus cinereus was synthesized chemically according to the yeast bias codon and integrated into Pichia pastoris GS115 genome by electroporation. SDS-PAGE analysis showed that the recombinant laccase has a molecular mass of approximately 56.8 kDa. Its biochemical properties was carried out using substrate 2-2^′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was showed that the optimum pH and temperature of the laccase is 3.0 and 55 °C, respectively. Except for copper ions, most metal ions inhibited the laccase activity at a high concentration about 10 mM. Sodium sulfite can also highly inhibit laccase activity whereas EDTA had no inhibitory effect on the laccase activity. The CcLCC5I have high ability to decolor not only azo but also aryl methane dyes. The recombinant laccase decolored 44.6 % orange G, 54.8 % Crystal Violet, and 87.2 % Malachite green at about 2.6 h. The novel laccase may be a good candidate for breeding engineering strains used in the treatment of industrial effluent containing azo and aryl methane dyes.     

Enhancing expression level of an acidophilic β-mannanase in Pichia pastoris by double vector system

An acidophilic β-mannanase-encoding gene (Auman5A) from Aspergillus usamii YL-01-78 was amplified and inserted into pPIC9K and pPICZαA vectors. The resulting recombinant vector, pPIC9K-Auman5A, was transformed into Pichia pastoris GS115. One strain having the highest recombinant β-mannanase activity of 54.6 U/ml, labeled GSKM4-8, was chosen from the first-batch P. pastoris transformants. Then, the pPICZαA-Auman5A was transformed into GSKM4-8 again. From the second-batch transformants, one strain (GSKZαM4-2) with the highest β-mannanase activity of 78.1 U/ml was obtained, and used to optimize expression conditions. As GSKZαM4-2 was induced under the optimized conditions (initial pH value 6.5, induction period 120 h, methanol concentration 1.5 %, and induction temperature 32 °C), β-mannanase activity reached 162.8 U/ml. Protein and carbohydrate assays showed that the β-mannanase, a glycoprotein with an apparent molecular weight of 49.8 kDa and a carbohydrate content of 21.3 %, was extracellularly expressed. It displayed the maximum activity at pH 3.0 and 70 °C, and was stable at a pH range of 3.0–7.0 and at 60 °C. Its activity was not significantly affected by metal ions tested and EDTA, but inhibited by Ag⁺ and Hg²⁺. Its most favorable substrate was locust bean gum, followed by konjac flour and guar gum. The K _m and V _max towards locust bean gum were 1.36 mg/ml and 415.8 U/mg, respectively. These results suggested that the β-mannanase can be expressed with higher level and possesses superior enzymatic properties, making it a good candidate in industrial processes.     

A thermophilic endo-1,4-β-glucanase from Talaromyces emersonii CBS394.64 with broad substrate specificity and great application potentials

Thermophilic cellulases are of significant interest to the efficient conversion of plant cell wall polysaccharides into simple sugars. In this study, a thermophilic and thermostable endo-1,4-β-glucanase, TeEgl5A, was identified in the thermophilic fungus Talaromyces emersonii CBS394.64 and functionally expressed in Pichia pastoris. Purified recombinant TeEgl5A exhibits optimal activity at pH 4.5 and 90 °C. It is highly stable at 70 °C and over a broad pH range of 1.0?10.0, and shows strong resistance to most metal ions, sodium dodecyl sulfate (SDS), and proteases. TeEgl5A has broad substrate specificity and exhibits high activity on substrates containing β-1,4-glycosidic bonds and β-1,3-glycosidic bonds (barley β-glucan, laminarin, lichenan, CMC-Na, carob bean gum, and birchwood xylan). Under simulated mashing conditions, addition of 60 U TeEgl5A reduced more viscosity (10.0 vs.7.6 %) than 80 U of Ultraflo XL from Novozymes. These properties make TeEgl5A a good candidate for extensive application in the detergent, textile, feed, and food industries.     

Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium,Bacillus pumilus

A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 °C, respectively. The enzyme retained 85 % of its activity at 5 °C. There was a significant difference between temperatures for maximal activity (20 °C) and for protein denaturation (approx. 45 °C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.     

In Vitro Characterization of a Recombinant AHL-Lactonase from Bacillus cereus Isolated from a Striped Catfish (Pangasianodon hypophthalmus) Pond

aiiA gene encoding AHL-lactonase was isolated from Bacillus cereus strain N26.2, originating from a striped catfish (Pangasianodon hypophthalmus) pond in Vietnam. This gene, abbreviated as aiiA(N26.2), was cloned and expressed in a competent Escherichia coli strain BL21(DE3)pLysS. The resulting protein, abbreviated as AiiA_N26.2, was highly active in the pH range of 6–8 and could retain 80 % of the maximum activity under storage for 5 days at 4 °C or for 3 days at 20 °C. These properties of AiiA_N26.2 protein confers its future application via feed supplementation, with the purpose of controlling aquaculture pathogens which regulate the virulence via a quorum sensing system.     

Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications

This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4–5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10–30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K^M vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K^M-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5–8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn²⁺, which caused a strong inhibition. The K _m and V _max, towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-β-1,4-d-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.     

Bench scale production of benzohydroxamic acid using acyl transfer activity of amidase from Alcaligenes sp. MTCC 10674

The acyl transfer activity of the amidase of Alcaligenes sp. MTCC 10674 has been applied to the conversion of benzamide and hydroxylamine to benzohydroxamic acid. The unique features of the acyl transfer activity of this organism include its optimal activity at 50 °C and very high substrate (100 mM benzamide) and product (90 mM benzohydroxamic acid) tolerance among the hitherto reported enzymes. The bench scale production of benzohydroxamic acid was carried out in a fed-batch reaction (final volume 1 l) by adding 50 mM benzamide and 250 mM of hydroxylamine after every 20 min for 80 min in 0.1 M potassium phosphate buffer (pH 7.0) at 50 °C, using resting cells equal to 4.0 mg dcm/ml of reaction mixture. From 1 l of reaction mixture 33 g of benzohydroxamic acid was recovered with 24.6 g l^?1 h^?1 productivity. The acyl transfer activity of the amidase of Alcaligenes sp. MTCC 10674 and the process developed in the present study are of industrial significance for the enzyme-mediated production of benzohydroxamic acid.     

Purification and characterization of a prolyl endopeptidase isolated from Aspergillus oryzae

A new fungal strain that was isolated from our library was identified as an Aspergillus oryzae and noted to produce a novel proly endopeptidase. The enzyme was isolated, purified, and characterized. The molecular mass of the prolyl endopeptidase was estimated to be 60 kDa by using SDS-PAGE. Further biochemical characterization assays revealed that the enzyme attained optimal activity at pH 4.0 with acid pH stability from 3.0 to 5.0. Its optimum temperature was 30 °C and residual activity after 30 min incubation at 55 °C was higher than 80 %. The enzyme was activated and stabilized by Ca²⁺ but inhibited by EDTA (10 mM) and Cu²⁺. The K _m and k _cat values of the purified enzyme for different length substrates were also evaluated, and the results imply that the enzyme from A. oryzae possesses higher affinity for the larger substrates. Furthermore, this paper demonstrates for the first time that a prolyl endopeptidase purified from A. oryzae is able to hydrolyze intact casein.     

Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production

In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349 U ml^?1, 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60 °C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8 h under optimal conditions, which included 400 g l^?1 inulin, an enzyme concentration of 40 U g^?1 substrate, 50 °C and pH 6.0, the inulooligosaccharide yield was 91 %. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.     

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1–10 °C in Luria–Bertani (LB) broth or 1–30 °C in Reasoner’s 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1–10 °C in LB medium. About 70–80 % of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60 % of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10–20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C₂–C₁₈). The highest hydrolytic activity was obtained with p-NP-butyrate (C₄) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C₁₂) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.     

Expression, purification and characterization of flavin reductase from Citrobacter freundii A1

Flavin reductase plays an important biological role in catalyzing the reduction of flavin by NAD(P)H oxidation. The gene that codes for flavin reductase from Citrobacter freundii A1 was cloned and expressed in Escherichia coli BL21(DE3)pLysS. In this study, we aimed to characterize the purified recombinant flavin reductase of C. freundii A1. The recombinant enzyme was purified to homogeneity and the biochemical profiles, including the effect of pH, temperature, metal ions and anions on flavin reductase activity and stability, were determined. This enzyme exhibited optimum activity at 45 °C in a 10-min reaction at pH 7.5 and was stable at temperatures up to 30 °C. At 0.1 mM concentration of metal ions, flavin reductase activity was stimulated by divalent cations including Mn²⁺, Sr²⁺, Ni²⁺, Sn²⁺, Ba²⁺, Co²⁺, Mg²⁺, Ca²⁺ and Pb²⁺. Ag⁺ was noticeably the strongest inhibitor of recombinant flavin reductase of C. freundii A1. This enzyme should not be defined as a standard flavoprotein. This is the first attempt to characterize flavin reductase of C. freundii origin.     

Solid-state cultivation of Bacillus thuringiensis R 176 with shrimp shells and rice straw as a substrate for chitinase production

In this study, shrimp shell powder, prepared by treating shrimp-processing waste by boiling and crushing, was used as a substrate for isolation of chitinase-producing microorganism. These organisms may have an important economic role in the biological control of rice and other fungal pathogens. Two hundred strains of bacteria with the ability to degrade chitin from shrimp shell waste were isolated from paddy soil, and of these, 40 strains showed chitinase activity in a solid state cultivation. One of the most potent isolates (strain R 176) was identified as Bacillus thuringiensis. Identification was carried out using morphological and biochemical properties along with 16S rRNA sequence analysis. This strain was able to produce high levels of extracellular chitinase in solid media containing shrimp shells as sole carbon source [1.36 U/g initial dry substrate (IDS)], which was 0.36-fold higher than the productivity in a liquid culture with colloidal chitin. The effects of medium composition and physical parameters on chitinase production by this organism were studied. The optimal medium contained shrimp shell mixed with rice straw in 1:1 ratio added with ball-milled chitin 0.5 % (w/v) and ammonium sulfate 0.5 % (w/v). The highest enzyme production (3.86 U/g IDS) by B. thuringiensis R 176 was obtained at pH 7, 37 °C after 14 days growth. With respect to the high amount of chitinase production by this strain in a simple medium, this strain could be a suitable candidate for the production of chitinase from chitinous solid substrates, and further investigations into its structure and characteristics are merited.