Purification and characterization of a thermophilic and acidophilic chitinase from Microbispora sp. V2

AIM: Purification and characterization of a chitinase from Microbispora sp. V2. METHODS AND RESULTS: The chitinase from Microbispora sp. V2 was purified to homogeneity by gel filtration chromatography with 4.6% recovery. It had a molecular weight of 35 kDa and showed maximum activity towards p-nitrophenyl-beta-d-N,N'-diacetylchitobiose, indicating a chitobiosidase activity. The enzyme had a pH optimum of 3.0 and temperature optimum of 60 degrees C. It was stable in a wide pH range from 3.0 to 11.0, retaining 61% activity at pH 3.0 and 52% activity at pH 11.0. It retained 71% activity at 30 degrees C and 45% activity at 50 degrees C, up to 24 h. The enzyme activity was not inhibited by any of the metal ions tested except Hg2+, in the presence of which only 10% activity was retained. CONCLUSIONS: The 35 kDa chitinase from Microbispora sp. V2 has an acidic pH optimum and a high temperature optimum. It is fairly stable and active, and degrades chitin efficiently, although the growth of the culture and enzyme production is slow. SIGNIFICANCE AND IMPACT OF THE STUDY: This report is the first detailed study of a chitinase from Microbispora sp. V2, isolated from hot springs. The chitinase from Microbispora sp. V2 may have potential applications in the recycling of chitinous wastes, particularly due to its thermophilic and acidophilic character. Studies at molecular level may provide further insight on the chitinolytic system of Microbispora spp. with respect to the number and types of chitinases and their regulation.     

Phospholipase A2 activity of rat stomach

Phospholipase A activity in rat stomach wall and in gastric content was studied using [1-14C]dioleoylphosphatidylcholine as substrate. The optimum activity of the stomach wall was found to take place at pH 7.0. During optimal phospholipase action about 40% of the [1-14C]oleic acid released was due to an active intracellular lysophospholipase. The gastric phospholipase required 5 mM Ca2+ for full activity and is inhibited by EDTA. It specifically hydrolyzed the sn-2 position of the phospholipid molecule. The enzyme was heat labile and inactivated by acidification at pH 3.0. The gastric content enzyme had a lower specific activity and an optimum pH of 8.0. It was heat stable and was not inactivated by acidification. These results indicate that gastric content phospholipase A is of pancreatic origin, via a duodenal reflux. By ligating the stomach we were able to further confirm that the gastric wall phospholipase was different from that of the gastric content. It originated from the stomach mucosa. Subcellular fractionation suggests that the gastric phospholipase A2 is essentially bound to the plasma membrane. About 6% of the activity was found to be soluble. Biopsies of human gastric mucosa displayed a phospholipase A activity which had similar properties to that of rat gastric enzyme. The physiological function of this enzyme is discussed in terms of prostaglandin synthesis via the release of arachidonic acid.     

One-step purification of chitinase from Serratia marcescens NK1, a soil isolate

AIMS: A simple single step technique of gel filtration was developed for the purification of chitinase from Serratia marcescens NK1. METHODS AND RESULTS: Chitinase from Ser. marcescens NK1 was purified to homogeneity by gel filtration chromatography with 9.2% recovery. The enzyme had a pH optimum of 6.2 and a temperature optimum of 47 degrees C. It was stable in a wide pH range of 3.0 to 10.0, retaining 60% activity at pH 3.0 and 65% activity at pH 10.5. It retained 70% activity at 28 degrees C after 72 h and nearly 50% activity at 50 degrees C up to 24 h. CONCLUSION: The chitinase from Ser. marcescens NK1 can be efficiently purified in a single step by gel filtration chromatography. The chitinase of Ser. marcescens NK1, a soil isolate, is highly stable and as active as that of other reported isolates of Ser. marcescens. SIGNIFICANCE AND IMPACT OF THE STUDY: This purification scheme is advantageous because of its simplicity and can therefore be applied for the purification of other enzymes. The yield is sufficient for initial characterization studies of the enzyme, and an improved resolution can be obtained if the chromatography is done under fast flow systems.     

The isolation and properties of a non-pepsin proteinase from human gastric mucosa.

1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D.     

Thiamine pyrophosphatase and nucleoside diphosphatase in rat brain

Two types of nucleoside diphosphatase were found in rat brain. One (Type L) had similar properties to those of the liver microsomal enzyme with respect to its isoelectric point, substrate specificity, Km values, optimum pH, activation by ATP and molecular weight. The other (Type B), which separated into multiple forms on isoelectric focusing, had lower Km values and a smaller molecular weight than the Type L enzyme, and was inhibited by ATP. The Type B enzyme catalyzed the hydrolysis of thiamine pyrophosphate as well as those of various nucleoside diphosphates at physiological pH, while Type L showed only nucleoside diphosphatase activity at neutral pH. These findings suggest that the two enzymes play different physiological roles in the brain.     

Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis

To investigate the roles of the active site residues in the catalysis of Bacillus thuringiensis WB7 chitinase, twelve mutants, F201L, F201Y, G203A, G203D, D205E, D205N, D207E, D207N, W208C, W208R, E209D and E209Q were constructed by site-directed mutagenesis. The results showed that the mutants F201L, G203D, D205N, D207E, D207N, W208C and E209D were devoid of activity, and the loss of the enzymatic activities for F201Y, G203A, D205E, W208R and E209Q were 72, 70, 48, 31 and 29%, respectively. The pH-activity profiles indicated that the optimum pH for the mutants as well as for the wildtype enzyme was 8.0. E209Q exhibited a broader active pH range while D205E, G203A and F201Y resulted in a narrower active pH range. The pH range of activity reduced 1 unit for D205E, and 2 units for G203A and F201Y. The temperature-activity profiles showed that the optimum temperature for other mutants as well as wildtype enzyme was 60°C, but 50°C for G203A, which suggested that G203A resulted in a reduction of thermostability. The study indicated that the six active site residues involving in mutagenesis played an important part in WB7 chitinase. In addition, the catalytic mechanisms of the six active site residues in WB7 chitinase were discussed.     

Human prostatic gastricsinogen: The precursor of seminal fluid acid proteinase

A gastricsinogen-like acid proteinase precursor has been purified by DEAE-cellulose, DEAE-Sephadex A-50, poly-l-lysine-Sepharose 4B, and N-acetyl-l-phenylalanyl-l-tyrosine-Sepharose 4B affinity chromatography from human prostates. The active enzyme hydrolyzes acid-denatured hemoglobin at pH 1.0 and 3.0, while two other active fractions only showed the pH 3.0 activity and resembled cathepsin D (EC 3.4.23.5). The pH optimum, milk-clotting activity, specificity toward synthetic substrates, inhibition by pepstatin, and molecular weight strongly suggest that the prostatic-derived enzyme is identical to seminal fluid and to gastric juice gastricsin.     

Proteinases for the gastric mucosa of the European sheatfish, Silurus glanis L

Three proteinases named as P1, P2 and P3, were isolated from European sheatfish (Silurus glanis L.) gastric mucosa by salting-out of (NH4)2SO4, gel-chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. Isoelectric points of isolated proteinases were determined by isoelectric focusing and were equal to 1.9, 3.2 and 4.75 respectively for P1, P2 and P3. The molecular weight of P1 was 39,800 Da and proteinases P2 and P3 had a molecular weight of 30,200 Da. The optimum pH for three peptidases isolated from sheatfish gastric mucosa and maximum stability of these enzymes were found at acidic pH. It allowed identifying these proteinases as pepsin-type enzymes of fish.     

Directed evolution of a Bacillus chitinase

Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito-oligosaccharide-based value-added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error-prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose at various pH in microtiter plates. From a medium-throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose as substrate, the overall catalytic efficiency, k_cat/K_m of the improved chitinase was 2.7- and 2.3-fold higher than the average k_cat/K_m of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications.     

Chitinase activity in the digestive tract of the cod, Gadus morhua (L.)

The study was made to determine if enzymatic degradation of chitin occurs in the digestive tract of the cod, Gadus morhua . The method employed corresponds to the end product measurement of Jeuniaux (1966), using 'native' chitin as the substrate. The following results were obtained.

• (1) 

  Chitinolytic enzyme of high activity is present in enzyme solutions from the stomach contents, gastric mucosa and intestinal contents.
• (2) 

  Lower chitinase activities are found in samples of the intestinal mucosa and the pyloric caeca.
• (3) 

  The optimum pH ranges for the action of the enzymes in the stomach and the intestine differ: 4.5–5.1 and 5.1–6.5, respectively.
• (4) 

  The role of chitin-decomposing bacteria is discussed, based on bacterial numbers and pH conditions in the digestive tract. The existence of two different enzyme systems is indicated.

     

Purification,characterization, and molecular cloning of chitinases from the stomach of the threeline grunt Parapristipoma trilineatum

Two chitinase isozymes, PtChiA and PtChiB, were purified from the stomach of the threeline grunt, Parapristipoma trilineatum. The molecular masses of PtChiA and PtChiB were estimated to be 50 and 60 kDa by SDS-PAGE, respectively. Both chitinases were stable at pH 3.0–6.0 (acidic) and showed the optimum pH toward both short and long substrates in the acidic region (pH 2.5–5.0). PtChiA and PtChiB preferentially degraded the second and third glycosidic bonds from the non-reducing end of N-acetylchitooligosaccharides, respectively. PtChiA and PtChiB exhibited wide substrate specificities toward crystalline chitin. Moreover, 2 cDNAs encoding PtChiA and PtChiB, PtChi-1 and PtChi-2, respectively, were cloned. The deduced amino acid sequences of both chitinase cDNAs comprised N-terminal signal peptides, glycoside hydrolase 18 catalytic domains, linker regions, and C-terminal chitin-binding domains. Phylogenetic tree analysis of vertebrate chitinases revealed that fish stomach chitinases including PtChi-1 and PtChi-2 form unique chitinase groups, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), which differ from the acidic mammalian chitinase (AMCase) group. The present results suggest that fish have a chitin-degrading enzymatic system in which 2 different chitinases, AFCase-1 and AFCase-2, with different degradation patterns are expressed in the stomach.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Enzymatic Properties of Phosphoenolpyruvate Carboxylase Isoforms in the CAM Plant Kalanchoe daigremontiana

Three isoforms (Types 1, 2 and 3) of phosphoenolpyruvate (PEP)carboxylase in young leaves of the Crassulacean acid metabolism(CAM) plant Kalanchoe daigremontiana were separated by DEAE-cellulosecolumn chromatography and preparative polyacrylamide-agarosegel electrophoresis, and their enzymatic properties were characterized. All three isoforms had similar molecular weights of about 234,000.At pH 8.0 Type 1 showed a high affinity to PEP, (K_m=0.08 mM),whereas Type 3 showed a low affinity (K_m=1.0mM). K_m values forMgCl₂ were 0.26 HIM in Types 1 and 3 and 0.5 nut in Type 2.All three types exhibited the same pH optimum at 8.0, but Type1 showed relatively low activity below pH 6.0, whereas Type3 showed high activity. Type 3 was more acid stable than theother forms. In the presence of glucose-6-phosphate, the K_mvalues of Types 1, 2 and 3 for PEP lowered to 0.027, 0.037 and0.044 mu at pH 8.0, respectively. Inhibition of activity byorganic acids such as malate and pyruvate was pronounced inType 3. Type 2 exhibited properties intermediate to Types 1and 3 with regard to pH curve, affinity to PEP and its effectof various metabolites. The physiological significance of PEPcarboxylase isoforms in CAM plants is discussed on the basisof these findings. ¹Present address: Agricultural Chemicals Research Lab., SankyoCo., Ltd., Yasu-cho, Yasugun, Shiga 520-23, Japan. (Received November 30, 1983; Accepted March 24, 1984)     

Molecular dynamics simulation of chitinase I from Thermomyces lanuginosus SSBP to ensure optimal activity

Abstract

The fungal chitinase I obtained from Thermomyces lanuginosus SSBP, a thermophilic deuteromycete, has an optimum growth temperature and pH of 323.15 K and 6.5, respectively. This enzyme plays an important task in the defence mechanism of organisms against chitin-containing parasites by hydrolysing β-1, 4-linkages in chitin. It acts as both anti-fungal and biofouling agents, with some being thermostable and suitable for the industrial applications. Three-dimensional model of chitinase I enzyme was predicted and analysed using various bioinformatics tools. The structure of chitinase I exhibited a well-defined TIM barrel topology with an eight-stranded α/β domain. Structural analysis and folding studies at temperatures ranging from 300 to 375 K using 10 ns molecular dynamics simulations clearly showed the stability of the protein was evenly distributed even at higher temperatures, in accordance with the experimental results. We also carried out a number of 20 ns constant pH molecular dynamics simulations of chitinase I at a pH range 2–6 in a solvent. This work was aimed at establishing the optimum activity and stability profiles of chitinase I. We observed a strong conformational pH dependence of chitinase I and the enzyme retained their characteristic TIM barrel topology at low pH.     

Proteinases from Gastric Mucosa of European Sheatfish, Silirus Glanis L.

Three proteinases (P1, P2, and P3) were isolated from the gastric mucosa of European sheatfish (Silurus glanis L.) by (NH₄)₂SO₄ precipitation, gel chromatography on Sephadex G-75, and ion-exchange chromatography on DEAE cellulose. Isoelectric focusing was used for determining the values of pI of the isolated proteinases, which were equal to 1.9, 3.2, and 4.75 (for P1, P2, and P3, respectively). The molecular weight of P1 was 39800 Da; P2 and P3 had equal molecular weights of 30200 Da each. The optimum pH for the three peptidases isolated from sheatfish gastric mucosa and the maximum stability of these enzymes were found to be at acidic pH. This allowed identification of these proteinases as pepsin-type enzymes of fish.     

Chitinolytic enzymes from<Emphasis Type="Italic">Clostridium aminovalericum</Emphasis>: Activity screening and purification

A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.     

Enzymatic sulfation of cholesterol by rat gastric mucosa

A sulfotransferase which catalyzes transfer of the sulfate group from 3'-phosphoadenosine-5'phosphosulfate to cholesterol has been demonstrated in the rat gastric mucosa. The product of the reaction was characterized as cholesterol sulfate by two-dimensional thin-layer Chromatographic behavior, and gas-liquid chromatography of cholesterol after acid solvolysis. The bulk of enzyme activity was found in the cytosol fraction. Sulfation of cholesterol did not require added Mg⁺², Mn⁺², or Ca⁺², and was unaffected by ethylenedia-minetetraacetate. Triton X-100 moderately enhanced the enzyme activity. A broad pH optimum from pH 6.0–9.0 was exhibited with a maximum at pH 7.0–7.5. The apparent Km for PAPS was 0.8 × 10^?6M. The possible function of cholesterol sulfate in gastric mucosa is discussed.     

Purification and partial characterization of two chitinases from the mycoparasitic fungus <Emphasis Type="Italic">Talaromyces flavus</Emphasis>

Chitinases were produced by Talaromyces flavus CGMCC 3.4301 when it was grown in the presence of chitin. Two chitinases from the culture filtrate of T. flavus were purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE–Sepharose and Phenyl–Sepharose hydrophobic interaction chromatography. By SDS–PAGE, the molecular weight of the two enzymes was estimated to be 41 and 32 kDa, respectively. The 41 kDa chitinase (CHIT41) had a 4.0 pH optimum; the 32 kDa chitinase (CHIT32) optimum activity was at pH 5.0. The optimum temperature for the two chitinase activities was 40 °C. The two chitinases had activity against cell wall of Verticillium dahliae, Sclerotinia sclerotiorum and Rhizoctonia solani, and inhibited spore germination and germ tube elongation of Alternaria alternata, Fusarium moniliforme, and Magnaporthe grisea.     

CHANGE OF ACID AGGLUTINATION OPTIMUM AS INDEX OF BACTERIAL MUTATION

A distinct difference in acid agglutination optimum for Type D (bacillus of rabbit septicemia) and its mutant form, Type G, has been observed. The optimum for Type D lies between pH 3.5 and pH 3.0. This changes during mutation, the resulting Type G mutants having in general an optimum lying between pH 4.7 and pH 3.8. The constancy of the optimum for Type D is very strict, while that for Type G is slightly less so. The variation is never so great as to cause an overlapping of optima and consequent failure of differentiation. These acid agglutination optima are in the nature of physical constants for the two types and would imply a fundamental difference in the chemical constitution of the organisms. Animal passage, far from causing a reversion of the mutant Type G to the primordial Type D form, brings about a still greater instability in the presence of H ions.