arastoo badoei

Cellulases are important industrial enzymes for the most bioconversion processes. In this study, Bacillus aerius isolated and identification as the best thermophilic cellulose degrading bacterium from Gorooh hot spring and was identified as Bacillus aerius AV10 based on 16S rRNA sequence homology and microbial- biochemical tests. Cultivation conditions of cellulase production by Bacillus aerius AV10 in solid-state fermentation (SSF) were investigated. The moisture content, yeast extract, MgSO4 and initial culture pH were identified by Plackett-Burman design (PBD) as the significant factors for endoglucanase, exoglucanase and filter paper (FPA) activities. Response surface Methodology and central composite design by 5-level and 4-factor were used for the optimization of enzyme. The optimal ranges of moisture content, yeast extract, MgSO4 and initial culture pH were 61.4%, 1.65%, 0.29% and 6.2, respectively. Under the optimized conditions, maximum CMCase production was 561.12 U/ml. The optimal pH and temperature of the enzyme for wheat straw hydrolysis were determined to be 7.0 and 60 °C.

Cancer cells are not capable of producing L-asparagine, and mainly are depended to the L-asparagine from the circulating plasma pools. L-Asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) catalysis the conversion of L-asparagine to L-aspartate and ammonium. L-asparaginase is the first enzyme with anti-leukemic activity to be intensively studied in human beings. Isolation and identification of L-asparaginase producing bacteria from Jiroft microflora was the aim of this study.
In this sectional study, soil and water samples were collected from citrus orchards and waste of milk factory in Jiroft, Kerman province. The bacterial-producing L-asparaginase was screened on agar medium supplied with L-asparagine and phenol red indicator dye. L-asparaginase activity was detected on the basis of pink color around the colony. The best strains were finally identified using PCR method.
16S rRNA sequencing results showed that the best isolate-producing L-asparaginase belonged to the Bacillus genus. DJK23 strain produced the maximum L-asparaginase, approximately 165 U/ml. The our analysis showed that L-asparaginase was active in a pH rang of 5 to 8, with maximal enzyme activity at pH 7. It is mentioned that this enzyme retains 36% of its initial activity at pH 5.
Although L-asparaginase from bacteria has been extensively characterized, a similar attention has not been paid to Bacillus. These results showed that Bacillus L-Asparaginase has desirable activity in the presence of neutral and acidic pH, which can be considered as a therapeutic and industrial enzyme.

The conversion of nitriles into amides or carboxylic acids by nitrilase has taken its application into consideration, as the scope of its applications has recently been extended.
In this study, P. aeruginosa RZ44 was isolated from sewage in the Kerman which has Nitrile-degradation activity. In order to improve the nitrilase production, several optimization were done on environmental condition. Nitrilase activity was characterized against different pHs, temperatures, ions, and substrates.
Enzyme activity was evaluated by determining the production of ammonia following to the modification of the phenol/hypochlorite method. Different factors that affect production of the enzyme by P. aeruginosa RZ44 were optimized and evaluated in the culture mediums.
The results showed that degradation of the acetonitrile by P. aeruginosa RZ44 increased the pH of the growth medium from the initial pH 7.0 to 9.37. Optimizing the medium for P. aeruginosa RZ44, it was found that glucose and starch (5 g.L-1) have strongly supported nitrilase production, compared to the control. As well, urea (5 g.L-1) and yeast extract (15 g.L-1) have favored an increased biomass and nitrilase production, as the nitrogen sources. These results show that nitrilase production increases in the pH range 5.0 to 7.0 and then start decreasing. Addition of the Mg2, Fe2 and Na has supported the biomass and nitrilase production. Co2, Mn2 and Cu2 were confirmed to inhibit cell growth and enzyme production. Enzyme characterization results show that, P. aeruginosa RZ44 nitrilase exhibits comparatively high activity and stability at pH 7.0 and 40°C. Nitrilase was completely inhibited by CoCl2 and CaCl2, whereas, the inhibition in the presence of MnSO4 and CuSO4 was about 60%. Time course analysis of the nitrile conversion by the resting P. aeruginosa RZ44 cells showed that nitrile substrates (i.e. acetonitrile) was hydrolyzed within 8 h.
these results indicate that P. aeruginosa RZ44 has the potential to be applied in the biotransformation of nitrile compounds.

L-Asparaginase can be effectively used for the treatment of lymphoblastic leukemia. The rapid growth of cancer cells are needed for L-asparagine abundant storage. L-asparaginase catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. The purpose of this study was to isolate and identify the L-asparaginase producing Erwinia strains from the potato farms of Jiroft.
Pectolytic Erwinia species isolated from crumbling potato in M9 medium. The desired L-asparaginase producing bacteria were isolated based on the color changes. Biochemical-microbial and the plant pathogenicity tests of these strains were also investigated with potato and geranium. The L-asparaginase production and molecular detection of these Erwinia strains were also investigated.
In this study, L-asparaginase producing Erwinia was isolated on the CVP and M9 mediums. The inoculation of Erwinia strains on the potato and geranium plants showed that Er8 and Er11 species have the ability to cause plant pathogenicity. Results showed that the maximum pathogenicity of Er8 and Er11 was observed after 48 and 15 h of inoculation in potato and geranium plants, respectively. 16S rDNA sequencing and phylogenetic analyses exhibited that Er8 and Er11 strains were similar to Erwinia chrysanthemi with 98% homology.
Discussion and Because of several applications of the Erwinia L-asparaginase in various fields, isolated Erwinia and their L-asparaginase can be suitable for applied utilization.

A thermophilic strain AMF-07, hydrolyzing carboxymethylcellulose (CMC) was isolated from Kerman hot spring and was identified as Bacillus licheniformis based on 16S rRNA sequence homology. The carboxymethylcellulase (CMCase) enzyme produced by the B. licheniformis was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 37 kDa. The CMCase enzyme was highly active and stable over broad ranges of temperature (40-80 ºC), pH (6.0-10.0) and NaCl concentration (10-25%) with an optimum at 70 ºC, pH 9.0 and 20% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high activity in the presence of cyclohexane (134%) and chloroform (120%). Saccharification of rice bran and wheat bran by the CMCase enzyme resulted in respective yields of 24 and 32 g L-1 reducing sugars. The enzymatic hydrolysates of rice bran were then used as the substrate for ethanol production by Saccharomyces cerevisiae. Fermentation of cellulosic hydrolysate using S. cerevisiae, reached maximum ethanol production about 0.125 g g-1 dry substrate (pretreated wheat bran). Thus, the purified cellulase from B. licheniformis AMF-07 utilizing lignocellulosic biomass could be greatly useful to develop industrial processes.

Proteases are one of the most important groups of industrial enzymes, constituting 65% of worldwide industrial enzyme marketing and around 25% of the total global enzyme production. Their use encompasses a great number of applications in different industrial sectors, such as detergent additives, in waste treatment processes, silver recovery and the food, leather, photographic and pharmaceutical industries. Protease producing bacteria were isolated from Kerman,s dairy industry sewage with clear zone surrounding colony in Skimmed milk agar plate. Its biochemically tests and 16S rRNA gene sequencing showed that it is Chryseobacterium indologenes strain BYK27. The extracellular protease secreted by this strain partially purified by a combination of ammonium sulfate precipitation, dialysis in Tris-HCl buffer 35 mM and ion-exchange chromatography. The temperature and pH optima for activity of produced protease were 40˚C, pH 8 and for stability were 40˚C, pH 8-9. This enzyme was activated by K, Triton X-100 and commercial detergent Tage. It was also active in 1% concentration of salinity. These results showed that protease BYK27 can be used in detergent industry, meat tenderization and peptide synthesis.

L- asparaginase is in an excessive demand in medical applications and in food treating industries, the request for this therapeutic enzyme is growing several folds every year.
In this study, a L- asparaginase gene from Pseudomonas aeruginosa strain SN4 was sequenced and cloned in E. coli. Primers were designed based on L- asparaginase from P. aeruginosa DSM 50071, which show high similarity to SN4 strain, according to 16S rRNA sequence. The L- asparaginase gene was exposed to restriction digestion with NdeI and XhoI enzymes and then ligated into pET21a plasmid. The ligated sample was transformed into competent E. coli (DE3) pLysS DH5a cells, according to CaCl2 method. The transformed E. coli cells were grown into LB agar plate containing 100 µg/ml ampicillin, IPTG (1 mM).
Recombinant L- asparaginase from E. coli BL21 induced after 9 h of incubation and showed high L- asparaginase activity about 93.4 IU/ml. Recombinant L- asparaginase sequencing and alignments showed that the presumed amino acid sequence composed of 350 amino acid residues showed high similarity with P. aeruginosa L- asparaginases about 99%. The results also indicated that SN4 L- asparaginase has the catalytic residues and conserve region similar to other L- asparaginases.
Discussion and This is the first report on cloning and expression of P. aeruginosa L- asparaginases in Escherichia coli. These results indicated a potent source of L- asparaginase for in vitro and in vivio anticancer consideration.

In this study, a Bacillus species was identified from the Dosarri mineral spring in Jiroft microflora. This strain produce clear halo in casein agar media. It has been identified as Bacillus Pumilus (KHB3) based on biochemical tests and 16S rRNA gene sequencing. To enzyme production, this strain was cultured in specific medium for 48 h. Supernatant was partially purified after precipitation with ammonium sulphate (85 %), dialysis and ion exchange chromatography (Q-Sepharose). KHB3 protease was characterized in the presence of different pHs, ions and detergents. Results indicated that the enzyme showed maximum activity and stability in pH 8.0. This enzyme retained about 100 % of its activity in the presence of 1.0 and 1.5 M NaCl. KHB3 protease showed 33 and 10 % increase in protease activity in the presence of MnSO4 and FeSO4. KHB3 protease retained at least 45 % of its activity and stability in the presence of commercial detergents. In addition, it show 12 % increase in enzyme activity in the presence of Banoo detergent. Activity and stability in alkaline pH, organic solvents and detergent compounds show that this protease has high value capacity in detergent industry.

Microbial phytases were applied mainly to animal and human foodstuffs in order to improve mineral bioavailability and food processing. In addition, phytases have potential biotechnological application in various other fields, such as environmental protection, aquaculture and agriculture. Bacillus sp. DM12, an isolate from a hot spring, produces phytase, which catalyzes the hydrolysis of phytic acid into myo-inositol and inorganic phosphates. Phytase from Bacillus sp. DM12 was purified using ammonium sulfate precipitation and dialysis, followed by anion exchange and gel filtration chromatography. Molecular weight of the purified phytase was estimated to be 28 kDa by SDS-PAGE. Km and Vmax values for sodium phytate were 0.177 mM and 1.126 μmol/min, respectively. The optimum temperature for phytase activity was found to be 50°C. The enzyme retained over 75% of its activity over a temperature range of 30 to 80°C. The highest phytase activity was observed at pH 4.5 and a decline of enzyme activity was observed on both sides of pH optimum. The enzyme was stable over the pH range of 3.0 to 6.0. The enzyme retained over 80% of its activity in the presence of 5 mM metal ions except CaCl2. It is also indicated that the enzyme retained over 65% of its activity over a 5 mM metal ions. These properties suggest that this phytase is a suitable enzyme for the hydrolysis of phytic acid and phytates in food and feed processing industries.

Organic solvent-tolerant bacteria are relatively novel extermophilic microorganisms, which can produce organic tolerant protease with capacity of being used in industrial biotechnology for producing high-value compounds. Therefore, finding of these bacteria has drawn much researchers attention nowadays. In this project, samples were collected from a hot spring, located in Jiroft. Samples were incubated in medium supplemented with cyclohexane and toluene for 3 days. Screening of protease producing bacteria was performed on the specific media, SKM (Skim milk agar), based on clear area diameter. The best bacterium was identified based on 16s rDNA gene. Protease activity was considered in different temperatures, pH and organic solvents. Sequence alignment and phylogenetic tree results showed that this bacteria was closely related to Bacillus niacini, with 97% homology. Enzymatic studies showed that, this enzyme was active at a wide range of temperatures, 20-90 °C and it,s optimal activity was in 60 °C. In addition, maximum protease activity was obtained in the 8-9 range of pH, and optimal stability was also at pH 9.0. Protease activity in the presence of methanol, toluene, isopropanol, cyclohexane and DMF ‏showed that, remaining activity was at least 80% compared to the control (without organic solvent) Discussion and Thermopilic capacity, being active in alkaline protease and high protease stability in the presence of organic solvents all herald a remarkable application for using in different industries.