Chemical profiling of Streptomyces sp. Al-Dhabi-2 recovered from an extreme environment in Saudi Arabia as a novel drug source for medical and industrial applications

Filamentous bacterial belonged to Streptomyces species were novel drug source for medical and industrial applications. However, the detailed identification of Streptomyces species from Saudi Arabian extreme environment for the identification novel drug source for medical and industrial applications were rarely studied. The Streptomyces strain Al-Dhabi-2 obtained from the thermophilic region kingdom of Saudi Arabia, exhibited antimicrobial potentials against the pathogenic microorganism were characterized. Biochemical and phylogenetic analysis confirmed that the strain was closely associated to the Streptomyces species. The chromatogram of GC-MS analysis of this ethyl acetate extract (EA) had diverse of chemical compounds namely benzene acetic acid (7.81%), acetic acid, methoxy-, 2-phenylethyl ester (6.01%) were the major compounds. EA of Al-Dhabi-2 showed inhibition zone ranged from 14 to 25 mm at 5 mg/well concentration against the tested microbial pathogens. Results revealed that the significant MIC values were observed against B. cereus, and E. faecalis by (less than 39 μg/ml) and against S. agalactiae with (78 μg/ml). Minimum inhibitory concentrations (MIC) for fungi: were also reported against Cryptococcus neoformans and Trichophyton mentagrophytes by (156 μg/ml), whilst Candida albicans and Aspergillus niger by (312 μg/ml). Results of this study showed that thermophilic actinobacteria could be promise source in the context of searching for unique antimicrobial agents with novel properties.     

Effects of oils and oil-related substrates on the synthetic activity of membrane-bound lipase from Rhizopus chinensis and optimization of the lipase fermentation media

The lipase from filamentous fungi Rhizopus chinensis, as a membrane-bound enzyme, possesses the excellent catalysis ability for esterification and transesterification reactions, and has a good potential in many industrial applications. In order to improve the synthetic activity of the lipase, the effects of oils and oil-related substrates on its production and the fermentation media optimization were investigated. Based on the results, it was suggested that oleic acid could be the important substrate for the lipase production. Among various oils and oil-related substrates, olive oil containing high content of oleic acid was the optimal one for the lipase production. Using orthogonal test and response surface methodology (RSM), the composition of fermentation media was further optimized. The optimized media for lipase synthetic activity and activity yield was composed of peptone 57.94 and 55.58 g L⁻¹, olive oil 21.94 and 22.99 g L⁻¹, maltose 12.91 and 14.34 g L⁻¹, respectively, with K₂HPO₄ 3 g L⁻¹, MgSO₄·7H₂O 5 g L⁻¹ and initial pH 6.0. Under the optimal conditions, the lipase activity and the activity yield were improved 61.5 and 93.4% comparing the results before optimization, respectively. The adequate models obtained had predicted the lipase production successfully.     

牦牛瘤胃中产脂肪酶微生物的分离与鉴定

【背景】脂肪酶是一类特殊的酯键水解酶,广泛应用于工业化生产中,微生物是工业脂肪酶的主要来源。瘤胃中微生物种类繁多、数量庞大,已有关于瘤胃微生物产纤维素酶的报道,尚无产脂肪酶瘤胃微生物的分离筛选报道。【目的】从牦牛瘤胃中分离筛选出能够产脂肪酶的微生物,并进行菌株鉴定及其酶学性质的研究。【方法】以橄榄油为唯一碳源,通过中性红油脂平板进行初步筛选后,用改进铜皂-分光光度法测定酶活力进行复筛;再经形态学观察、生理生化实验和16S rRNA基因序列分析进行菌种鉴定;研究3种脂肪酶的最适作用温度、pH值及金属离子、有机溶剂和表面活性剂对酶活力的影响。【结果】筛选出6株酶活力较高的菌株,其中3株为液化沙雷氏菌,2株为白地霉,1株为卷枝毛霉。脂肪酶的酶学性质研究表明:液化沙雷氏菌、白地霉和卷枝毛霉所产脂肪酶的最适作用温度为45、35和40°C;最适pH为8.0、7.0和7.0;Ca2+和Mg2+对3种脂肪酶均有激活作用;Zn2+对3种脂肪酶有不同程度的抑制作用,EDTA、SDS可使3种脂肪酶失活;3种脂肪酶对丙三醇的耐受力较高,卷枝毛霉脂肪酶对甲醇、乙醇、丙酮的耐受力较高。【结论】从牦牛瘤胃中分离出3种产脂肪酶的微生物,且证实瘤胃微生物在脂肪酶研究方面具有较高的价值。     

Thermophilic protease production by Streptomyces sp. 594 in submerged and solid-state fermentations using feather meal

AIMS: Protease production by Streptomyces sp. 594 in submerged (SF) and solid-state fermentation (SSF) using feather meal, an industrial poultry residue, and partial characterization of the crude enzyme. METHODS AND RESULTS: Streptomyces sp. 594 produced proteases in SF (7.2 +/- 0.2 U ml(-1)) and SSF (15.5 +/- 0.41 U g(-1)), with pH increase in both media. Considering protease activity, values obtained in the liquid extract after SSF (6.3 +/- 0.17 U ml(-1)) were lower than those from SF. The proteases, which belong to serine and metalloproteinase classes, were active over a wide range of pH (5.0-10.0) and high temperatures (55-80 degrees C). Strain 594 was also able to degrade feather in agar and liquid media. Keratinase activity (80 U l(-1)) also confirmed the keratin degrading capacity of this streptomycete. CONCLUSIONS: Proteases produced using residues from poultry industry have shown interesting properties for industrial purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as we are concerned, this is the first contribution towards the production of thermophilic protease by a streptomycete in SSF using a keratinous waste.     

Response surface method to optimize the production and characterization of lipase from Penicillium verrucosum in solid-state fermentation

Current studies about lipase production by solid-state fermentation involve the use of agro-industrial residues towards developing cost-effective systems directed to large-scale commercialization of enzyme-catalyzed processes. In this work, lipase production and partial characterization of the crude enzymatic extracts obtained by Penicillium verrucosum using soybean bran as substrate was investigated. Different inductors were evaluated and the results showed that there is no influence of this variable on the lipase production, while temperature and initial moisture were the main factors that affected enzyme production. The optimized cultivation temperature (27.5 °C) and initial moisture of substrate (55%) were determined using the response surface methodology. Kinetics of lipase production was followed at the optimized growth conditions. Optimum lipase yield was 40 U/g of dry bran. The crude enzymatic extract showed optimal activity in the range from 30 to 45 °C and in pH 7.0.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: compatibility study with detergent ingredients and washing performance

An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol? XP80 and Triton? X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1h) while the protease activity was enhanced by the addition of Triton? WR1339 and Tween? 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%.     

Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis protease (Subtilisin Carlsberg)

Alcalase 2T, a commercial preparation of Subtilisin Carlsberg, was covalent immobilized onto physiochemically characterized silica supports. The effect of mean pore diameter and surface chemistry on enzyme activity in the hydrolysis of casein has been examined. Two sets of chemically distinct silica supports were used presenting terminal amino (S_APTES) or hydroxyl groups (S_TESPM-pHEMA). The percentage of immobilized protein was smaller in S_APTES (31–39%) than in S_TESPM-pHEMA (62–71%), but presented higher total and specific activity. Silicas with large pores (S₁₀₀₀, 130/1200 Å) presented higher specific activities relative to those with smaller pore sizes (S₃₀₀, 130/550 Å). The influence of glutaraldehyde concentration and the time of enzyme coupling to the S₁₀₀₀S_APTES supports was examined. The apparent K_m value for the S₁₀₀₀S_APTES immobilized enzyme is lower than the soluble one which may be explained by the partitioning effects of the substrate. No intraparticle diffusion limitations were observed for the immobilized enzyme and therefore the substrate diffusion does not influence the observable kinetics. Finally, the optimum pH, optimum temperature, thermal stability, operational stability, and storage stability of the immobilized and freely soluble enzymes were compared.     

Short communication: Production of l-glutamate oxidase by Streptomyces sp. N1 in submerged fermentation

In submerged fermentation of Streptomyces sp. N1 in a shake flask, glucose (3% w/v) and (NH₄)₂SO₄ (0.6% w/v) were found to be suitable for extracellular l-glutamate oxidase (GluOx) (EC.1.4.3.11) production. GluOx production was higher with the addition of further KCl or MgCl₂ to the medium within the range of 0 to 0.12% (w/v). The effect of inoculum type, that is, spore inoculation or mycelium inoculation on GluOx biosynthesis was also investigated, and the maximum GluOx production obtained was 2.7 U/ml after 33h fermentation with mycelium inoculation. The results demonstrated a much higher GluOx production and productivity compared with those reported previously.     

Improved thermal stability and activity in the cold-adapted lipase B from Candida antarctica following chemical modification with oxidized polysaccharides

In order to improve the thermal stability (t_1/2) and activity of lipase B from cold-adapted Candida antarctica (CALB), amino groups of the enzyme were chemically linked to a range of oxidized polysaccharides using a range of reducing agents. By chemically modifying CALB using 0.1% dextran (250 kDa) at pH 8.6 for 10 days using borane–pyridine complex as reducing agent, increased thermal stability (t_1/2, 168 min at 70°C) and activity (65% higher specific activity) was achieved compared to the unmodified enzyme (t_1/2, 18 min at 70°C). Improvements in thermostability were generally better with high molecular weight polymers such as dextran (40 and 250 kDa) or ficoll (70 and 400 kDa) in comparison to low molecular weight inulin (5 kDa). The shape of the polymer also appeared to be important with elongated, elipsoidal-shaped dextran providing better thermostabilization than spherical-shaped ficoll. Borane–pyridine complex was found to be a good, non-toxic reducing agent for improving thermostability, compared with sodium borohydride and sodium cyanoborohydride. An interesting finding was that, in all cases, specific activity of the modified enzymes increased with a concomitant increase in thermostability. This response defies the general principle of a trade-off between activity and stability, and demonstrates that chemical modification provides new avenues for improving the thermal stability of enzymes from psychrophiles without sacrificing their activity.     

华根霉液态培养生产脂肪酶不同形态菌体的转录组差异分析

【背景】作为发酵工业中一类重要的生产菌株,丝状真菌目的产物的形成与菌体形态有着紧密的联系。华根霉(Rhizopus chinensis)CCTCC M201021是从我国传统酿造浓香型白酒大曲中筛选到的一株丝状真菌,其生成的包括脂肪酶在内的酶蛋白具有较高的工业应用价值。【目的】华根霉(Rhizopus chinensis)CCTCC M201021在脂肪酶液态发酵中形成两种不同形态菌体,发酵表现差异明显。本研究考察华根霉不同菌体形态及其细胞代谢在转录组水平的内在差异。【方法】基于RNA-Seq高通量转录组测序,分别对液态培养获得的不同形态华根霉菌体高表达和显著差异表达的转录基因进行功能分析。【结果】两种形态菌体转录组存在明显的差异。利用RPKM值对表达量最高的前20个基因进行分析,聚集态菌体高表达基因主要为不同类别的核糖体蛋白,而分散态菌体中与细胞形态相关的几丁质酶及与信号传导相关的基因也是高表达基因。在两种形态菌体显著差异表达的20个基因中,除了涉及代谢的基因有明显不同外,分散态菌体中也有一些涉及"细胞过程与信号"的基因上调表达显著。两种形态菌体中独有表达基因总体表达量均较低,但聚集态菌体独有表达基因在基因种类和表达量上都要明显高于分散态菌体。同时,转录分析表明,华根霉脂肪酶在聚集菌体中较高的生产水平与脂肪酶基因的高水平转录有关。【结论】菌体形态的差异显著影响了华根霉的转录组,不仅不同形态菌体高表达基因和显著差异表达基因有明显不同,而且功能相同的蛋白在不同菌体形态下也多是由不同基因表达,它们可能承担着不同的作用。总体而言,华根霉聚集态菌体中存在更为复杂的生理过程,而分散菌体中受到信号的传导和调控似乎更多。菌体形态的改变可能是细胞分化的结果,伴随着菌体对细胞微环境改变的一种响应。研究结果为深入了解丝状真菌形态分化的内在机制及其影响提供了一些线索。     

Isolation and characterization of an extracellular lipase from Mucor sp strain isolated from palm fruit

During our screening of lipolytic fungus which may play a role in the acidification of palm oil, we have recently isolated a Mucor sp strain. Culture conditions were optimized and the highest lipase production amounting to 57 U/ml was achieved after 6 days of cultivation. The extracellular lipase was purified 1050-fold by ammonium sulfate precipitation, carboxymethyl–sephadex chromatography and Sephadex G75 gel filtration to a final specific activity of 6600 IU/mg. The molecular weight of the homogenous lipase was determined about 42 kDa by gel filtration and SDS–polyacrylamide gel electrophoresis. The purified lipase was determined as a glycoprotein with a pI of 6.2. The Nt sequence was determined as AspGluIleGluThrValGlyXPheThrMetAspLeuProProAsnProPro and showed no homology with the sequences of the known lipases suggesting that the enzyme may be a new lipase. The purified lipase hydrolyzed both synthetic and natural triglycerides with the optimal activity recorded on trioctanoin and sunflower oil, respectively. Its activity was strongly inhibited by Triton X-100 and SDS. Metal ions such as Fe³⁺, Fe²⁺ and Hg²⁺ also decreased the lipase activity.     

Physical crosslinking of lipase from Rhizomucor miehei immobilized on octyl agarose via coating with ionic polymers: Avoiding enzyme release from the support

Lipase from Rhizomucor miehei (RML) was immobilized on octyl-agarose (OC) at different loadings. Using low enzyme loadings (1/7 of the maximum loading), the incubation of the enzyme with polyethylenimine (PEI) increased the resistance to enzyme desorption in the presence of Triton X-100. However, more than 10% of the enzyme activity could be released from the OC-RML-PEI. The same treatment using fully loaded biocatalyst reduced the enzyme desorption to less than 5%. Further treatment with dextran sulfate (DS) of the PEI treaded immobilized enzyme fully avoids the enzyme desorption even in presence of a Triton X-100 concentration higher than that required for the complete enzyme release from OC-RML. This treatment produced a high stabilization of OC-RML in thermal or organic solvent inactivations, reducing the enzyme release under these drastic conditions. Nevertheless, the support could be recovered by incubation under adequate conditions, and reused in several adsorption/desorption cycles. Thus, the strategy permitted to avoid enzyme desorption, very likely by physical intermolecular crosslinking improving enzyme stability, while still maintaining the reversibility of the immobilization.     

Screening for tyrosinase inhibitors from actinomycetes; identification of trichostatin derivatives from Streptomyces sp. CA-129531 and scale up production in bioreactor

In the course of a primary screening of 614 microbial actinomycete extracts for the discovery of tyrosinase inhibitors, the EtOAc extract of the fermentation broth of the strain Streptomyces sp. CA-129531 isolated from a Martinique sample, exhibited in cell free and cell-based assays the most promising activity (IC₅₀ value of 63 μg/mL). Scaled-up production in a bioreactor led to the isolation of one new trichostatic acid analogue, namely trichostatic acid B (1), along with six known trichostatin derivatives (2–7), four diketopiperazines (8–11), two butyrolactones (12–13) and one hydroxamic acid siderophore (14). Among them, trichostatin A (4) showed a Ki value of 6.1 μM and six times stronger anti-tyrosinase activity (IC₅₀ 2.18 μΜ) than kojic acid (IC₅₀ 14.07 μΜ) used as a positive control. Deoxytrichostatin A (6) displayed also strong inhibitory activity against tyrosinase (IC₅₀ 19.18 μΜ). Trichostatin A production in bioreactor started together with the exponential phase of growth (day 4) and the maximum concentration was reached at day 9 (2.67 ± 0.13 μg/mL). Despite the cytotoxicity of some individual components, the EtOAc extract showed no cytotoxic effect on HepG2, A2058, A549, MCF-7 and MIA PaCa-2 cell lines, (IC₅₀ >2.84 mg/mL) and against BG fibroblasts at the concentrations where the whitening effect was exerted, reassuring its safety and great tyrosinase inhibitory potential.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.     

Isolation of bioactive peptides from tryptone that modulate lipase production in Yarrowia lipolytica

In this work the effect of several organic nitrogen sources on lipase production in Yarrowia lipolytica LgX64.81 overproducing mutant was studied. Among them, tryptone and peptone showed the most prominent stimulatory effect. Interestingly, only tryptic and peptic casein digest were found to highly induce lipase biosynthesis while lipase production was very limited in the presence of casein digest from papain and pronase-catalysed hydrolysis and absent in case of chymotryptic digest. It was also demonstrated that the stimulatory peptides should be present in the culture medium at specific proportions and molecular size to match the physiological requirement of Yarrowia lipolytica strain for lipase biosynthesis.     

Isolation and characterization of biosurfactant production under extreme environmental conditions by alkali-halo-thermophilic bacteria from Saudi Arabia

Twenty three morphologically distinct microbial colonies were isolated from soil and sea water samples, which were collected from Jeddah region, Saudi Arabia for screening of the most potent biosurfactant strains. The isolated bacteria were selected by using different methods as drop collapse test, oil displacement test, blue agar test, blood hemolysis test, emulsification activity and surface tension. The results showed that the ability of Virgibacillus salarius to grow and reduce surface tension under a wide range of pH, salinities and temperatures gives bacteria isolate an advantage in many applications such as pharmaceutical, cosmetics, food industries and bioremediation in marine environment. The biosurfactant production by V. salarius decreased surface tension and emulsifying activity (30 mN/m and 80%, respectively). In addition to reducing the production cost of biosurfactants by tested several plant-derived oils such as jatropha oil, castor oils, jojoba oil, canola oil and cottonseed oil. In this respect the feasibility to reusing old frying oil of sunflower for production rhamnolipids and sophorolipids, their use that lead to solve many ecological and industrial problems.     

Molecular identification and diversity analysis of dental bacteria in diabetic and non-diabetic females from Saudi Arabia

Periodontal disease is a chronic infectious disease, which is characterized by the damaged dental hard tissue by lactic acid generated by microorganisms after the fermentation of carbohydrates rich diet. The risk of periodontal disease is known to be higher in diabetic patients. We compared the diversity of five commonly occurring dental bacteria including Porphyromonas gingivalis, Tannerella forsythia, Capnocytophaga ochracea, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans in 14 type-2 diabetic patients and equal numbers of healthy controls. The subgingival samples were collected using sterile paper points. We used 16S rRNA sequence specific primers for PCR-based identification of dental bacteria. Our results showed that A. actinomycetemcomitans was completely absent in control subjects but present in 43% of diabetic patients. C. ochracea was highly prevalent in diabetic patients (100%) as compared to controls (28.5%). The frequency of other three bacterial species was also higher in diabetic patients than control subjects. These findings indicate that dental bacteria are highly prevalent in subgingival pockets of diabetic patients. Therefore, proper monitoring of diabetic patients for dental care is important to prevent bacterial growth and its sequela in risky individuals. Further case-control studies using larger sample size would help in validating the association between oral diseases and diabetes.     

Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125

Response surface methodology (RSM) was employed to optimize culture medium for production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was used to evaluate the effects of different components in the culture medium. Soybean oil, soybean powder and K₂HPO₄ have significant influences on the lipase production. The concentrations of three factors were optimized subsequently using central composite designs and response surface analysis. The optimized condition allowed the production of lipase to be increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l fermenter reached 9600 IU/ml.