Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain KSM-LD1 contains a record number of subtilisin-like serine proteases genes

The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337–340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.     

Restricting detergent protease action to surface of protein fibres by chemical modification

Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1′-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (ΔE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres.     

Growth Inhibitors of Lettuce Seedlings From Bacillus cereus EJ-121

The ethyl acetate extract of the Bacillus sp. EJ-121 culture broth exhibited growth inhibitory activity on a lettuce (Lactuca sativa L.) seedlings assay. Bacillus sp. EJ-121 was identified as Bacillus cereus by the morphological characteristic and nucleotide sequence of the 16S rDNA. The bioassay-guided fractionation of the ethyl acetate extract led to the isolation of two compounds. Their structures were deduced by spectroscopic methods and determined as sodium vanillate (1) and 2-aminobenzoic acid (2). Both compounds 1 and 2 inhibited more than 90% of root length at 50 ppm (0.26 and 0.36 mM, respectively) while they had a limited effect on shoot growth at the same concentration level. Roots and shoots of lettuce seedlings showed severe deterioration at 100 ppm. In order to study the fundamental structure–activity relationship, several structurally related benzoic acid derivatives were also assayed. The existence of a polar carboxyl moiety seemed to be responsible for the stronger activity.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Purification and characterization of a propanol-tolerant neutral protease from Bacillus sp. ZG20

Abstract

A propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655?U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29?kDa. The optimal temperature and pH of the protease were 45?°C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg²⁺, Zn²⁺, K⁺, Na⁺ and Tween-80 could improve its activity. The calculated K_m and V_max values of the protease towards α-casein were 12.74?mg/mL and 28.57?µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20.     

Surface modification of wool with protease extracted polypeptides

Polypeptides were extracted from wool protein fibres using the serine type protease Esperase 8.0L (EC 3.4.21.62), a subtilisin from Bacillus sp., in a reducing solution. The extracted polypeptides, in aqueous liquor, were then applied to modify the fibre surface of wool fabric with or without additional protease. The treated wool fabric was subsequently treated with the cross-linking agent, glycerol diglycidyl ether, and then underwent a curing process to affix the polypeptide to the fibre. The resulting knitted fabric showed a very high level of shrink-resistance to machine washing, without excessive fibre damage. Shrinkage of 1-2% could be achieved after 5 times 5A washes with minimal (<1%) weight loss due to washing and a burst strength of 317 kPa.     

Use of response surface methodology to optimize protease synthesis by a novel strain of Bacillus sp. isolated from Portuguese sheep wool

Aims: To investigate the influence of yeast extract, peptone, temperature and pH upon protease productivity by Bacillus sp. HTS102 – a novel wild strain isolated from wool of a Portuguese sheep breed (Merino). Methods and Results: A 2⁴ full factorial, central composite design together with response surface methodology was used to carry out the experiments and analyse the results, respectively. Among the individual parameters tested, temperature and peptone concentration produced significant effects upon protease productivity. A high correlation coefficient (R² = 0·994, P < 0·01) indicated that the empiric second‐order polynomial model postulated was adequate to predict said productivity, with the optimum loci characterized by: temperature of 43°C, peptone content of 1·4 g l^?1, pH of 5·1 and yeast extract concentration of 10·0 g l^?1. Conclusions: Protease synthesis depends chiefly on temperature and peptone level. The maximum protease activity was more than twice that obtained with the basal medium, so the experimental design and analysis undertaken were effective towards process optimization. Significance and Impact of the Study: Rational choice of processing conditions for maximum protease productivity will be relevant if an economically feasible fermentation process based on Bacillus sp. HTS102 is intended.     

Combined use of mild oxidation and cutinase/lipase pretreatments for enzymatic processing of wool fabrics

A cutinase from Thermobifida fusca WSH04 and two lipases, L3126 and Lipex 100L, were applied to the enzymatic pretreatment of wool fabrics followed by protease treatment, aiming at hydrolyzing the outmost bound lipids on the wool surface. A mild oxidation with 2 g/L hydrogen peroxide (30%) was selectively carried out before the enzymatic treatments. The cooperative actions of mild oxidation, cutinase and lipase pretreatments during wool processing were investigated. The results showed that lipase pretreatment alone had less impact on the wettability and anti‐felting ability of wool fabrics than cutinase treatment. Combined use of cutinase and lipase pretreatments did not evidently improve the properties of the wool fabric compared with the individual cutinase pretreatment. By contrast, mild oxidation slightly enhanced the activity of cutinase toward the wool surface and promoted the subsequent proteolytic reactions. The wetting time and contact angle of the protease‐treated fabric deceased to 1.2 min and 55°, respectively; the area shrinkage decreased to 3.1%, with an acceptable strength loss from 489 to 418 N. The changes in the cuticle scales of the wool fibers, confirmed by scanning electron microscopy, further proved the cooperative actions of mild oxidation and cutinase pretreatment during enzymatic wool processing.     

Purification and Characterization of a Thermo- and Organic Solvent-Tolerant Alkaline Protease from Bacillus sp. JER02

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. K _m and V_max of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H₂O₂ (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.     

Some Properties of Protease from Bacillus R-4 Which Lyses Rhizopus Cell Wall

A strain of Bacillus sp. (Bacillus R-4) produces a protease and a chitosanase which have the ability of lysing Rhizopus cell wall. Some enzymatic properties of the protease purified to a homogeneous state were examined.

The molecular weight of the enzyme was estimated as 19,000 and the isoelectric point as pH 8.65. The protease appeared to have a relative wide range of substrate specificity, hydrolyzing various proteins, such as gelatin, hemoglobin and protamine, and synthetic peptides, such as Z-Gly-Try-NH₂, Z-Gly-Ala-NH₂, Z-Ala-Leu-NH₂ and Z-Gly-Leu-NH₂. The activity lost by EDTA and by Hg²⁺ was restored by Zn²⁺ and reduced glutathione, respectively.     

Dyeing and antimicrobial characteristics of chitosan treated wool fabrics with henna dye

Chitosan, a naturally available biopolymer which is now increasingly being used as a functional finish on textile substrates to impart antimicrobial characteristics and increase dye uptake of fabrics was applied on wool fabrics. Henna a natural dye with proven bactericidal properties was applied on wool fabrics along with chitosan to impart antimicrobial characteristics. The effect of chitosan application on the dyeing properties of wool fabrics was studied by measuring the K/S values of the treated substrates at various concentrations of chitosan and the dye. The antimicrobial properties of chitosan and natural dyes both when applied independently and collectively on fabrics were assessed. The results proved that the chitosan treated wool fabrics showed increase dye uptake of fabrics. The treated fabrics were found to be antimicrobial and the chitosan treatment enhances the antimicrobial characteristics of the dyes. Fastness properties of the applied finish to washing, rubbing and perspiration have also been discussed.     

Molecular Cloning,Nucleotide Sequence,and Expression of the Structural Gene for Alkaline Serine Protease from Alkaliphilic Bacillus sp. 221

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus suhtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6—61.70/0). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

A new cuticle scale hydrolysing protease from Beauveria brongniartii

From a screening for the production of new proteases specific for cuticle scales, Beauveria brongniartii was selected producing an alkaline Ca⁺⁺ dependent protease. The purified had a molecular weight of 27 kDa and a pI value of 8.0. Substrate specificities of model substrates (wool with partially removed cuticles treated with SDS) were analyzed by protein release, dissolved organic carbon (DOC) and nitrogen analysis. The C/N ratio of released material turned out to be a good parameter to determine the site of action of proteases on fibres. Compared to other enzymes, the fungal protease preferentially hydrolyzed cuticle scales and has thus a potential for anti-shrinking pre-treatment of wool fabrics.     

一株深海热液口嗜热芽孢杆菌SY27F代谢产物活性的研究

【目的】对一株来源于深海热液口嗜热芽孢杆菌的次生代谢产物进行抑菌活性和抗肿瘤活性的初步研究。【方法】采用纸片法和微量肉汤稀释法检测嗜热芽孢杆菌SY27F次生代谢产物的抑菌活性,采用CCK-8法测定其次生代谢产物的抗肿瘤活性。【结果】抑菌实验表明,嗜热芽孢杆菌代谢产物对大肠杆菌、金黄色葡萄球菌均有抑菌作用,其最低抑菌浓度分别为1.56 mg/mL和3.13 mg/mL;细胞实验表明,其代谢产物对肿瘤细胞A549、HepG2、HeLa、MCF-7均有一定的抑制作用,其半致死浓度分别为0.390、0.451、0.704、1.105 mg/mL;与人肝肿瘤细胞(HepG2)相比,其对人正常肝细胞(L02)表现出良好的生物相容性。【结论】嗜热芽孢杆菌SY27F次生代谢产物具有一定的抑菌和抗肿瘤活性,可为寻找新型抑菌抗肿瘤活性物质提供优质资源。     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.     

Efficient production of poly γ-d-glutamic acid from the bloom-forming green macroalgae,Ulva sp., by Bacillus sp. SJ-10

Numerous studies on poly γ-d -glutamicacid (γ-PGA) production have investigated terrestrial renewable sources for reducing production costs, but there are no studies using waste marine resources so far. We aimed to develop a cost-effective production method of γ-d -PGA by Bacillus sp. SJ-10 using green macroalgae (Ulva sp.) as a major substrate without hydrolysis pretreatment. The SJ-10 was shown to not only cause immediate tissue degradation of the Ulva membrane but also grew well as a sole substrate. The γ-d -PGA yield was 6.29 ± 0.34 g/L under optimized conditions via the response surface method, and the produced γ-d -PGA had a thermal decomposition temperature of 310°C and molecular weight of 250–1780 kDa. The calculated cost efficiency for the final yield was 32% when compared with complex media. Therefore, the present study provided a strategy for promoting an ecofriendly and cost-effective means to produce γ-d -PGA via a marine renewable resource.     

Isolation and characterization of polyphenol oxidase- and peroxidase-producing <Emphasis Type="Italic">Bacillus</Emphasis> strains from fully fermented tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Sixteen aerobic endospore-forming Bacillus spp. were isolated from fully fermented tea leaf samples from 10 tea factories in Lahijan and Langrod cities (Gillan province, Iran). Bacillus spp. isolates were characterized using phenotypic characteristics, antibiotic susceptibility and cellular fatty acid (CFA) patterns. Based on the data obtained, five isolates of tea Bacillus spp. (TB): TB2, TB4, TB6, TB10 and TB12 belonged to the species B. subtilis. Two isolates, TB1 and TB14 were recognized as B. licheniformis. Two Bacillus spp. isolates, TB9 and TB 16 were identified as B. sphaericus. Two isolates, TB5 and TB13 were shown to be B. pumilus. Two isolates, TB7 and TB15 belonged to B. cereus. Amongst the isolates, Bacillus sp. TB3, Bacillus sp. TB8 and Bacillus sp. TB11 showed different phenotypic traits, distinct antibiotic sensitivity and fatty acid profiles, and they may represent novel species. The isolates showed polyphenol oxidase (tyrosinase) and peroxidase activities. The highest polyphenol oxidase and peroxidase activities were observed for Bacillus sp. TB3 and B. licheniformis TB14, respectively, where values of 5.48 and 3.73 units mL⁻¹ were observed.     

Bacillus sp. mutant for improved biodegradation of Congo red: Random mutagenesis approach

This study presents the improved biodegradation of Congo red, a toxic azo dye, using mutant Bacillus sp. obtained by random mutagenesis of wild Bacillus sp. using UV and ethidium bromide. The mutants obtained were screened based on their decolorization performance and best mutants were selected for further studies. Better decolorization was observed in the initial Congo red concentration range 100–1000 mg/l for wild species whereas mutant strain was found to offer better decolorization up to 3000 mg/l. Mutant strain offered 12–30% reduction in time required for the complete decolorization by wild strain. The optimum pH and temperature were found to be 7.0 and 37 °C, respectively. Two efficient strains such as Bacillus sp. ACT 1 and Bacillus sp. ACT 2 were isolated from the various mutants obtained. Bacillus sp. ACT 2 showed improved enzymatic production and Bacillus sp. ACT 1 showed improved growth compared to wild strain. The enzyme responsible for the degradation was found to be azoreductase by SDS–PAGE and about 53% increased production of enzyme was achieved with mutant species. The experimental data were modeled using growth and substrate inhibition models.     

A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol

Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35°C, pH 7.5 with glucose and NH₄Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.