Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15~C and 30~C, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35~C and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride （PMSF） and hydrochloride 4-（2-aminoethyl）-benzenesulfonyl fluoride （AEBSF）, indicating that it was a serine protease. The presence of Cae＋ and Mnz＋ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid （AA） residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.

Properties of Alkaline Protease Produced by Strain Ⅰ13

[Objective] This study aimed to investigating properties of alkaline protease produced by strain Ⅰ 13.[Method] Crude enzyme of alkaline protease was obtained from alkaline protease produced by strain I 13,while effects of temperature and pH value on enzyme activity were also investigated in this study.[Result] The optimal temperature of alkaline protease produced by strain Ⅰ 13 was 40 ℃,while enzyme activity maintains a higher level from 30 to 60 ℃ and over 40% of the largest enzyme activity still maintained within the range from 20 to 30 ℃.The optimal pH value was 10.5,and over 90% of the largest enzyme activity still maintained within the range from 8.0 to 11.0,which had broader pH value spectrum.[Conclusion] This alkaline protease has huge potential to be developed into washing-powder additive.

Optimized production and properties of thermostable alkaline protease from Bacillus subtilis SHS-04 grown on groundnut (Arachis hypogaea) meal

Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme production (1616.21 U/mL) was achieved using groundnut meal (0.75%) as nitrogen source and 0.5% glucose as carbon source at 48 h cultivation period, pH 9, 45 ° C and 200 rpm. The yield was 348% increase over comparable control samples. The alkaline protease had optimum temperature of 60 ° C and remarkably exhibited 80% relative activity at 70 ° C. It was highly thermostable showing 98.7% residual activity at 60 ° C after 60 minutes of incubation at pH 9.0 and was stable in the presence of organic solvents studied. These properties indicate the viability of the protease for biotechnological and industrial applications. The optimized yield of enzyme achieved in this study establishes groundnut meal as potential low-cost substrate for alkaline protease production by B. subtilis SHS-04.

Regioselective Synthesis of Polymerizable Vinyl Guaifenesin Esters Catalyzed by an Alkaline Protease of Bacillus subtilis

Three polymerizable vinyl guaifenesin esters with different acyl donor carbon chain lengths (C4,C6,C10) were regioselectivly synthesized by an alkaline protease from Bacillus subtilis in pyridine at 50C for 1, 3, 5 days respectively.

Levels of antioxidant enzymes and alkaline protease from pulp and peel of sunflower

Objective:The activity of enzymes participating in the systems of antioxidant protection was assayed in the peel and pulp of sunflower.The essential roles of proteases in food stimulate research to find other sources of the enzyme especially from non-conventional sources.In the present work,we study several biochemical parameters in the pulp and peel of sunflower.Methods:Pulp and peel of sunflower was extracted,antioxidant enzymes and nonenzymatic antioxidant were measured.Alkaline protease was measured and purified from pulp in sunflower.Results:High carbohydrate concentration,beta-carotene,catalase and ascorbate peroxidase activities,free radical scavenging capacity and free flavonoid content were observed in the peel of sunflower.Whereas,MDA and ceruloplasmin activities were high in the pulp of sunflower.Conclusions:The present study concluded that peel in sunflower are strong radical scavengers and can be considered as good sources of natural antioxidants for medicinal and commercial uses.Further analysis showed that protease activity was a significantly high in the pulp compared to the peel.

Alkaline Protease Production by a Strain of Marine Yeasts

Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45 ℃. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH 6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min -1 and agitation speed 150 r min -1. Under the optimal conditions, 623.1 U mg -1 protein of alkaline protease was reached in the culture within 30 h of fermentation.

Study on enzymatic hydrolysis of soybean β-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and antigenicity of its hydrolysates

Due to its beneficial health effects,the use of soybean protein has shown a continuous increase,but concerns regarding the allergenicity of soybean antigenic protein have also increased.This study aimed to evaluate the hydrolytic effects of a non-commercial alkaline protease isolated from the Bacillus subtilis ACCC 01746 on soybeanβ-conglycinin and the allergenicity of its hydrolysates.Alkaline protease of the strain was separated by precipitation method of organic solvents,and theβ-conglycinin was separated by alkali-solution and acid-isolation and purified by use of gel column.Using the degree of hydrolysis(DH)and inhibition rate as evaluation indexes,the enzymatic hydrolysis parameters ofβ-conglycinin was optimized by single factor and L_(9)(3^(4))orthogonal tests,so as to explore the effect of the protease on the hydrolysis degree and the antigenicity ofβ-conglycinin hydrolysates.The results showed that the native enzyme existed as an 18.3 kDa monomer with a 430 U/g maximum activity.The purity ofβ-conglycinin was 84.8%.The single-factor test results showed that DH showed the oppostie trendency with the inhibition rate,and the increase of protein concentration causedmonotone increasing and monotone decreasing of the inhibition rate and the DH,and the optimal protein concentration was 30 mg/mL.The optimization results showed that pH had the largest impacts on both DH and the inhibition rate,followed by enzyme dosage,hydrolysis temperature and hydrolysis time.Under the optimum hydrolysis conditions of protein concentration 30mg/mL,enzymedosage0.7%,hydrolysis time40min,temperature 55°C and pH8.5,the DH reached the highest of 76.28%,and the inhibition rate was the lowest of 27.03%,which was reduced greatly compared with that before optimization.These results suggested that alkaline protease appeared to show a relatively high effeciency in lowering soybean allergenicity,making it possible to produce low-allergenicity soybean protein.

Kinetic study of alkaline protease 894 for the hydrolysis of the pearl oyster Pinctada martensii

A new enzyme (alkaline protease 894) obtained from the marine extremophile Flavobacterium yellowsea (YS-80-122) has exhibited strong substrate-binding and catalytic activity, even at low temperature, but the characteristics of the hydrolysis with this enzyme are still unclear. The pearl oyster Pinctada martensii was used in this study as the raw material to illustrate the kinetic properties of protease 894. After investigating the intrinsic relationship between the degree of hydrolysis and several factors, including initial reaction pH, temperature, substrate concentration, enzyme concentration, and hydrolysis time, the kinetics model was established. This study showed that the optimal conditions for the enzymatic hydrolysis were an initial reaction pH of 5.0, temperature of 30°C, substrate concentration of 10% (w/v), enzyme concentration of 2 500 U/g, and hydrolysis time of 160 min. The kinetic characteristics of the protease for the hydrolysis of P. martensii were obtained. The inactivation constant was found to be 15.16/min, and the average relative error between the derived kinetics model and the actual measurement was only 3.04%, which indicated a high degree of fitness. Therefore, this study provides a basis for the investigation of the concrete kinetic characteristics of the new protease, which has potential applications in the food industry.

Production of Alkaline Protease by Bacillus Licheniformis

In this research the results of studies on optimization of alkaline protease production by Bacillus licheniformis are reported. The parameters, which were taken into consideration, are pH, temperature, time course of enzyme production, stirring rate and kinetics parameters. The effect of various carbon and nitrogen sources in culture medium compound on enzyme production was also considered The result of optimization revealed that maximum protease production was obtained at 37 ℃, pH equivalent tol 0.0 and with 150 rpm will occur after 72 hours. By comparing the effect of 5 carbon sources （maltose, glucose, starch, casein and lactose） in enzyme production, it has been known that using lactose will increase about 1.5 times enzyme production, compared to condition in which maltose is used. The result of studies on the effect of five nitrogen sources （i.e., peptone, tryptone, ammonium sulfate, urea and corn steep liquor） shows that corn steep liqour increases enzyme production more than others, while peptone can also be considered as a good nitrogen source; but, ammonium sulfate and urea reduce enzyme production considerably. It was concluded that protease production occurs in the stationary phase of growth. Studying the kinetics parameters resulted that the best model for the enzyme above is Lineweaver-Burk model according to which Km is 0.64 mmol and Vmax is 88 lamol/min.

Enzymatic kinetics ofβ-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and analysis of antigenicity of hydrolyzed peptide

β-Conglycinin,the main protein of soybean,is a key allergen that causes soybean allergies,and hydrolysis is usually applied to lower its antigenicity.We evaluated the enzymolysis characters ofβ-conglycinin from the perspective of enzymolysis kinetics using alkaline protease from B.subtilis ACCC 01746.A dynamic model describing the hydrolysis ofβ-conglycinin was proposed using the initial substrate concentration,enzyme dosage(enzyme to substrate ratio)and hydrolysis time as variables to illustrate the kinetic behavior of enzymatic hydrolysis.The hydrolysis of soybeanβ-conglycinin was carried out at 60 g/L protein concentration,0.6%enzyme dosage,55℃ and pH 8.5 to observe the peptides with anti-enzymatic activities.The hydrolysates were gradually fractionated by ultrafiltration through cut-off membranes with molecular weights of 40,30,20,and 10 kDa,and their antigenicities were evaluated using indirect competitive enzyme-linked immunosorbent assay.The results showed that the degree of hydrolysis(DH)ofβ-conglycinin decreased as theβ-conglycinin concentration(S0)increased,but increased with enzyme dosage(E0)increasing.Thus,the enzymatic hydrolysis ofβ-conglycinin followed the first-order kinetics model.The hydrolysis rate(V)was(527.89C_(E0)-2.5533C_(S0))exp(-0.022DH),the DH-hydrolysis time was 45.454ln[1+(11.614C_(E0)/C_(S0)-0.0562)t],and the correlated kinetic constants k2 and kd were 527.89 min^(−1)and 8.6126 min^(−1),respectively.The hydrolysis behavior ofβ-conglycinin varied considerably among theα',α,andβsubunits.Faster hydrolysis rates were observed for theα'andαsubunits compared to theβsubunit.The relative molecular weights of the intercepted peptides from the hydrolysates were 14.8-40.1 kDa,and the antigenicity of the peptides with smaller molecular weight was reduced,but not removed completely.However,the alkaline protease from the strain appeared to effectively reduce the allergenicity ofβ-conglycinin.Therefore,it is possible to produce less allergenic soybean proteins using enzymatic hydrolysis.Additionally,the microbial alkaline protease may serve as a potential novel food enzyme and should be evaluated for the development of hypoallergenic foods.

Regiospecific Addition of Uracil to Acrylates Catalyzed by Alkaline Protease from Bacillus subtilis

Michael addition reactions of uracil to acrylates were catalyzed by an alkaline protease from Bacillus subtilis in dimethyl sulfoxide at 55 ℃ for 72 h. The adducts were determined by TLC, IR and 1H NMR.

Hydrolysis of Fish Protein by Analkaline Protease

Cod muscle protein was hydrolyzed by an alkaline protease in our study. The influences of hydrolysis temperature,fish protein concentration,and ratio of protease addition to protein amount on its degree of hy- drolysis (DH) of protein were studied in details by applying dual quadratic rotary combinational design. The final results showed that more than 84% cod muscle protein could be hydrolyzed and recovered. Cod protein hydrolysate thus obtained had a balanced amino acid composition and mainly consisted of small peptides with molecule weight less than 6900 dalton.

Screening of Strains Producing Alkaline Protease from Soil and Study on the Conditions for Enzyme Production

[Objective] The aim was to screen strains producing alkaline protease from soil and study the conditions for enzyme production.[Method] Eight strains producing alkaline protease were isolated from soil through plate isolation,and the ability of enzyme production was measured by filter paper and Folin-phenol method.The strain with the strongest ability of enzyme production was screened as a candidate strain,then the factors influencing the ability of enzyme production was studied,finally the conditions for enzyme production was optimized through orthogonal test.[Result] No.5 strain was screened as a candidate strain due to its strong ability of enzyme production(6.00 U/ml),which accounted for 134.1% of that of Bacillus licheniformis,and it was gram-positive bacterium belonging to Clostridium.Orthogonal test showed that the optimal condition for producing protease was an environment with pH=11,0.3% of sucrose and 0.8% of peptone in the fermentation medium,and inoculation amount was 105 cfu/ml.In addition,peptone had significant impact on the level of enzyme production.[Conclusion] The study could provide theoretical references for the screening of strains producing alkaline protease.

Thermostable alkaline protease production from Bacillus pumilus D-6 by using agro-residues as substrates

Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available proteases lack desired properties;?therefore, search for better and efficient thermostable alkaline proteases are?always on.?Bacillus pumilus?D-6, isolated from dairy plant soil sample, in the?current study produced protease which showed activity and stability at high?alkaline?pH (8 - 12) and high?temperatures (70。C- 100。C). Enzyme activity remained unfazed even in presence?of inhibitors like Pb2+and Hg2+which are considered?universal inhibitors of enzyme activity. Besides, the organism successfully?utilized crude agriculture based substrates as carbon and nitrogen source and?produced substantial enzyme titre.

Mutagenesis of Alkaline Protease-Producing Marine Strains and Enzymatic Properties

[ Objective] The aim of the research was to select alkaline pmtease-producing strains and provide basis for the application in production. [ Method ] An alkaline protease-preducing strain was isolated from the sea water and sea mud collected from Qingdao coastal area. After ultraviolet mutagenesis and microwave mutagenesis, a target strain ZR-58-3-1-3 was obtained. The protease activity and enzymatic properties of the strain were determined preliminarily. [ Result ] Prote- ase activity of the selected strain reached 145 U/ml. After the compound mutagenesis, protease activity of the fermentation liquid of strain ZR-58-3-1-3 reached 775 U/m｜, which was about 4.3 times higher than that of the original strain. The optimal temperature for alkaline protease produced by strain ZR-58-3-1-3 was 45℃, and the optimal pH was 8.5, suggesting it belongs to moderate temperature protease with relatively high thermal stabihty. [ Conclusion] Strain ZR-58-3-1-3 has stable alkaline protease production performance and can be used as an alkaline protease-producing mutant strain.

The selection of alkaline protease-producing yeasts from marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains （HN3.11, Nllb, YF04C and HN4.9） capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, lssatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N1 lb was 10.0. The optimal temperature for protease activity was 45℃for strains HN3.11, N11b, and YF04C, and 50℃ for strain HN4.9. After digestion of shrimp （Penaeus vannamei） protein and spirulina （Arthospira platens＆） protein with the four crude alkaline proteases, the filtrate from spirulina （Arthrospira platensis） powder digested by the crude alkaline protease of strain HNYl 1 was found to have the highest antioxidant activity （61.4%） and the highest angiotensin I converting enzyme （ACE）-inhibitory activities （68.4%）. The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.

Relationship Study between the Alkaline Protease Production and the Growth Phases of <i>Pseudomonas aeruginosa</i>Isolated from Patients

This study was conducted in Diyala University Laboratories collaboration with the Directorate of Diyala Health. Occurrence of Pseudomonas aeruginosa was investigated in 161 samples from different clinical sources included Swabs from wounds, burns, ear, eye and samples from Urine and sputum which were collected from patients. Depending on the cultural and micro features and biochemical tests 49 isolated items of this bacteria have been diagnosed and all the isolates showed the proteolytic activity by using skim milk agar through forming clear zone around the growing colonies, and tested the isolates ability of alkaline protease production by quantitative methods, the local isolation P. aeruginosa AP3 had been selected based on the higher productivity of enzyme comparing to other isolates and thus it was used in the current study. Studied the relationship between the production of alkaline protease enzymes and growth phases of P. aeruginosa to determine the time of the enzyme production and the results showed that the local isolation P. aeruginosa AP3 began production of the enzyme in the later stages of the log phase and increased production significantly in the stationary phase reaching amaximum after 48 hours as estimated the enzyme activity 159.2 units/ml in the farm leaky and keep the enzyme fully functional almost in the stationary phase.

Isolation of enriched-yielders and fed-batch production of alkaline protease from the newly isolated <i>Bacillus</i>sp. BHA

An alkalophilc and thermophilic Bacillus sp. BHA that produced a thermostable alkaline protease was isolated from decaying protein substrates. The isolate was found to grow in pH range 7 - 11 with an optimum pH 9.0 and temperature up to 55℃. The activity of alkaline protease of Bacillus sp. BHA (68.98 APU/ml) was found higher than the standard strains of Bacillus amyloliquefaciens MTCC 610 (8.98 APU/ml) and Bacillus subtilis MTCC 8349 (12.14 APU/ml, used in this study, and was comparable (68.98 APU/ml, equivalent to 30.38 APU/mg) to the activity of the commercially produced standard protease procured from Novo Nordisk, Denmark (30.35 APU/mg). Hence, the proteolytic activity produced by this isolate was further investigated in batch and fed-batch process. Sucrose was the best carbon source for the production of protease activity by that isolate. Different organic nitrogen sources (casein, peptone and beef extract) at 1% (w/v) with varying levels of sucrose (1% - 4% w/v) initially repress enzyme synthesis. The duration and extent of repression decreased with increased concentration of sucrose. Maximum protease activity was found in basal medium with 4% (w/v) sucrose and 1% (w/v) yeast extract. Yeast-extract was thought to be an inducer of enzyme synthesis. Further, the basal medium was unique with respect to the enzyme production, as protease production was growth associated with the peak enzyme production being detected at the time of maximum growth. Interestingly, a rise in 34.2% (104.86 APU/ml) of protease activity was detected at incubation temperature of 50℃ and when culture filtrate was assayed at 60℃, signifying a high temperature stability of the produced protease by this isolate. Additional studies on the enzyme characterization were resulted in recognition of highly significant properties of the activity towards casein at pH 9.0 and stability at high temperature with retention of 96% the enzyme activity at 60℃. The parametric study under feed intervals had enabled improvement in the maximum protease activities attainable from batch cultures in excess of 21.78% and 26.32% via two feeding strategies. A small continual increase in enzyme activity (132.46 APU/ml during 24 h - 120 h) and enhancement in protease production in excess of 36.84% was observed by fed-batch process than the batch experiment.

Green recycling of chicken feather and sheep wool using the partially purified alkaline protease from <i>Bacillus circulans</i>L.

The previously optimized crude alkaline protease from the haloalkaliphilic Bacillus circulans L. was partially purified using ammonium sulphate fractionation and dialysis. The best specific activity (27.7 U/mg protein) was obtained at 80% saturation. The optimum reaction temperature and reaction pH was 47℃ and 9, respectively. The enzyme activity was enhanced with Ca and K chlorides but suppressed with HgCl2 and EDTA. The partially purified protease showed strong proteolytic activity on sheep wool and chicken feather. Also, the enzyme was compatible with the common detergent Tide and could improve its cleaning power in removing blood stain. These findings support the application of the present alka-line protease in biotechnological industries.

Screening of protease-producing marine yeasts for production of the bioactive peptides

Over 400 yeast strains from seawater and sediments were obtained, but only five strains named HN2 -3, N13d, N13C, Mb5 and HN3 - 2 among them could form clear zones around their colonies on the double plates with 2.0% casein. Peptides in the hydrolysate produced by the proteases from strains HN2 -3 and N13d had higher angiotensin I-converting-enzyme （ACE）-inhibitory activity. The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. After purification of the proteases from the two marine yeast strains, it was found that the optimal pH for them was both 9.0, both of them were serine alkaline protease. However, the optimal temperature for the protease from the strain HN2 -3 was 52℃ while that from strain N13d was 48℃. ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2 -3 was the highest while antioxidant activity in the hydrolysate of spirulina protein produced by the purified protease from the strain N13d was the highest.