Maximum Enzyme Activity Research Articles

Safety and efficacy of HOSTAZYM® X (endo-1,4-beta-xylanase) as a feed additive for rabbits for fattening.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of HOSTAZYM ® X as a feed additive for rabbits for fattening. HOSTAZYM ® X contains endo‐1,4‐beta‐xylanase produced by a strain of Trichoderma citrinoviride and is available in liquid and solid formulations. It is authorised as a feed additive for chickens for fattening, turkeys for fattening, laying hens, minor poultry species for fattening and laying, weaned piglets, pigs for fattening and carps. The applicant has now requested to extend the authorisation to rabbits for fattening. The FEEDAP Panel considered that the new use of the additive would not change the previous conclusions regarding the safety for the consumer, user and environment. There are no concerns for consumer safety and no risks for the environment are expected from the use of the additive as a feed additive. Regarding the safety for the user, the additive should be considered a potential skin and eye irritant, and a potential skin and respiratory sensitiser. A subchronic oral toxicity study in rats was submitted to support the safety of the additive when administered to rabbits for fattening. The maximum safe enzyme activity in feed for the rabbits for fattening was derived from the data obtained in the subchronic oral toxicity study in rats; considering the maximum safe intake derived for rabbits from the toxicological data, the FEEDAP Panel concluded that the additive is safe for rabbits for fattening at the recommended use level. Two efficacy studies were submitted to support the efficacy of the additive; a significant effect of the additive in a relevant parameter was found in only one trial. Due to the insufficient evidence the Panel could not conclude on the efficacy of the additive in rabbits for fattening.

Enzyme immobilization on quercetin capped gold and silver nanoparticles for improved performance

Recent development in the field of nanobiotechnology has gained importance due to its industrial applications, wherein the surface modification of nanoparticles with enzymes is of special interest. The enzyme immobilization strategies may advance the overall utility of the enzyme with improved performance. In this study, we illustrate the immobilization of enzyme “α-amylase” onto quercetin capped gold and silver nanoparticles without using any cross-linkers or additional surface alteration. The optimum pH and temperature for maximum enzyme activity for the immobilized amylase were estimated. The optimum pH for enzyme immobilized gold nanoparticles was found to be 6.5 and for silver nanoparticles it was found to be 4.5 and 8, whereas the ideal temperature for maximum amylase activity was 40°C and 50°C for gold and silver nanoparticles, respectively. The nanoparticles immobilized enzyme could retain over 50% of enzyme activity compare to 18% activity of free enzyme after six cycles. The outcome of the study indicates the ability of quercetin to reduce metal ions to form gold and silver nanoparticles with suitable surface corona to anchor amylase, and upon enzyme immobilization these nanoparticles can improve the performance of immobilized amylase toward pH, temperature and reusability compare to free enzyme

Enhanced Production of Salinity-Induced Proteases from <i>Aspergillus flavus</i> and <i>Aspergillus niger</i>

Proteases are important industrial enzymes that account for about 60% of the total enzyme market globally due to their large application in food, feed, textile and pharmaceutical industries. The effect of salt stress on protease production was evaluated on Aspergillus flavus and Aspergillus niger. The enzyme production was enhanced by stepwise optimization of the culture parameters, notably, carbon source, nitrogen source, pH, and temperature of the submerged fermentation process while using a minimal salt media and casein as substrate for the protease activity. The fungi species were found to be good producers of both acid and alkaline proteases under 4% salt stress condition. The optimum culture conditions for alkaline protease production by Aspergillus flavus were sucrose 4%, peptone 1%, pH 8 at 40°C with maximum enzymatic activities of 8.85 mM/min/mg protein, 5.22 mM/min/mg protein, 3.75 mM/min/mg protein, and 1.64 mM/min/mg protein, respectively. Lactose 4%, peptone 1%, pH 6 at 50°C were the optimum culture conditions for acid protease production by Aspergillus flavus with maximum enzymatic activities of 4.59 mM/min/mg protein, 2.06 mM/min/mg protein, 1.24 mM/min/mg protein, and 1.23 mM/min/mg protein, respectively. For Aspergillus niger, the optimum culture conditions for alkaline protease production were corn starch 4%, yeast extract 1%, pH 6 at 40°C with maximum enzymatic activities of 5.99 mM/min/mg protein, 3.85 mM/min/mg protein, 6.18 mM/min/mg protein, and 3.72 mM/min/mg protein, respectively. While lactose 4%, yeast extract 1%, pH 6 at 50°C were the best culture conditions for acid protease production by Aspergillus niger with maximum enzymatic activities of 4.81 mM/min/mg protein, 0.93 mM/min/mg protein, 5.71 mM/min/mg protein, and 3.34 mM/min/mg protein, respectively.

Immobilization of trypsin onto large-pore mesoporous silica and optimization enzyme activity via response surface methodology

Tannic-acid-templated large pore mesoporous silica nanoparticle (LMSN) was synthesized using one-pot procedure and the external surface was further modified with APTES (LMSN-N) to immobilize trypsin. After modification, trypsin was difficult to be immobilized on the outer surface of the LMSN due to the electrostatic repulsion. Naturally, study of the trypsin immobilized in the interior pores could be implemented solely. Meanwhile, the activity of immobilized trypsin was optimized by response surface method (RSM), and the ANOVA was employed to explore the interaction between independent variables. The maximum enzyme activity (12.28 U/mg) was obtained under a certain condition that the quality of T-LMSN-N-272 was 153 μg, the pH was 7.9, and the temperature was 40.4 °C. Simultaneously, the immobilized enzyme was proved to possess better thermal stability, storage stability and recycling performance than free enzyme.

Extracellular Production of a Potent and Chemically Resistant Nattokinase in Immobilized Escherichia coli Using Response Surface Methodology

Nattokinase is a potent fibrinolytic protease, which is used as a drug for treatment or a supplement for preventing thrombosis besides various industrial applications. The present study aimed to produce a soluble nattokinase in low cost media, which has with high activity and resistance to metal ions, detergents, and organic solvents, and can be easily used in medicines or as detergents. Generally, most of the native extracellular proteins, such as nattokinase from Bacillus subtilis, are lysed by secretory proteases. One way for solving this problem is to employ other hosts for nattokinase production. For producing secretory form of nattokinase from Bacillus subtilis, different factors such as a suitable host, optimized media for the maximum enzyme activity and easy purification are important. These factors are studied in this investigation. Escherichia coli BL21 (DE3), as a reliable host was selected for a high yield production of an extracellular recombinant nattokinase. A mature nattokinase gene from Bacillus subtilis 1023, was cloned in the expression vector pET22b by which the host was transformed. The recombinant nattokinase was expressed through induction with IPTG. The expressed protein was confirmed by SDS-PAGE, and its fibrinolytic activity was assayed on the fibrin plates. Afterwards, the enzyme was purified by Ni-NTA native affinity column. Different media components were evaluated for maximum nattokinase production and activity. The highest enzyme activity of 883.107 U/ml was obtained, when a medium approximately consisting of yeast extract (4.38 g/L), tryptone (4 g/L), K2HPO4 (1.61 g/L) and CaCl2 (0.01 g/L) was used. Entrapping the transformed host in calcium alginate could lead to more enzyme activity and decrease media cost.

Effect of lignocellulosic enzymes on the treatment of mature landfill leachate

The inherent necessity to remediate refractory contaminants from the toxic problematic wastewater like mature landfill leachate (MLL) has become a global challenge. This study investigated the effect of a potentially sustainable technological approach, i.e. lignocellulosic enzymatic activities (lignin-peroxidase, manganese-peroxidase and laccase), produced from six selected fungi on the removal efficiency of chemical oxygen demand (COD) and soluble COD (sCOD) from the MLL. The COD/sCOD removal percentage was significantly increased with higher enzymatic activities. Tyromyces chioneus was revealed to be the first ever fungi that produced significant amount of all three enzymes. Penicillium sp. and Tyromyces chioneus were the most effective strains, which removed 66% and 59% of COD, and 64% and 57% of sCOD, respectively. The maximum lignin-peroxidase, manganese-peroxidase and laccase enzymatic activities were 19.3 and 26.9 U/L by Tyromyces chioneus, and 249.8 U/L by Penicillium sp, respectively. It was concluded that lignocellulosic biomass could be a sustainable and advanced biological treatment option to remove refractory components from MLL.

Prospects of using extremely low doses of physical factors impact in food biotechnology

The paper deals with urgent issues of development of resource-saving methods of biotechnological processes intensification. The main purpose of the work is to show the efficiency of using extremely low doses of physical factors impact in food biotechnology. Acoustic and electric treatment was in various modes. Impact capacity did not exceed 10-4 W/kg. The duration of exposure ranged from 5 to 15 min. Barley grains enzymes and lactic acid microorganisms were subjected to treatment. Impact of vibration as a physical factor, its frequency is considered as a priority in controlling growth and biochemical processes in biological objects. Impact frequency in the range of 50–10000 Hz influences the activity of hydrolytic enzymes in bimodal way. It is presented in the article. The coincidence of the frequency ranges of the maximum activity of enzymes in the model reactions with the ones in the grain of barley under the treatment of alternating current and sound is noted. Improvement of all indexes of germinating barley and improvement of the quality of the finished malt were observed in these ranges. Low-intensity acoustic treatment at a frequency of 2000 Hz contributed to an increase in β-galactosidase activity of the CT-95 Str. thermophilus strain. Selected ST-95 Str. thermophilus strain was used in the starter composition for the experimental sample production of fermented melted milk. The fermentation process intensification was observed. The lactose content of this product is 30% less in comparison with the feedstock. The use of traditional starter without a selected strain can reduce the lactose content by an average of 7.5%. The results of biotechnological processes modification by means of the treatment of enzymes and extremely low doses of physical factors impact used in meat technologies are observed. Thus, the prospects of using low-intensity physical effects of vibration in the development of innovative food technologies are substantiated. It is also promising to use these technologies in making absolutely new food products with different qualities.

Production of highly thermo-tolerant laccase from novel thermophilic bacterium Bacillus sp. PC-3 and its application in functionalization of chitosan film

In this study, a novel thermophilic bacterial strain was isolated from Tattapani hot spring located in the Chhattisgarh state of India. The laccase was produced via submerged fermentation and purified by ammonium sulfate precipitation and anion exchange chromatography up to 13.7 fold. The 16S rRNA gene sequence and biochemical analysis revealed that the bacterial isolate is Bacillus sp. strain PC-3. The activity of extracellular crude laccase was determined to be 11.2 U/mL using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate. The SDS-PAGE revealed that the enzyme consists of single subunit with molecular size of 36kDa. The laccase exhibited the maximum enzyme activity at temperature of 60°C and pH 7. Moreover, the laccase retained 99.1% of its original activity for 180min and exhibited half-life of 3.75hat 60°C. Similarly, the laccase retained 95% activity at pH 7 for 240min and displayed significant activity at wider pH range. In addition, the laccase was used for functionalization of chitosan film and characterized for antioxidant and antimicrobial activity. Interestingly, the functionalized chitosan film showed the improved antioxidant and antimicrobial activity.

Metabolic Network Modelling of Chinese Hamster Ovary (CHO) Culture Bioreactors Operated as Microbial Cell Factories

Chinese hamster ovary (CHO) epithelial cells are one of the most used therapeutic medical lines for the production of different biopharmaceutical drugs. They have a high consumption rate with a fast duplication cycle that makes them an ideal biological clone. The higher accumulated amounts of toxic intracellular intermediates may lead to lower organism viability, protein productivity and manufactured biosimilar, so a careful optimal balance of medium, bioreactor operational parameters and bioprocess is needed. A precise phenomenological knowledge of metabolism's chemical transformations can predict problems that may arise during batch, semi-continuous fed batch and continuous reactor operation. For a better detailed understanding (and relations), future performance optimization and scaling, mechanistic model systems have been built. In this specific work, the main metabolic pathways in mammalian structured CHO cultures are reviewed. It starts with organic biochemical background, controlling associated phenomena and kinetics, which govern the sustaining conversion routes of biology. Then, individual turnover paths are described, overviewing standard mathematical formulations that are commonly applied in engineering. These are the core of black box modeling, which relates the substrates/products in a simplified relationship manner. Moreover, metabolic flux analysis (MFA)/flux balance analysis (FBA), that are traditionally characterizing mechanisms, are presented to a larger portion extent. Finally, similarities are discussed, illustrating the approaches for their structural design. Stated variables' equations, employed for the description of the growth in the controllable environmental conditions of a vessel, the researched reaction series of proliferating dividing CHO population, joint with the values of maximal enzymatic activity, and solutions are outlined. Processes are listed in a way so that a reader can integrate the state-of-the-art. Our particular contribution is also denoted.

Characterization, expression and application of a zearalenone degrading enzyme from Neurospora crassa

A gene named zenc, encoding a zearalenone lactonase from Neurospora crassa, was over-expressed in Pichia pastoris. The zenc gene is 888-bp in length, encoding a 295-residue polypeptide. Purified ZENC has maximal activity at pH 8.0 and 45 °C, and is highly stable at pH 6.0–8.0 for 1 h at 37 °C. The activity of the secreted enzyme in shaken-flask fermentation was 40.0 U/ml. A high-density fermentation of the ZENC-producing recombinant strain was performed in a 30-l fermenter and the maximal enzyme activity reached 290.6 U/ml. The Km, Vmax and specific activity toward zearalenone are 38.63 μM, 23.8 μM/s/mg and 530.4 U/mg, respectively. ZENC can resist metal ions and inhibitors to some extent. We applied the enzyme into three different kinds of animal feed. On addition of ZENC (800 U) to distillers dried grains with solubles (DDGS), maize by-products and corn bran (25 g), the concentration of zearalenone was reduced by 70.9%, 88.9% and 94.7% respectively. All these properties of ZENC are promising for applications in the animal feed and food industries.

Enhanced Efficiency of an Enzyme Cascade on DNA-Activated Silica Surfaces.

In nature, compartmentalized and spatially organized enzyme cascades are utilized to increase the efficiency of enzymatic reactions. From a technologically relevant perspective, synthetic enzyme systems have to be optimized with emphasis on enzyme activity, productivity, scalability, and ease of use. But the underlying principles and relevant parameters that lead to an enhancement of the activity of enzyme cascades through spatial organization are still under debate. Here, we report on the 10-fold activity enhancement of the GOx-HRP enzyme cascade for the oxidation of luminol, when the enzymes are colocalized on micron-scaled solid scaffolds. Both enzymes were initially assembled and concentrated on DNA origami rectangles and finally further concentrated on the surface of silica particles. We show that each particular component of the designed system contributes to the activity enhancement. Furthermore, we measured an influence of the silica particle length scale on the total productivity by a factor of 5-10, but to a lesser extent on the maximum enzyme activity. Our findings demonstrate that micrometer-sized scaffolds can be used to enhance the efficiency of enzyme-cascades by at least a magnitude and that solid-phase scaffolds enable scalability for technological applications.

Effect of temperature on growth, digestion and immunity of green, white and purple sea cucumbers,Apostichopus japonicus(Selenka)

Three color morphs (white, green and purple strains) of Apostichopus japonicus (Selenka) were cultured in artificial seawater, for approximately 90 days, in three temperature ranges: 27–22°C (high), 22–17°C (mid) and 17–12°C (low). All strains grew in all temperature ranges. Temperature significantly affected growth rate, digestibility, digestive enzymes and immune-related enzymes. Highest specific growth rates were exhibited in 4-month-old sea cucumbers at mid and high temperatures, and in 16-month-old sea cucumbers at mid and low temperatures. Specific growth rates of green and purple strains were not significantly different, but were significantly higher than that of the white strain at mid temperatures. The digestibility of each strain was significantly higher at 27°C, 22°C and 17°C than at 12°C. Green-strain digestibility was higher than that of purple and white strains at specific temperatures. Protease and amylase activities of all strains followed bell-shaped temperature curves with maximum digestive enzyme activity at 17°C. The activities of alkaline and acid phosphatases were higher in the guts of the green strain than in the white or purple strains at the same temperature. Superoxide dismutase activity was higher in the purple strain than in white and green strains.

Effects of Royal Jelly Administration on Endurance Training-Induced Mitochondrial Adaptations in Skeletal Muscle.

We investigated the effect of royal jelly (RJ), a natural secretion from worker bees, on the endurance training-induced mitochondrial adaptations in skeletal muscles of ICR mice. Mice received either RJ (1.0 mg/g body weight) or distilled water for three weeks. The mice in the training group were subjected to endurance training (20 m/min; 60 min; 5 times/week). There was a main effect of endurance training on the maximal activities of the mitochondrial enzymes, citrate synthase (CS), and β-hydroxyacyl coenzyme Adehydrogenase (β-HAD), in the plantaris and tibialis anterior (TA) muscles, while no effect of RJ treatment was observed. In the soleus muscle, CS and β-HAD maximal activities were significantly increased by endurance training in the RJ-treated group, while there was no effect of training in the control group. Furthermore, we investigated the effects of acute RJ treatment on the signaling cascade involved in mitochondrial biogenesis. In the soleus, phosphorylation of 5′-AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were additively increased by a single RJ treatment and endurance exercise, while only an exercise effect was found in the plantaris and TA muscles. These results indicate that the RJ treatment induced mitochondrial adaptation with endurance training by AMPK activation in the soleus muscles of ICR mice.

Optimization of Enzymatic Hydrolysis of Viscera Proteins of Rainbow Trout (<em>Oncorhynchus mykiss</em>)

This study aimed to optimize the enzymatic hydrolysis of rainbow trout ( Oncorhynchus mykiss) viscera with the enzyme alcalase® 2.4 L to found the highest the Degree of Hydrolysis (DH). In vitro evaluation of the optimum conditions (pH and temperature) was performed to maximize the Proteolytic Activity (PA) of alcalase® 2.4 L with the rainbow trout viscera. The optimal conditions for maximum enzymatic activity were a pH of 8.5 and a temperature of 60°C. Response Surface Methodology (RSM) was used to evaluate the effect of the enzyme/substrate ratio (10-30%), substrate concentration (2.0-6.0%) and temperature (45-65°C) on DH. A substrate concentration of 5.53% and an enzyme/substrate level of 30% were found to be the optimum conditions to obtain the highest DH (27.6%). The Michaelis-Menten plot indicated that these conditions were not in the saturation area.

Genome-wide analysis of superoxide dismutase genes in Larix kaempferi

Superoxide dismutase is a key enzyme that scavenges superoxide anion and plays vital roles in plant antioxidant system. This study identified six SOD genes from the deciduous conifer Larix kaempferi, which is widely distributed across the cooler regions of the northern hemisphere. These six SOD genes were classified into three types: Cu/Zn-SOD (LkSOD1, 2, 3 and 4), Fe-SOD (LkSOD5) and Mn-SODs (LkSOD6). Three Cu/Zn-SOD proteins (LkSOD1, 3 and 4) were cytosolic-localized, while the other three proteins (LkSOD2, 5 and 6) were chloroplast-localized. Larix SOD proteins displayed catalytic activities toward superoxide anion, and retained >55% of its maximum enzymatic activity between 10 °C and 40 °C. Over expressions of three Larix SOD genes (LkSOD2, 4 and 6) in Arabidopsis thaliana, respectively, showed increased germination rates and root lengths during salt stress. LkSOD5 gene could rescue pale green and dwarf phenotype of Arabidopsis atfsd2-2 mutant. Taken together, this study provided comprehensive insight into the gymnosperm SOD gene family.

Optimization of Cellulase Production by Aspergillus niger ITBCC L74 with Bagasse as Substrate using Response Surface Methodology

Cellulase is a very important enzyme for lignocelluloses based ethanol production. Bagasse contains mainly cellulose (57.76%), hemicellulose (12.44%), lignin (21.34%), and others (7.96%). Lignocellulosic material has been considered as the good option for cellulase production because it is cheap and already available in a huge amount. The objective of this research was to produce cellulase enzyme and to optimize it by using response surface methodology. The bagasse with water content of 80% was incubated with 2 ml inoculum of Aspergillus niger ITBCC L74 in a 250 ml Erlenmeyer flask. After reaching the specified time the enzyme was extracted and then determined for its activity. Effect of process parameters such as pH, urea and MgCl2 addition were studied. The optimal cellulase activity was achieved at urea concentration of 4.5% (w/w), MgCl2 concentration of 1 mM and pH of 3.5, with maximum enzyme activity was 0.630 U/gr.

Thermotolerant lipase from Penicillium sp. section Gracilenta CBMAI 1583: Effect of carbon sources on enzyme production, biochemical properties of crude and purified enzyme and substrate specificity

In this study, Penicillium sp. section Gracilenta CBMAI 1583 was used to produce lipase under submerged conditions. The enzyme was purified and the biochemical properties of both the crude and purified enzymes were evaluated. Maximum lipase production (1.62 U mL−1) was obtained using olive oil 0.5% (w v−1) after 72 h of cultivation, representing a 90% increase in the lipase initially produced. The enzyme was purified using hydrophobic interaction chromatography (phenyl Sepharose) under conditions which allowed its interfacial activation. The partially purified sample showed an enzyme with esterase activity (65.4 kDa) on α- and β-naphthyl acetate and other with lipase activity (52.9 kDa) on octyl oleate. Optimum activity of crude and purified lipase was observed at pH 4.0 and 70 °C. The purified lipase was activated by NaCl, BaCl2, NH4Cl, MnSO4 and MgSO4; it also presented high stability in organic solvents such as hexane, 2.2.4-trimethylpentane, acetone, DMSO and toluene. Maximum enzyme activity was observed with p-nitrophenyl decanoate as substrate; and the enzyme kinetics showed to be directly affected by Triton X-100. The enzyme shows potential application in processes that operate in acid pH, such as treatment of dairy and industry effluents, resolution of esters in the pharmaceutical industry or in the food industry, as well in synthesis reaction under non-aqueous conditions.

Enzymatic CO2 reduction to formate by formate dehydrogenase from Candida boidinii coupling with direct electrochemical regeneration of NADH

Enzymatic conversion of CO2 to formate was carried out in the cathodic cell of a two-chamber electrochemical apparatus where NAD+ was reduced on the surface of a Copper foam electrode. Formate dehydrogenase (FDH) was used as the biocatalyst in both free form and immobilized on the modified electrospun polystyrene nanofibers (EPSNF). The fabricated EPSNF were modified by a multistage procedure including acid treatment, silanization followed by activation with glutaraldehyde. The effects of regenerated NADH concentration and time of enzymatic reaction on the formate production in the both systems were studied. The results indicated that the EPSNF immobilized FDH had a desirable activity, long-term storage stability (41% after 20 days) and reusability after eight cycles of successive reactions (53% of the initial activity). Moreover, it was revealed that the increase of cofactor concentration at the early times of reaction was favorable to the formate production. However, an inhibitory effect was observed at higher concentrations of NADH, and the optimum values of 0.45 mM and 0.51 mM were obtained for the maximum enzyme activity by the free and immobilized enzymes respectively. The produced formate at the optimum cofactor concentration after 300 min was 0.61 mM and 0.31 mM for the free and immobilized enzyme systems. Finally, it can be concluded that the presented process is a promising approach to the enzymatic conversion of CO2.

Effect of Physical and Chemical Factors in Production of Alkaline Protease Enzyme by <i>Bacillus</i> Strains

Proteases is family of enzymes and it has crucial role due to their physiological roles and very valuable commercial applications. Alkaline protease are produced by Bacillus species are particular importance because of their thermal stability and stability at different pH values. This study aimed to investigate the effect of physical and chemical factors in production of alkaline protease enzyme fermentation by members of the genus Bacillus. In this study, alkaline protease enzyme production were evaluated in submerged fermentation by Bacillus strains which were isolated from alkaline soils of Guilan province. Factors incubation were optimized such as time, pH, amount of inoculation and ammonium sulfate in alkaline protease enzyme production whit using response surface methodology (RSM) in culture. The maximum enzymatic activity was observed in incubation time of 36 hours, pH=9, inoculation amount of 15% (V) and ammonium sulfate 1.5% (W/V). Factors had significant effect on the production of alkaline protease enzyme such as pH and ammonium sulfate.

Effect of Physical and Chemical Factors in Production of Alkaline Protease Enzyme by <i>Bacillus</i> Strains

Proteases is family of enzymes and it has crucial role due to their physiological roles and very valuable commercial applications. Alkaline protease are produced by Bacillus species are particular importance because of their thermal stability and stability at different pH values. This study aimed to investigate the effect of physical and chemical factors in production of alkaline protease enzyme fermentation by members of the genus Bacillus. In this study, alkaline protease enzyme production were evaluated in submerged fermentation by Bacillus strains which were isolated from alkaline soils of Guilan province. Factors incubation were optimized such as time, pH, amount of inoculation and ammonium sulfate in alkaline protease enzyme production whit using response surface methodology (RSM) in culture. The maximum enzymatic activity was observed in incubation time of 36 hours, pH=9, inoculation amount of 15% (V) and ammonium sulfate 1.5% (W/V). Factors had significant effect on the production of alkaline protease enzyme such as pH and ammonium sulfate.