In Silico Study of Alkaline Serine Protease and Production Optimization in Bacillus sp. Khoz1 Closed Bacillus safensis Isolated from Honey

International Journal of Peptide Research and Therapeutics - Protease is a kind of enzyme with different applications in medicine and industry. The limitation of the enzymes in industry is lack of...     

产碱性蛋白酶嗜碱芽孢杆菌的筛选及其研究

利用造纸黑液对土样进行富集,筛选出3株碱性蛋白酶酶活力较高的嗜碱芽孢杆菌X1、X2、X3。对它们的生长曲线,产酶曲线,在不同C、N源、pH值、盐浓度下的产酶活力进行的研究表明:3株嗜碱芽孢杆菌(Bacillussp.JBX1、X2、X5)酶活力较高(达到100U/mL),X2最高酶活可达140U/mL。最适pH值为9.5,碳源中的蔗糖,氮源中的酵母浸提物和硝酸钠均利于产酶。X1、X5两株嗜碱芽孢杆菌均表现出较强的耐盐耐高渗透压的能力。X1在11%的NaCl浓度下生长良好,酶活仍然达到80U/mL以上。而X2和X5对温度的耐受性比较强,在经70℃处理15min后依然保持了80%以上的酶活力,所产蛋白酶为高温碱性蛋白酶,从而为进一步的应用和研究奠定了基础。     

地衣芽孢杆菌D-1产碱性蛋白酶培养条件的优化

为了对产碱性蛋白酶的地衣芽孢杆菌D-1的培养条件进行优化,利用10 L发酵罐,采用正交设计19(34)试验,对培养温度、pH值、搅拌转速、通气量4条件进行优化,得到地衣芽孢杆菌D-1发酵产碱性蛋白酶的最优培养条件为:培养温度37.0℃,pH值7.5,通气量4L/min,搅拌转速300r/min.利用最优条件组合进行验证...     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。     

Biodegradation of Shrimp Biowaste by Marine Exiguobacterium sp. CFR26M and Concomitant Production of Extracellular Protease and Antioxidant Materials: Production and Process Optimization by Response Surface Methodology

Twelve marine bacterial cultures were screened for extracellular protease activity, and the bacterium CFR26M which exhibited the highest activity on caseinate agar plate was identified as an Exiguobacterium sp. Significant amount of extracellular protease (5.9?±?0.3 U/ml) and antioxidant materials, measured as 2,2′-diphenyl picrylhydrazyl (DPPH) radical scavenging activity (44.4?±?0.5 %), was produced by CFR26M in submerged fermentation using a shrimp biowaste medium. Response surface methodology (RSM) was employed to optimize the process variables for maximum production of protease and antioxidant materials by CFR26M. Among the seven variables screened by two-level 2**(7–2) fractional factorial design, the concentration of shrimp biowaste, sugar, and phosphate was found to be significant (p?≤?0.05). The optimum levels of these variables were determined by employing the central composite design (CCD) of RSM. The coefficient of determination (R ²) values of 0.9039 and 0.8924 for protease and antioxidant, respectively, indicates the accuracy of the CCD models. The optimum levels of shrimp biowaste, sugar, and phosphate were 21.2, 10.5, and 2.3 % (w/v) for production of protease and 28.8, 12, and 0.32 % (w/v) for production of antioxidant material, respectively. The concentration of shrimp biowaste, sugar, and phosphate had linear and quadratic effect on both protease and antioxidant productions. RSM optimization yielded 6.3-fold increases in protease activity and 1.6-fold in antioxidant material production. The crude protease of CFR26M had a maximum activity at 32?±?2 °C with pH 7.6. This is the first report on the use of marine Exiguobacterium sp. for concomitant production of protease and antioxidant materials from shrimp biowaste.     

丛梗孢酵母发酵产赤藓糖醇的响应面优化

为了提高丛梗孢酵母发酵产赤藓糖醇的产量,在前期单因素实验结果的基础上,利用Plackett-Burman实验设计对影响其产赤藓糖醇的发酵条件进行评估并筛选出了影响显著的3个因素:葡萄糖、初始pH和温度.采用响应面法进行实验方案设计,利用SAS软件对其结果进行二次回归分析,确定了优化后的发酵条件为:葡萄糖260g/L、酵...     

固定化芽孢菌产碱性蛋白酶的研究

本文报导了用海藻酸钙固定地衣芽孢杆菌(Bacilluslicheniformis),发酵生产碱性蛋白酶的研究。固定化细胞颗粒在低浓度的玉米粉、黄豆饼粉为原料的培养基中发酵24h,酶活高达1724u/ml,充分利用并节省了原材料,缩短了发酵周期。发酵液菌浓度的测定结果表明,固定化细胞凝胶粒细菌的渗漏程度较低,有利于提取工艺的简化。     

Production of Alkaline Protease from n-Paraffins by a Kabicidin Resistant Mutant Strain of Fusarium sp.

An antagonistic fungus to Valsa ceratosperma was isolated from soil, and identified as Fusarium solani. The fungus was found to produce at least two antifungal substances in stationary culture. These two substances were isolated from their culture filtrate as chromatographically homogeneous, amorphous solids. Their examined physico-chemical properties appeared to be identical with cyclosporin A and C, obtained from the fermentation broth of Trichoderma polysporum, and they showed a pronounced inhibitory effect on growth of V. ceratosperma.     

Production and Purification of Alkaline Protease Inhibitors of Streptomyces sp.

N-Acetyl-d-glutamate deacetylase and N-acetyl-d-aspartate deacetylase were found in cell extracts from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. N-Acetyl-d-glutamate deacetylase was produced inducibly by N-acetyl-d-glutamate and was highly specific to N-acetyl-d-glutamate. N-Acetyl-d-aspartate deacetylase was produced inducibly by N-acetyl-d-aspartate and was highly specific to N-acetyl-d-aspartate.     

Ectoine Production by Halomonas boliviensis: Optimization Using Response Surface Methodology

Two cultivation steps were used for production of biomass and ectoine by Halomonas boliviensis, respectively. The optimization of some nutrient parameters in each step was investigated by using response surface methodology. Twenty and 12 experiments were performed to attain optimal conditions for biomass and ectoine production, respectively. The model predicted a maximum biomass concentration of 3.34 g/L on optimization of NH₄Cl, K₂HPO₄, and MgSO₄•7H₂O concentrations during the first cultivation, while a maximum ectoine concentration of 1.27 g/L was predicted on optimizing NaCl and monosodium glutamate concentrations in the second cultivation. The experimental values obtained (3.36 g biomass/L and 1.25 g ectoine/L) were in good agreement with the predicted values. The optimized conditions were also used for two-step 1.5-L fed-batch fermentations. In the first step, biomass concentration of 28.7 g/L was obtained while in the second step biomass concentration increased to 63 g/L. Ectoine concentration of 9.2 g/L was obtained, and the overall ectoine productivity was 6.3 g/L/day, being among the highest reported so far.     

Optimization of Protease Extraction from Horse Mango (Mangifera foetida Lour) Kernels by a Response Surface Methodology

The thermophilic protease aqualysin I (AQI) gene (aqul), derived from Thermus aquaticus YT-I, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JMI09 (DE3), one carrying and one lacking an F’ episome, which carries the lacI_q gene. Upon cultivation the strain carrying an F’ episome produced AQI as an insoluble fusion protein (74 kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F’ episome was used as a host cell for aqul expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.     

侧孢短芽胞杆菌产生线虫侵染性蛋白酶的优化

采用单因素试验确定侧孢短芽胞杆菌G4产线虫侵染性蛋白酶的最佳碳氮源,通过Placket-Burman设计筛选影响蛋白酶活力的主效因子,最陡坡试验和Box-Behnken设计获得主效因子的最佳水平,建立线虫侵染性蛋白酶的最佳生产体系:葡萄糖9.78 g/L、牛肉膏16.65 g/L、磷酸氢二钾0.75 g/L、可溶性淀粉12.5 g/L、氯化钠0.75 g/L、硫酸镁0.5 g/L、初始pH值自然、装液量50 mL,37℃摇瓶培养32 h,蛋白酶活力可达12 379.41 U/mL,较优化前的2 476.3 U/mL提高了4倍。     

Optimization of Ethanol Production From Microfluidized Wheat Straw by Response Surface Methodology

In this study, wheat straw was pretreated with a microfluidizer to improve its enzymatic hydrolysis and ethanol yields. The pretreatment was performed at various pressures (500, 1000, and 1500 bar) and solid loadings (1, 2, and 3%). The microfluidized biomass was then subjected to hydrolysis and simultaneous saccharification and co-fermentation (SSCF) experiments at different enzyme loadings (5, 10, and 15 FPU/g dry wheat straw) using a mutant yeast. The results indicated that the microfluidization method alters the structure of biomass and leads to a reduction in lignin content. The samples pretreated at 1% solid loading contained the minimum lignin concentration and provided the maximum sugar and ethanol yields. These results signified that the microfluidization method is more effective on biomass at low solid loadings. The process conditions were optimized for higher ethanol and sugar yields using response surface methodology (RSM). The optimum pressure and solid and enzyme loadings were found as 1500 bar, 1%, and 15 FPU/g dry wheat straw, respectively. The yields obtained at this condition were 82%, 94%, and 65% for glucose, xylose, and ethanol, respectively. High sugar yields implied that microfluidization is an effective pretreatment method for cellulosic ethanol production. On the other hand, low ethanol yield may indicate that the microorganism was sensitive to inhibitory compounds present in the fermentation medium.     

Optimization of Protease Extraction from Horse Mango (Mangifera foetida Lour) Kernels by a Response Surface Methodology

Protease is one of the most important industrial enzymes with a multitude of applications in both food and non-food sectors. Although most commercial proteases are microbial proteases, the potential of non-conventional protease sources, especially plants, should not be overlooked. In this study, horse mango (Mangifera foetida Lour) fruit, known to produce latex with a blistering effect upon contact with human skin, was chosen as a source of protease, and the effect of the extraction process on its protease activity evaluated. The crude enzyme was extracted from the kernels and extraction was optimized by a response surface methodology (RSM) using a central composite rotatable design (CCRD). The variables studied were pH (x(1)), CaCl(2) (x(2)), Triton X-100 (x(3)), and 1,4-dithryeitol (x(4)). The results obtained indicate that the quadratic model is significant for all the variables tested. Based on the RSM model generated, optimal extraction conditions were obtained at pH 6.0, 8.16 mM CaCl(2), 5.0% Triton X-100, and 10.0 mM DTT, and the estimated response was 95.5% (w/w). Verification test results showed that the difference between the calculated and the experimental protease activity value was only 2%. Based on the t-value, the effects of the variables arranged in ascending order of strength were CaCl(2)< pH < DTT < Triton X-100.     

Optimization of Phosphatidylinositol-specific Phospholipase C Production Using Response Surface Methodology

Summary Response surface methodology was employed in optimizing the nutrient levels needed towards the optimal production of phosphatidylinositol-specific phospholipase C enzyme by Bacillus thuringiensis serovar. kurstaki. A 2³ factorial central composite experimental design was used. The multiple regression equation, relating the enzyme activity to the nutrient medium, was used to find the optimum values of glucose, peptone and dipotassium hydrogen phosphate. The optimum values of these variables for maximal enzyme production were found to be: glucose, 6.5 g l⁻¹; peptone, 5.38 g l⁻¹ and dipotassium hydrogen phosphate, 6.36 g l⁻¹ with the predicted enzyme activity of 0.96 U ml⁻¹.     

利用响应面法优化Bacillus natto TK-1产脂肽发酵培养基

用响应面法对Bacillus natto TK-1发酵产脂肽的培养基进行了优化。首先用Plackett-Burman法筛选出三个影响较大的重要因素,分别为：蛋白胨、酵母粉、CaCl2 ,然后进行最陡爬坡实验逼近最佳响应面区域,最后通过Box-Behnken设计,利用SAS软件进行回归分析,得到各因素的最佳浓度。在优化培养基条件下,发酵液的溶血圈直径较初始培养基提高了29.3 %,脂肽的产量提高了约30%。     

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.     

Production of Alkaline Protease from Acetic Acid

The use of acetic acid as a carbon source in alkaline protease fermentation was examined. Acetic acid was a good carbon source and yielded a great deal of alkaline protease.

Acetic acid has advantages over ordinary carbon sources such as starch and glucose in that it can be supplied to culturing liquid as much as needed to perform the fermentation efficiently, that it has a function to control the pH of culturing liquid at a constant level and that it was obtained at lower price.

The maximum proteolytic activity attained was 1.6 × 10⁴ units/ml (11.4 mg-enzyme/ml).