Improved elastase production by Bacillus sp. EL31410─further optimization and kinetics studies of culture medium for batch fermentation

INTRODUCTION Elastase is an enzyme that attacks and solubi-lizes elastin. As elastase can degrade elastin (Mori-hara, 1967) that other proteases cannot; it has broad applications in medical therapy, food processing and daily use chemicals industry. Considerable eff- orts were made to screen the elastase-producing strains, to study its pathogen effect and its charac-terizations (Tsuzuki and Oka, 1965; Tsai et al., 1988; Sharon et al., 1997; Ozaki and Shiio, 1975). Shiio, 1975). Reporte…     

Improved elastase production by<Emphasis Type="Italic">Bacillus</Emphasis> sp. EL31410 —Further optimization and kinetics studies of culture medium for batch fermentation

An efficient culture medium producing a bacterial elastase with high yields was developed further following preliminary studies by means of response surface method. Central composite design (CCD) and response surface methodology were applied to optimize the medium constituents. A central composite design was used to explain the combined effect of three medium constituents, viz, glucose, K₂HPO₄, MgSO₄·7H₂O. The strain produced more elastase in the completely optimized medium, as compared with the partially optimized medium. The fitted model of the second model, as per RSM, showed that glucose was 7.4 g/100 ml, casein 1.13 g/100 ml, corn steep flour 0.616 g/100 ml, K₂HPO₄ 0.206 g/100 ml and MgSO₄·7H₂O 0.034 g/100 ml. The fermentation kinetics of these two culture media in the flask experiments were analyzed. It was found that the highest elastase productivity occurred at 54 hours. Higher glucose concentration had inhibitory effect on elastase production. At the same time, we observed that the glucose consumption rate was slow in the completely optimized medium, which can explain the lag period of the highest elastase production. Some metal ions and surfactant additives also affected elastase production and cell growth. Project (No. 300024) supported by the Natural Science Foundation of Zhejiang Province, China     

樟芝真菌发酵产三萜培养基优化研究

以L18（37）正交试验设计优化了樟芝真菌发酵产三萜类化合物培养基条件.适宜于樟芝真菌发酵产三萜类化合物的培养基条件为：葡萄糖40,黄豆粉20,酵母膏2,MgSO4 0.2,K2HPO4 1.5,VB1 0.01（g/L）.经验证,在此条件下,樟芝真菌摇瓶发酵后的菌体产量为383.6 mg/L,三萜类化合物为27.85 mg/g.经对比,樟芝真菌菌体产量和三萜类化合物产量不成正比.     

Optimization of Riboflavin Production from ccpA Mutant Bacillus subtilis 24A1/pMX45

Bacillus subtilisis widely used for the productionof vitamins and other products including industrial en-zymes such as amylases, proteases and lipase. Butsyntheses of those products are repressed by the pres-ence of glucose, which is viewed as carbon catabolicrepression (CCR). InBacillus subtilis,CCR is main-ly mediated by the global regulator protein CcpAwhich was encoded byccpAgene[1]. It is therefore of crucial importance to relievingCCR ofBacillus subtilisfor industrial production. I…     

大豆根瘤菌AWCS 13－4菌株培养条件的筛选与优化

研究了大豆根瘤菌AWCS13-4菌株在YMA、TY、PA、BSE 4种培养基中的生长情况,并通过单因素和正交试验确定最佳培养条件。结果表明,AWCS13-4菌株在YMA培养基中生长较好,蔗糖为最佳碳源,酵母粉为最佳氮源,最佳p H为7.0,最佳接种量为3%,最佳培养温度为30℃。在单因素的基础上,采用正交试验,对4个因素进行优化,其最佳培养组合为:每升培养基中添加蔗糖10 g,酵母粉3 g,Mg SO4·7H_2O 0.2 g,K2HPO40.5 g,NaCl 0.1 g,CaCl_2·5H_2O 0.05 g,Rh微量元素液4 m L;p H为7.0,接种量为3%,培养温度为30℃。     

Effective extraction of elastase from Bacillus sp. fermentation broth using aqueous two-phase system

INTRODUCTION Aqueous two-phase system (ATPS) is widely used in biochemistry and biotechnology for purifica- tion of proteins (Diamond and Hsu, 1992; Balasubramaniam et al., 2003), enzymes (Pan et al., 2001; Bim and Franco, 2000), amino acids (Li et al., 2002), antibiotics (Yang et al., 1994; Guan et al., 1994), nucleic acids (Ohlsson et al., 1978; Kimura, 2000), aroma compounds (Marco et al., 2000) and lactic acid (Planas et al., 1998). ATPS is mainly composed of two-incompatible p…     

假丝酵母Candidasp．发酵生产脂肪酶培养条件的优化

研究了碳源和氮源种类、碳源浓度以及起始pH值对假丝酵母Candidasp。菌株发酵生产脂肪酶的影响。结果显示,最适碳源为橄榄油,氮源为酵母膏,橄榄油浓度为2％,起始pH值为6.0。应用响应面试验设计对酵母膏、橄榄油和起始pH值进行培养基优化,结果表明,优化后的培养基配方为：橄榄油2.582％,酵母膏0.276％,K2HPO4 0.1％,MgSO4·7H2O 0.01％,NaCl 0.05％,pH6.28。在此条件下,假丝酵母Candidasp.液体发酵液中脂肪酶的酶活力可达36250U·mL^-1。     

Improvement of xylanase production by Aspergillus niger XY-1 using response surface methodology for optimizing the medium composition

Objective: To study the optimal medium composition for xylanase production by Aspergillus niger XY-1 in solid-state fermentation (SSF). Methods: Statistical methodology including the Plackett-Burman design (PBD) and the central composite design (CCD) was employed to investigate the individual crucial component of the medium that significantly affected the enzyme yield. Results: Firstly, NaNO3, yeast extract, urea, Na2CO3, MgSO4, peptone and (NH4)2SO4 were screened as the significant factors positively affecting the xylanase production by PBD. Secondly, by valuating the nitrogen sources effect, urea was proved to be the most effective and economic nitrogen source for xylanase production and used for further optimization.Finally, the CCD and response surface methodology (RSM) were applied to determine the optimal concentration of each sig-nificant variable, which included urea, Na2CO3 and MgSO4. Subsequently a second-order polynomial was determined by mul-tiple regression analysis. The optimum values of the critical components for maximum xylanase production were obtained as follows: x1 (urea)=0.163 (41.63 g/L), x2 (Na2CO3)=-1.68 (2.64 g/L), x3 (MGSO4)=1.338 (10.68 g/L) and the predicted xylanase value was 14374.6 U/g dry substrate. Using the optimized condition, xylanase production by Aspergillus niger XY-1 after 48 h fermentation reached 14637 U/g dry substrate with wheat bran in the shake flask. Conclusion: By using PBD and CCD, we obtained the optimal composition for xylanase production by Aspergillus niger XY-1 in SSF, and the results of no additional expensive medium and shortened fermentation time for higher xylanase production show the potential for industrial utilization.     

黑皮鸡枞菌液体培养基优化

试验比较不同pH条件及培养基配方对黑皮鸡枞菌丝体（球）生长的影响以探讨最佳液体培养黑皮鸡枞最佳的pH及配方条件．通过预实验筛选出玉米粉、麸皮、大豆粉、K2HPO4、MgSO4为碳源、氮源及微量营养因子;用四因素四水平正交表进行试验,以菌丝体的干物质量为基准比较．结果表明：液体培养条件下,获得最高黑皮鸡枞菌丝体生物量的最佳配方为：麸皮14g／L·玉米粉11g／L·大豆粉2g／L·KH：PO4 1.2g／L·MgSO40．9g／L;最适pH为6．0．     

无机盐对酵母菌产麦角固醇的影响

研究了PO4^3-、Mg^2 和Fe^2 对酵母菌细胞生长及麦角固醇合成的影响。结果表明：3种无机盐在所测试的浓度范围内均能促进酵母菌细胞生长，提高生物量，对麦角固醇的合成也有显著的促进作用。当培养基中分别加入2.0g／L的K2HPO4、1．5g／L的MgSO4和0.05g／L的FeSO4时，刺激麦角固醇合成作用最强，麦角固醇含量分别增加了39．7％、9.5％和35．2％。     

纳豆菌液态产酶条件的优化

实验采用单因素试验优化纳豆菌液体发酵条件。通过蛋白凝块溶解时间法测定纳豆激酶活力,筛选出最佳培养条件。液态发酵选用甘油、乳糖以及木糖与葡萄糖的混合糖代替基础培养基中的麦芽糖;用酵母膏、干酪素、胰蛋白胨和黄豆汁代替基础培养基中的麸皮进行发酵产酶试验,筛选出最佳碳氮源,并在此基础上变换不同的碳、氮源浓度,筛选出最佳的碳、氮比。试验结果表明：液态发酵最佳条件为,甘油10％,酵母膏2％,明胶0．5％,NaCl 0．5％,KH2PO4 0．1％,K2HPO4 0．4％,MgSO4 0．05％,初始pH7．0。在此条件下培养,测得的纳豆激酶活力相当于尿激酶1081．22IU／mL。与此前报道结果800．50IU／mL相比有了明显的提高。     

Enhanced production of elastase by Bacillus licheniformis ZJUEL31410： optimization of cultivation conditions using response surface methodology

Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cul- tures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.     

Influence of medium components on elastase production using crude sources by<Emphasis Type="Italic">Bacillus</Emphasis> sp. EL31410

A newly isolated strain EL31410, producing elastase (E. C3. 4. 4. 7) with high elastolytic activity was identified asBacillus sp. In the medium optimization, it was found that wheat bran and soybean flour hydrosate were the best crude carbon and nitrogen source for enzyme production, respectively. Addition of corn steep flour can affect the bacterium growth and elastase production. A fractional factorial design was applied to study the main factors that affect the enzyme production, and central composite experimental design and response surface methodology were adopted to derive a statistical model for the effect of wheat bran and soybean flour hydrosate on elastase production. The experimental results showed that wheat bran had positive effect but soybean flour hydrosate had negative effect, on enzyme production. An initial concentration of 3.4% (w/v) wheat bran and 9.4% (v/v) soybean flour hydrosate were found to be optimal for enzyme production in batch culture. The time course of elastase production in the optimized medium composition was also described. Project (No. 300024) supported by the Zhejiang Provincial Natural Science Foundation of China     

Optimization of elastolysis conditions and elastolytic kinetic analysis with elastase from Bacillus licheniformis ZJUEL31410

INTRODUCTION Elastases are heterogeneous with differing sub-strate specificities and catalytic mechanisms. In fact, enzymes with elastolytic activity can be found in most of the major proteolytic families, including serine, thiol, aspartic, and metallo-enzymes. Despite the differences in catalytic mechanisms, all of these elastases share a common specificity for cleaving peptide bonds associated with hydrophobic or aro-matic amino acids (Hall and Czerkawaki, 1961). Bacterial elastases b…     

机械化学法合成羟基磷灰石

以CaCO3 和CaHPO4 ·2H2 O为原料 ,研究羟基磷灰石合成过程中混合料在水介质及高速球磨条件下发生的机械化学作用 .混合料球磨 1h可合成结晶良好、含碳酸根及磷酸氢根的羟基磷灰石 ,其化学组成与由骨水泥水化而成的羟基磷灰石相同 ,颗粒呈球状或短棒状 ,粒径分布范围为 2 0— 10 0 nm .     

粗十水碱半干法制备过碳酸钠的研究

采用半干法新工艺,以粗十水碳酸钠为原料替代工业碳酸钠,制备得过碳酸钠产品,可充分利用十水碳酸钠的溶解热使反应物料温度很快从室温(25℃)降至低温(70C—8℃),实现节能降耗。经正交试验优化得适宜的制备工艺条件为每40克粗十水碳酸钠用30%H_2O_216ml,各0.2gEDTA 和 Na_2SiO_3复合使用为内稳定剂,各0.2g,MgSO_4和 Na_2HPO_4复合使用为外稳定剂,产品质量符合部颁合格标准,一次性产率迭89.5%。     

Medium Optimization for Antifungal Active Substance Production from <Emphasis Type="Italic">Streptomyces Lydicus</Emphasis> Using Response Surface Methodology

Response surface methodology was used to optimize the medium for antifungal active substance production from Streptomyces lydicus E12 in flask cultivation. Initially, the component factors, which influence antifungal substance production, were studied by varying one factor at a time. Starch, soybean cake powder, K₂HPO₄·3H₂O and MgSO₄·7H₂O were found to have a significant effect on the production of antifungal substances by the traditional design. Then, a Box–Behnken design was applied for further optimization. A quadratic model was found to fit antifungal active substance production. The analysis revealed that the optimum values of the tested variable were starch 84.96 g/L, soybean cake powder 4.13 g/L, glucose 5 g/L, MgSO₄·7H₂O 1.23 g/L, K₂HPO₄·3H₂O 2.14 g/L and NaCl 0.5 g/L. The test result of 67.44% antifungal inhibition agreed with the prediction and increased by 14.28% in comparison with the basal medium.     

Influence of medium components on elastase production using crude sources by Bacillus sp. EL31410

A newly isolated strain EL31410, producing elastase (E.C3.4.4.7) with high elastolytic activity was identified as Bacillus sp. In the medium optimization, it was found that wheat bran and soybean flour hydrosate were the best crude carbon and nitrogen source for enzyme production, respectively. Addition of corn steep flour can affect the bacterium growth and elastase production. A fractional factorial design was applied to study the main factors that affect the enzyme production, and central composite experimental design and response surface methodology were adopted to derive a statistical model for the effect of wheat bran and soybean flour hydrosate on elastase production. The experimental results showed that wheat bran had positive effect but soybean flour hydrosate had negative effect, on enzyme production. An initial concentration of 3.4%(w/v) wheat bran and 9.4%(v/v) soybean flour hydrosate were found to be optimal for enzyme production in batch culture. The time course of elastase production in the optimized medium composition was also described.     

Effect of temperature on batch elastase production by <Emphasis Type="Italic">Bacillus</Emphasis> sp. EL31410

The production of elastase by Bacillus sp. EL31410 at various temperatures was investigated. In order to study the effect of temperature on elastase fermentation, different cultivation temperatures, ranging from 39 °C to 28 °C, were evaluated in shake flask. The result indicated that 37 °C was best for cell growth at earlier stage; while maximum elastase activity was obtained when the cells were cultivated at 30 °C. This result was verified by batch fermentation in 5-L bioreactor under 37 °C and 30 °C temperature, respectively. The specific cell growth rate at 37 °C was higher than that at 30 °C during earlier stage of cultivation. The maximum value [5.5 U/(h·g DCW)] of elastase formation rate occurred at 24 h at 30 °C compared to 4.6 U/(h·g DCW) at 30 h at 37 °C. Based on these results, two-stage temperature shift strategy and oscillatory temperature cultivation mode were evaluated in the next study. When compared to single temperature of 37 °C or 30 °C, both two-stage temperature shift strategy and oscillatory temperature strategy improved biomass but did not yield the same result as expected for elastase production. The maximum biomass (both 8.6 g/L) was achieved at 30 h at 37 °C, but at 42 h using two-stage temperature cultivation strategy. The highest elastase production (652 U/ml) was observed at 30 °C in batch process. It was concluded that cultivation at constant temperature of 30 °C was appropriate for elastase production by Bacillus sp. EL31410. Project (No. 20276064) supported by the National Natural Science Foundation of China     

凯氏定氮法测定虾壳中氮含量

自组一套简易凯氏定氮装置,对提高装置气密性方法进行研究,并采用单次单因子法对加入双氧水测定虾壳中氮含量的消化条件进行了研究,通过与国标法进行对比,确定该方法测定虾壳中氮含量的可行性。结果表明,加氢氧化钠的漏斗采用水封能提高蒸馏装置的气密性;最佳消化条件为:虾壳0.2 g,五水硫酸铜0.2 g,硫酸钾0.6 g,浓硫酸8 mL,双氧水4 mL,消化40 min。所测虾壳氮含量为(63.48±0.88)mg/g(P=0.95),加标回收率在95%~100%。该法准确度与国标法相当,且省时、经济、环保,适用于实验教学、科研和生产实践。