Purification and characterization of keratinase from a new Bacillus subtilis strain

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50℃ was 8.5 and the optimum temperature at pH 8.5 was 55℃. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT),mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO)stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.     

Effect of cultivating conditions on α-galactosidase production by a novel Aspergillus foetidus ZU-G1 strain in solid-state fermentation

The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain Aspergillusfoetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60%initial moisture of medium, 28 ℃ incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0～6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2037.51 U/g dry matter near the 144th hour of fermentation.     

Effect of cultivating conditions on α-galactosidase production by a novel Aspergillusfoetidus ZU-G1 strain in solid-state fermentation

The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain ,Aspergillusfoetidus ZU-GI in solid-state fermentation （SSF）. Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60% initial moisture of medium, 28 ℃ incubation temperature, 18^h cultivation period of seed, 10% inoculum volume, 5.0-6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2037.51 U/g dry matter near the 144th hour of fermentation.     

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.     

Optimization of cultural conditions for thermostable β-1, 3-1, 4-glucanase production byBacillus subtilis ZJF-1A5

The optimization of cultural conditions for β-glucanase production byBacillus subtilis ZJF-1A5 was investigated in flask trials. Temperature had great effect on β-glucanase production which maximized at optimal temperature of 37°C and decreased significantly when temperature was over 37°C. Charge quantity affected β-glucanase production significantly. Adding oxygen vector N-dodecane or acetic ether benefited β-glucanase production, but it depended on the concentration and charge quantity. The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting β-glucanase production and the cultivation time span to reach the highest β-glucanase activity. The optimal cultural conditions for β-glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82% (v/v)), shaking speed 210 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37°C for 50 h. Repeated experimental results accorded with those predicted by a second-order polynomial model. The amount of β-glucanase, α-amylase and neutral protease produced byB subtilis ZJF-1A5 was associated partially with cell growth. Those three enzymes' activities increased following the cell growth and increased significantly when cells entered the stationary phase. Project (No. B0608) supported by the National Natural Science Foundation of China and a grant (2001121B25) from Hangzhou Science and Technology Development Project of Zhejiang Province, China     

纤维素酶产生菌CMC-Z的筛选及产酶条件的优化

用刚果红染色羧甲基纤维素钠平板筛选方法,从土壤中筛选到一株具有高效产纤维素酶的菌株CMC-Z,经菌落形态观察及镜检,初步鉴定为放线菌.采用单因素及正交设计等试验对CMC-Z培养基、发酵条件进行了优化,碳源、氮源最佳添加量分别为可溶性淀粉1%、明胶2%;最佳发酵条件：接种量7.5%,培养基初始pH 7.0,装量100 mL,培养温度30℃,培养时间72 h.通过方差和极差分析,表明影响菌株CMC-Z纤维素酶产生的因素主次顺序为：可溶性淀粉浓度〉初始pH〉装量〉明胶浓度〉接种量〉温度〉发酵时间.经优化后,其酶活可达到38.37 u.mL-1.     

Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design

研究目的：优化获得血红密孔菌（P.sanguineus）的最佳培养基组成,提高耐热漆酶的产量。创新要点：获得了目前文献报道的最高水平的漆酶活力。研究方法：通过单因素试验研究了不同培养基（番茄汁、麦麸、麦芽提取物和葡萄糖细菌蛋白胨培养基）和不同组合诱导剂（大豆油、阿魏酸、没食子酸、二甲基苯胺、酸性蓝62和活性蓝19分别与硫酸铜组合诱导剂）对P.sanguineus产耐热漆酶的影响。在此基础上采用中心组合试验设计,进一步研究了番茄汁培养基结合硫酸铜和大豆油组合诱导剂对P.sanguineus产耐热漆酶的影响。利用SAS10.0和响应面分析方法对试验结果进行了统计分析和建立回归模型。重要结论：通过中心组合设计优化得出P.sanguineus产耐热漆酶的最优培养基条件：以36.8%番茄汁为培养基,以3 mmol/L硫酸铜和1%大豆油作为组合诱导剂。该条件下在10 L搅拌槽生物反应器中漆酶活力达到了143 000 IU/L（2,2＇-联氮双（3-乙基苯并噻唑啉-6-磺酸）为底物,pH值为3.0）。     

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

ＩＮＴＲＯＤＵＣＴＩＯＮＡｎｉｎｃｒｅａｓｉｎｇｄｅｍａｎｄｆｒｏｍｃｏｎｓｕｍｅｒｓｆｏｒｈｅａｌｔｈｙａｎｄｌｏｗｃｏｓｔｍｅｄｉｃａｌｓｈａｓｌｅｄｔｏｔｈｅｄｅ ｖｅｌｏｐｍｅｎｔｏｆａｌｔｅｒｎａｔｉｖｅｍｅｄｉｃａｌｔｈｅｒａｐｙａｇｅｎｔｓｕ     

锦纶6粗旦中弹DTY丝的开发

国内生产锦纶6DTY的企业较多，但100D以上的DTY的生产却是个空白。通过对前、后纺各项工艺参数的摸索，成功地在原绦纶纺丝装置上生产出锦纶6粗旦中弹DTY丝。     

Improved production of spiramycin by mutant Streptomyces ambofaciens

INTRODUCTION which the hydroxyl group is acetylated, and spi- ramycin III in which the same position is propio- Spiramycin belongs to the macrolide antibi- nylated (Omura et al., 1979). The addition of short-otics group. The molecular structure of spiramycin chain fatty acids stimulates the production of spi-consists of a 16-membered branched lactonic ring …     

Improved production of spiramycin by mutant Streptomyces ambofaciens

Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S. ambofaciens XC 2-37 were studied. The potency of S. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation. The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.     

高产柠檬酸突变菌株以玉米粉为原料的生物反应器扩大生产研究

研究目的：研究高产黑曲霉突变菌株以玉米粉为原料的生物反应器扩大发酵,以期获得适合于工业化生产柠檬酸的发酵工艺。 创新要点：以玉米粉为原料,系统地研究了筛选得到的高产菌株在50L生物反应器中不同糖浓度发酵生产柠檬酸的特性,最终优化出适合于工业化生产柠檬酸的发酵工艺。 研究方法：（1）利用淀粉酶对粉碎后的玉米进行液化,然后过滤,最终得上清液;（2）以50L生物反应器作为发酵设备,对筛选得到的高产柠檬酸菌株进行扩大培养;（3）通过测定不同培养时期中积累的柠檬酸含量和剩余的残总糖,最终优化出高效率生产柠檬酸的发酵工艺。 重要结论：以不同糖浓度的液化玉米粉上清液作为碳源,突变菌株H4002能积累177．7--196．0g,L的柠檬酸,效率能达到2．96～3．27g/（L·h）,尤其当糖浓度为210g／L,H4002菌株表现出最佳的柠檬酸生产水平,如柠檬酸积累187．5g/L,生产效率达3．13g／（L·h）。上述结果说明了突变菌株H4002拥有快速生产柠檬酸的能力。     

Effects of proteinase a on cultivation and viability characteristics of industrial Saccharomyces cerevisiae WZ65

Proteinase A (PrA), encoded by PEP4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. It was observed that the lag phase of cell growth of partial PEP4 gene deletion mutant (36 h) and PrA-negative mutant (48 h) was significantly ex-tended, compared with the wild type strain (24 h) (P<0.05), but PrA had no effect on glucose metabolism either under shaking or steady state cultivations. The logistic model was chosen to evaluate the effect of PrA on S. cerevisiae cell growth, and PrA was found to promote cell growth against insufficient oxygen condition in steady state cultivation, but had no effect in shaking culti-vation. The effects of glucose starvation on cell growth of partial PEP4 gene deletion strain and PrA-negative mutant were also evaluated. The results show that PrA partial deficiency increased the adaption ofS. cerevisiae to unfavorable nutrient environment, but had no effect on glucose metabolism under the stress of low glucose. During heat shock test, at 60 ℃ the reduced cell viability rate (RCVR) was 10% for the wild type S. cerevisiae and 90% for both mutant strains (P<0.01), suggesting that PrA was a negative factor for S. cerevisiae cells to survive under heat shock. As temperatures rose from 60 ℃ to 70 ℃, the wild type S. cerevisiae had significantly lower relative glucose consumption rate (RGCR) (61.0% and 80.0%) than the partial mutant (78.0% and 98.5%) and the complete mutant (80.0% and 98.0%) (P<0.05), suggesting that, in coping with heat shock, cells of the PrA mutants increased their glucose consumption to survive. The present study may provide meaningful information for brewing industry; however, the role of PrA in industrial S. cerevisiae physiology is complex and needs to be further investigated.     

益生素生产菌——产乳酸芽孢杆菌JY-LZ培养条件的优化

摇瓶中对益生素生产菌———产乳酸芽孢杆菌JY-LZ培养条件进行了研究,在发酵培养基培养时,得到最适培养条件为接种量5%~10%,温度30~40℃,灭菌前pH值7~9,100 mL摇瓶装液量30 mL,48 h培养密度大于1010/mL,芽孢率大于50%,可以进一步开发应用于生产实际.     

吉林省体育产业发展状况的调查研究

阐述了吉林省体育产业现状和发展状况,并针对当前吉林省在体育产业发展过程中存在的一些问题,提出了体育产业化改革、经营、政策扶植、人才培养、发挥地域特色等措施,为吉林省体育产业的发展提供了参考。     

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.     

明清时期江南与福建广东的经济联系

明清时期,江南与广东福建两大经济区之间的经济发展,有着极强的互补性.江南需要广东或福建的木材、纸张、染料、烟叶、食糖、花果、铁货、洋货、南货等商品,广东或福建需要江南的棉花、棉布、生丝、丝绸等大宗工业原料和产品.通过经由浙东或江西或安徽的三条内地商道和沿海的一条商道,江南与广东福建之间保持着极为密切的经济联系,而且华南与华北之间的商品流通,通常经由江南转输,从广州出口在国际市场享有盛誉的丝绸、棉布等大宗商品,也主要来自江南地区.而沟通两大区域之间经济联系的主体,来自广东福建各地的地域商帮有着压倒其它地域商帮的绝对优势.     

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     

烤烟红花大金元巨型变异株系的发现、选育及可用性研究

2006年9月在昆明市安宁禄脿乡种植的红花大金元品种田间发现了一棵巨型变异株,经培育次年获得首批种子,2008~2010年在昆明烟区多点多年开展了系统选育、性状考察、区域试验、配套生产技术及烘烤特性探索、抗病性鉴定、生产示范和种子繁殖技术等一系列试验研究.经过多年系统选育,该新品系性状独特优异、适应性较广、农艺性状可调控阈值大、产量产值高、烟叶品质良好、可用性强,多项指标已近似或超过其亲本红花大金元,被命名为YK01.根据其研究利用现状,重点探讨了品种特征特性、烟叶质量、栽培烘烤技术、植保技术和种子繁殖技术等5个方面,为进一步开发利用提供科学依据与参考.     

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