产弹性蛋白酶菌种的筛选及发酵条件优化研究

从合肥肉联厂附近的土壤和污水中分离得到19株产弹性蛋白酶菌株,初步鉴定该菌株属于假单胞菌属.经过发酵复筛有三株产酶能力超过15u/mL.实验对菌株最佳产酶发酵条件进行了优化2%干酪素、0.5%葡萄糖、0.4%酵母膏、0.2% K2HPO4、0.01% MgSO4·7H2O;起始pH值7.0;最适发酵温度为30℃;装液量为25mL/250mL;该菌株在28h左右产弹性蛋白酶的量达18u/mL.     

纹藤壶附生菌的分离鉴定及蛋白酶菌株的产酶性质研究

目的：研究纹藤壶附生菌的分离鉴定及蛋白酶菌株的产酶性质。方法：黄渤海潮间带海域纹藤壶中分离到54株菌株,经酪蛋白酶实验,筛选得到4株高产蛋白酶的菌株,并对其进行菌种鉴定、产蛋白酶的发酵条件优化及产酶活性测定。结果：经筛选,得到4株高产蛋白酶的菌株,编号分别为X8、X18、X21、X48。通过16S rDNA序列分析,结合透射电镜及形态学观察、生理生化测试结果,初步鉴定X8为河豚毒素假交替单胞菌、X18为乔治亚海杆菌、X21为杀鱼假交替单胞菌、X48为康氏菌。通过测定菌株的生长曲线,确定菌株X8发酵量最高的培养时间为18h、确定菌株X18发酵量最高的培养时间为18h、确定菌株X21发酵量最高的培养时间为21h、确定菌株X48发酵量最高的培养时间为18 h。在最适培养时间的基础上,通过单因素多水平试验设计,可得产蛋白酶菌株X8的最佳发酵条件：可溶性淀粉5.0‰、初始pH 7.0、培养基盐度10‰、培养温度25℃;X18最佳发酵条件：可溶性淀粉5.0‰、初始pH 7.0、培养基盐度15‰、培养温度30℃;X21最佳发酵条件：蔗糖5.0‰、初始pH 7.0～8.0、培养基盐度15‰、培养温度25℃;X48最佳发酵条件：可溶性淀粉5.0‰、初始pH 7.0、培养基盐度15‰、培养温度25℃。结论：在优化发酵条件下,X8、X18、X21、X48菌株产蛋白酶酶活力分别达到707.06、240.00、441.18、328.22 U/mL。该研究为探索潜在的高产蛋白酶菌株提供了科学依据。     

腐乳毛霉蛋白酶菌株紫外线诱变育种

目的：选育酶活力高、耐热性好的毛霉蛋白酶菌株,用于腐乳发酵实验或生产。方法：从腐乳中分离毛霉蛋白酶菌株,对其出发菌株进行紫外线诱变,并经控温培养,选育优良变株。结果：获得一株酶活力高、耐热性强的优良变株SCLG—毛霉16,该菌株在32℃条件下,生长茂盛,其HE值为1.39、蛋白酶活力为180.375u/g,分别较出发菌株高出7.8%和51.4%。结论：将此优良变株用于腐乳发酵实验或生产,在缩短发酵时间,提升腐乳质量,延长生产季节等方面具有一定的应用价值。     

高温蛋白酶产生菌的筛选及其产酶条件和酶学性质分析

从徂徕山温泉附近土样中分离到9株产高温蛋白酶菌株,选取一株碱性蛋白酶高产菌株L7为出发菌株,进行显微形态、16S rRNA基因序列分析,将其初步鉴定为短芽孢杆菌(Brevibacillus sp.)。研究该菌株发酵条件,确定产酶的最佳培养基组成为葡萄糖40 g/L,蛋白胨20 g/L,磷酸氢二钠1.4 g/L,氯化钙0.6 g/L,硫酸镁0.4 g/L,通过培养基优化,酶活达到103.08 U/mL。最佳培养条件为250 mL三角烧瓶中装液量50 mL、pH8.0、培养温度为55℃、培养时间为24 h。对该菌株酶学性质研究,L7菌株所产高温蛋白酶的最适温度为55℃,最适pH为10,并且具有良好的温度稳定性和pH稳定性,酶活性受PMSF强烈抑制。     

1株产单宁酶细菌的分离鉴定及产酶条件研究

从富含单宁酸的土壤中分离筛选出1株产单宁酶的细菌,经过菌落形态观察和16S rDNA分子生物学鉴定,该细菌为肺炎克雷伯菌。对该菌所产胞外单宁酶的发酵条件进行了初步研究,得出最佳产酶条件：培养温度37℃,培养时间36 h,培养转速180 r/min,单宁酸含量2 g/L,最佳碳源为葡萄糖,最佳氮源为氯化铵,此时所产单宁酶活力为0.8 U/mL。     

元阳豆豉中高产蛋白酶乳酸菌的筛选及其产酶条件的研究

从云南省元阳地区采集豆豉样品,并从中分离得到62株乳酸菌菌株。通过脱脂乳平板试验,从中初步筛得到21株具有高蛋白酶活力的菌株,采用茚三酮法测定其蛋白酶活力,复筛出高产蛋白酶菌株YY-1-6L,并对其产酶条件进行研究。结果表明,YY-1-6L在以葡萄糖为碳源、多聚蛋白胨为氮源、起始pH 5.0的明胶诱导培养基中,接种量为3%,35℃发酵培养36 h,其蛋白酶活力高达32.50 U/mL,且K2HPO4、MgSO4能促进蛋白酶产生。     

红树林微生物DH-2胞外蛋白酶的性质及产酶条件优化

从大亚湾红树林土壤样品中分离得到产蛋白酶菌株,鉴定所产胞外蛋白酶的酶学性质以及菌株的最佳发酵培养条件。采用平板透明圈法筛选菌株,福林酚显色法测定蛋白酶的酶活,通过单因素和正交试验确定其最佳发酵培养基以及发酵条件。从壤样品中分离得到一株产蛋白酶的枯草芽孢杆菌DH-2,该菌株分泌的蛋白酶最适反应pH和温度分别为8.0和65℃,50℃保温处理60 min后,剩余酶活仍保留80%以上。该蛋白酶对多种金属离子、有机溶剂及表面活性剂均有较好的耐受性。确定该菌株产蛋白酶的最适条件:1%(m/V,下同)可溶性淀粉,1%胰蛋白胨、1%NaCl,初始pH 5.5及7%的接种量,40℃培养36 h。在最适条件下测得其发酵液的酶活为236.30 U/mL,约为初筛时的酶活的8倍。该蛋白酶具有较为广阔的作用温度和pH范围,金属离子、有机溶剂及表面活性剂耐受性好,酶的性质比较稳定。     

三种霉菌产纤维素酶能力分析与培养条件优化

对实验室现有3种真菌产纤维素酶能力的分析及培养条件优化。比较了3种菌在刚果红培养基上的透明圈大小、并分析产纤维素酶酶活;通过单因素与响应面分析的方法优化毛酶产纤维素酶的培养条件。通过试验得出3种真菌均能产纤维素酶,毛霉能产较多的纤维素酶。毛霉产纤维素酶的最佳条件为:pH 5.0,转速220 r/min,发酵时间47 h,发酵温度35℃,纤维素酶活为6.99U/mL。毛霉、青霉、曲霉均产纤维素酶,毛霉能降解玉米芯纤维素。     

一株降解纤维素的放线菌的筛选及其产酶条件的研究

从食草动物的粪便中经筛选分离到1株能降解纤维素的放线菌,经初步鉴定为Streptomycesspp.。对其在以秸秆为惟一碳源的培养基上的产酶条件进行研究,结果表明,最适摇瓶发酵产酶条件为以硫酸铵为氮源,采用种龄72 h的菌液接种,在接种量10%、培养基装量1/10、培养温度30℃时,发酵60 h CMCase活力可达4.5 u/mL。     

一株脂肪酶产生菌的筛选及产酶条件优化研究

通过利用溴甲酚紫显色培养基初筛和酶活测定法复筛得到产脂肪酶的一株细菌HP2,经形态学观察和生理生化测定初步鉴定该菌株为不动杆菌属。并对该菌株的摇床培养产酶条件进行了初步研究,采用正交试验对HP2菌株发酵产脂肪酶的条件进行了优化,得到最佳发酵条件为初始pH为7.7,培养温度为35℃,接种量(V/V)为1.5%,发酵周期为48 h,酶活力达到129.7 U/mL。     

极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

Summary A chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.     

一株中性蛋白酶产生茵的筛选及产酶条件优化

从酒曲中筛选出一株高产蛋白酶的菌株,命名为OPY6,初步鉴定为假单胞菌Pseudomonassp．,经Plackett．BurmanDesign设计和Box．Behnken响应面设计对其产酶培养基做了优化,最优产酶条件为：葡萄糖2．83％,淀粉0．87％,酵母膏0．55％,氯化钙0．001％,氯化钠0．5％,黄豆粉1％,初始pH=7．0,在最佳培养基添加量下蛋白酶活提高到134．718U／mL;又对该酶在水产蛋白降解过程中的酶解效果进行了评价。     

Optimization of growth and hydrolytic enzymes production by Fusarium culmorum using response surface method

Production of hydrolytic enzymes by a phytopathogenic fungus Fusarium culmorum was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing starch or soybean meal as a carbon source. The amylolytic and lipolytic activities were not found. Response surface modeling was applied to shake-flask culture of the fungus to determine the optimum concentration of carbon source and optimal culture time for growth and protease production. The results indicated that the maximum yield of protease production corresponded to the concentration of soybean meal of 1.4?g/ml and culture time of 4.5?days. The fungus growth depends on the concentration of carbon source in the medium whereas the enzyme production was also influenced by the culture time and interaction between these two variables.     

Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and beta-xylosidase while maintaining low protease production

A growth medium was developed for maximal production in batch culture of extracellular xylanase and beta-xylosidase by Aspergillus awamori CMI 142717 and a mutant (AANTG 43) derived from the wild-type strain. The optimum pH for the production of xylanase and beta-xylosidase was 4.0. The best temperature of xylanase production was 30 degrees C; 35 degrees C was optimal for beta-xylosidase. Protease production was never completely suppressed under any of the conditions tested. However, protease titre was 3.5-fold less than the control in medium in which proteose peptone and yeast extract were omitted: the level of xylanase was not affected (8.6 U mL(-1)) but beta-xylosidase titre was increased 4.7-fold to 1.5 U mL(-1). When corn steep liquor was used as the sole nitrogen source, xylanse and beta-xylosidase titres were further increased by 1.5- and 1.9-fold, respectively. Of the carbon sources investigated, ball-milled oat straw or oat spelt xylan produced the highest titres of xylanse and beta-xylosidase. None of the soluble carbon sources investigated produced the high titres of xylanase or beta-xylosidase induced by either oat straw for xylanse and beta-xylosidase was 2% and the optimum spore inoculum was between 10(6) and 10(7) spores/mL(-1) final concentration. The level of xylanse activity obtained in the culture filtrates of the mutant was a remarkable 820 U mL(-1) when the reducing sugar released was measured by the dinitrosalicylic acid method. This enzyme titre would appear to be the highest reported so far. The xylanases system contained the correct balance of enzymes to effect extensive hydrolysis of oat spelt xylan. The protease titre was very low.     

Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease

While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated. Strain SD8 was initially classified to belong to genus Pseudomonas by morphological, physiological and biochemical characterizations, and then through 16S rDNA sequence it was identified to be likely Pseudomonas hibiscicola. In addition, the culture mediums, carbon sources and culture conditions of strain SD8 were optimized for maximum production of alkaline protease. Optimum enzyme production (236U/mL when cultured bacteria being at 0.75 mg dry weight/mL fermentation broth) was obtained when the isolate at a 3% inoculum size was grown in LB medium at 20 mL medium/100mL Erlenmeyer flask for 48h culture at 30°C with an initial of pH 7.5. This was the first report of strain Pseudomonas hibiscicola secreting alkaline protease, and the data for its optimal cultural conditions for alkaline protease production has laid a foundation for future exploration for the potential use of SD8 strain for alkaline protease production.     

Volatile factor involved in the dimorphism of Mucor racemosus.

Both hyphal and yeastlike development of Mucor racemosus and M. rouxii were demonstrated under 100% N2. Under standardized conditions in yeast extract-peptone-glucose medium, the morphology depended on the N2 flow rate and not on the glucose concentration. The effect was related to the rate of flushing of the atmosphere over the culture medium. The results indicate that a volatile compound produced by Mucor is involved in morphogenesis.     

中国冰川1号产适冷蛋白酶耐冷菌的分离鉴定及产酶条件

从中国冰川 1号样品分离获得一株产适冷蛋白酶耐冷菌株SYP- A2 - 3,鉴定为蜡状芽孢杆菌 (Bacilluscereus)。该菌生长温度范围为 0～ 38℃ ,最适生长温度 2 5℃ ,而最适产酶温度为 15℃。所产蛋白酶为中性金属蛋白酶 ,最适催化温度为 4 2℃ ,低温催化活力较高 ,适宜作用pH为 7. 0～ 8 .5 ,SDS PAGE测定的分子量为 34 2kD。SYP A2 3产酶条件的研究结果显示酪蛋白是较好的氮源 ,葡萄糖、淀粉是较好的碳源 ,产酶最佳pH为 6. 5～ 7. 0 ,在优化的条件下 ,15℃摇瓶产酶达到 380 0U mL ,5L发酵罐通气培养产酶达 4 80 0U mL。     

Efficient production of cellulase in the culture of Acremonium cellulolyticus using untreated waste paper sludge

Cellulase was produced by Acremonium cellulolyticus using untreated waste paper sludge (PS) as the carbon source. The clay present in PS did not show any inhibitory effect on cellulase production but did alter the pH during fermentation. On the flask scale, the maleate buffer concentration and pH were key factors that affected the efficiency of cellulase production from PS cellulose. Optimum cellulase production in a 3-L fermentor of working volume 1.5 L was achieved by controlling the pH value at 6.0 using 2 M NaOH and 2 M maleic acid, and the productivity reached 8.18 FPU/mL. When 40.89 g/L PS cellulose, 2.2 g/L (NH(4) )(2) SO(4) , and 4.4 g/L urea were added to a 48-h culture, the cellulase activity was 9.31 FPU/mL at the flask scale and 10.96 FPU/mL in the 3-L fermentor. These values are ～80% of those obtained when pure cellulose is used as the carbon source. The method developed here presents a new route for the utilization of PS.     

Production of milk clotting protease by a local isolate of Mucor circinelloides under SSF using agro-industrial wastes

Agro-industrial residues, a cheap source of energy have high potential in the area of fermentation for the production of enzymes. Twenty agro-industrial residues were evaluated to check the possibility of potential utilization of substrates in SSF for milk clotting enzyme protease production by Mucor circinelloides. In this study, dhal husk holds the greatest promise for cost effective production of the milk clotting enzyme. The dhal husk supported maximum milk clotting protease production, and yield was improved with the supplementation of sucrose and yeast extract as carbon and nitrogen source, respectively. Among all the physico-chemical parameters tested, the best results were obtained in a medium having moisture content of 20% at pH 7.0, when inoculated with 30% of spore suspension and incubated at 30°C for 5 days. The activity was increased further on addition of Ca²⁺, Cu²⁺, and Mg²⁺ ions. The purified milk-clotting protease obtained from M. circinelloides was successfully applied and compared with commercial rennet in the manufacture of a cheddar cheese.