利用天然纤维废弃物发酵生产L-乳酸的研究

为了降低L-乳酸的生产成本,更好的实现生物质秸秆的资源化,利用天然纤维素依次接种经离子注入诱变处理的木聚糖酶高产菌黑曲霉P602和米根霉RL6041高产菌进行固、液体二次发酵的方法,将其转化成用于工业生产的L-乳酸。结果表明:本实验条件下,未经过任何化学预处理的秸秆等物质接种黑曲霉P602进行固体发酵,产生的木聚糖酶活力为6 320 IU/g干(培养)基,纤维素酶活力为29 IU/g干基;加入100 mL水浸提后,产生的还原糖浓度为14.07 g/L,纤维物质糖化率为79.45%。取滤液接入米根霉RL6041进行液体发酵后,生成乳酸的量为7 g/L,糖酸转化率为47.6%,以(NH4)2SO4作为氮源时,最佳氮源浓度为3 g/L。     

木霉菌株T6木聚糖酶固态发酵条件和酶学性质研究

研究了碳源和氮源、起始pH、接种量及温度等条件对一野生型木霉Trichodermasp.T6菌株固态发酵产木聚糖酶的影响。在28℃培养4d后,酶活力可达1918IU／g干培养物。酶的最适反应温度为50℃,最适反应pH4．5。不同温度保温1h后,测定酶的半失活温度为47．7℃,酶的pH稳定性也进行了研究。     

黑曲霉与里氏木霉固态混合发酵产β-葡萄糖苷酶

对黑曲霉NL02与里氏木霉RUT-C30固态混合发酵产β-葡萄糖苷酶的发酵培养基进行优化,研究培养基含水率、C源、N源、接种量、温度和2种菌种不同延长接种时间与接种比例对β-葡萄糖苷酶活力的影响。研究表明：麸皮17.5 g、玉米芯7.5 g、（NH4）2SO4 0.40 g、尿素0.37 g、黑曲霉孢子接入量为107个接种到250 mL三角瓶中,温度30 ℃、摇床转速100 r/min时,里氏木霉以105个孢子与黑曲霉同时接入,每克干曲所得β-葡萄糖苷酶的活力为132.45 IU,较黑曲霉单独培养时的104.35 IU提高了26.94%。     

木霉菌株T6木聚糖酶固态发酵条件和酶学性质研究

研究了碳源和氮源、起始pH、接种量及温度等条件对一野生型木霉Trichoderma sp.T6菌株固态发酵产木聚糖酶的影响。在28℃培养4d后,酶活力可达1918IU/g干培养物。酶的最适反应温度为50℃,最适反应pH4.5。不同温度保温1h后,测定酶的半失活温度为47.7℃,酶的pH稳定性也进行了研究。     

木霉菌株T6木聚糖酶固态发酵条件和酶学性质研究*

研究了碳源和氮源、起始pH、接种量及温度等条件对一野生型木霉Trichoderma sp.T6菌株固态发酵产木聚糖酶的影响。在28℃培养4d后,酶活力可达1918IU/g干培养物。酶的最适反应温度为50℃,最适反应pH4.5。不同温度保温1h后,测定酶的半失活温度为47.7℃,酶的pH稳定性也进行了研究。     

甘蔗渣固态发酵产纤维素酶

以甘蔗渣和麸皮混合作为固态发酵产酶培养基,采用单因素优化实验对里氏木霉固态发酵产纤维素酶进行优化。结果表明,在50 m L体系培养基中,在底物绝干原料5.2 g、甘蔗渣与麸皮质量比7∶3、氮源((NH4)2SO4)7.5 g/L、产酶诱导物1.6 g/L、表面活性剂(聚乙二醇PEG6000)0.1 g、发酵起始p H 4.4、培养基中里氏木霉孢子接入量5×105个的条件下,温度30℃时发酵120 h,里氏木霉固态发酵产纤维素酶的酶活达76.39 IU/g,是起始优化前20.29 IU/g的3.76倍。     

黑曲霉产木聚糖酶发酵条件的研究

将经过诱变选育高产木聚糖酶并具有Nystatin抗性的黑曲霉,分别在不同条件下进行固体发酵培养,探讨最佳产酶条件。结果显示:在以质量分数1%木糖为附加碳源,以质量分数2%NH4NO3为氮源,无机盐为质量分数1%NaCl,加水比例为1:1.3,接种量为1.5%,装料量为5g/300m l三角瓶,培养温度为30℃,培养周期为72 h的培养条件下,菌株产木聚糖酶活力提高至7285.4 IU.g-1。比出发菌株提高了20%。酶活力测定采用3,5-二硝基水杨酸(DNS)法。     

利用农业废弃物生产嗜热真菌（T．1anuginosus）耐热木聚糖酶的固体发酵研究

研究了嗜热真菌(Thermomyces lanuginosus CBS288．54-M18)生产木聚糖酶的碳氮源组成、料水比、培养基初始pH值、发酵温度及接种量等的影响。结果表明,以麦麸和玉米芯粉(8：2)作为复合碳源,酵母膏和胰蛋白胨(1：1)作为复合氮源时,菌株所产的木聚糖酶量相比未优化碳氮源的培养基提高幅度高达200％。其最佳产酶的料水比为1：3,培养基初始pH7．0为最适。菌株在50℃条件下发酵5d,能够得到活力高达15023U／g干基碳源的木聚糖酶制剂,且该酶制剂不合纤维素酶和蛋白酶活性。     

里氏木霉与黑曲霉混合发酵产纤维素酶及其水解特性

研究了利用里氏木霉和黑曲霉混合培养产纤维素酶,以黑曲霉孢子悬浮液的不同活化浓度及不同的活化时间来寻找2个菌种发挥最大协同作用的结合点以及所产纤维素酶的水解特性。以里氏木霉单一培养和黑曲霉单一培养为参照进行对比研究。底物为农林废弃物之一的玉米秸秆,经过蒸气爆破预处理后,用作产酶C源。结果表明:黑曲霉孢子悬浮液活化浓度为10个/mL,活化时间为12 h时,滤纸酶比酶活最高,达3.32 U/mL,高于里氏木霉单一培养的2.25 U/mL,β-葡萄糖苷酶比酶活达1.32 U/mL,高于里氏木霉单一培养的0.57 U/mL。为进一步验证混合菌产纤维素酶的水解效果,利用混合菌产纤维酶的酶液及里氏木霉产纤维素酶的酶液进行酶水解实验,当酶用量为20 U/g绝干纤维素,底物质量浓度为100 g/L条件下水解48 h,混合菌所产酶液酶解得率达70.00%,高于里氏木霉所产酶液的酶解得率63.05%。实验表明里氏木霉与黑曲霉混合培养产酶是可行的,并优于单一菌种培养。     

里氏木霉与米根霉混合固态发酵产纤维素酶

以里氏木霉及米根霉单菌固态发酵为对象,考察不同混合发酵形式对里氏木霉与米根霉混合固态发酵产纤维素酶的影响。结果表明:同时接种里氏木霉与米根霉,试验考察的两菌种接种量比1∶1(以孢子个数计)及5∶1条件下,两菌未产生明显协同产酶作用。米根霉延时(24 h)接种且菌种量比5∶1以及米根霉延时(48 h)接种且菌种量比1∶1,2种发酵形式产酶情况类似,滤纸酶活(FPA)及羧甲基纤维素酶(CMCase)酶活相对米根霉单菌发酵有所提高,而β-葡萄糖苷酶(β-GA)酶活相对里氏木霉单菌固态发酵结束时分别增加4.66及4.40倍,可以发现两菌产生一定协同作用。在米根霉延时(48 h)接种且菌种量比5∶1的发酵形式下,FPA及CMCase在发酵第7天酶活分别达到44.04 IU/g、627.14 U/g(以1 g干曲计),分别是里氏木霉固态单菌发酵产酶达到稳定期时酶活的1.36和1.63倍,两菌产生了有效的协同作用。     

离子注入选育高产木聚糖酶黑曲霉及其发酵条件研究

以黑曲霉A3为出发菌,利用离子注入技术选育出一株遗传性状稳定的木聚糖酶高产突变株AN497,其产酶水平较出发菌从野生型A3菌株的405.6IU/ml提高到586.2IU/ml,即酶产量增加了44.5%;对高产菌进行发酵条件优化,发现以玉米芯粉为主要碳源、用蔗糖代替葡萄糖作为附加碳源,对木聚糖酶的发酵具有明显的促进作用;采用复合的无机氮源 (NH4)2SO4和NaNO3,(1: 2)浓度以10g/L为宜;菌株对发酵通氧量具有较高的要求,摇瓶转速在230r/min时的产酶水平较200r/min要高;通过发酵条件的优化,高产菌株的产酶活力最高可达671.1IU/mL,比出发菌株的产酶量提高了65.5%。     

Sorghum straw for xylanase hyper-production by Thermomyces lanuginosus (D2W3) under solid-state fermentation

This paper reports the production of very high levels of cellulase free xylanase and associated hemicellulases by an indigenous thermophilic isolate of Thermomyces lanuginosus (D(2)W(3)) using solid-state fermentation. Sorghum straw, an inexpensive and abundant source of carbon supported maximal xylanase activity (11,855 units/g dry substrate). Culturing T. lanuginosus D(2)W(3) on sorghum straw and optimizing other culture conditions (media types, particle size of carbon source, inoculum level, inoculum age and additives), yielded increased levels of xylanase (39,726 units/g dry substrate). Further optimization of enzyme production was carried out using Box-Behnken design of experiments with three independent variables (inoculum level, glycerol and ammonium sulphate concentrations) which resulted in very high levels of xylanase, 48,000+/-1774 units/g dry substrate, and 2.6+/-0.2, 13.4+/-0.56, 68+/-1.7, 1.4+/-0.08, 1.2+/-0.05 (units/g dry substrate) of beta-xylosidase, alpha-galactosidase, pectinase, beta-mannosidase and alpha-L-arabinofuranosidase, respectively.     

Production of phytase by Mucor racemosus in solid-state fermentation

Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.     

黑曲霉AF-98固体发酵产纤维素酶的产酶条件研究

通过单因子及正交试验,对黑曲霉AF-98固体发酵产纤维素酶的产酶条件进行了探讨。其优化的产酶条件为:甘蔗渣3g,麸皮2g,加含尿素为0.15%的Mandels营养液25mL(加水比1:5),调初始pH5.0,28℃发酵72h。在此优化条件下,纤维素酶活力可达7.56u/g干曲。     

Response surface optimization of fermentation conditions for producing xylanase by <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">niger</Emphasis> SL-05

Fermentation conditions were statistically optimized for producing extracellular xylanase by Aspergillus niger SL-05 using apple pomace and cotton seed meal. The primary study shows that culture medium with a 1:1 ratio of apple pomace and cotton seed meal (carbon and nitrogen sources) yielded maximal xylanase activity. Three significant factors influencing xylanase production were identified as urea, KH(2)PO(4), and initial moisture content using Plackett-Burman design study. The effects of these three factors were further investigated using a design of rotation-regression-orthogonal combination. The optimized conditions by response surface analysis were 2.5% Urea, 0.09% KH(2)PO(4), and 62% initial moisture content. The analysis of variance indicated that the established model was significant (P < 0.05), "while" or "and" the lack of fit was not significant. Under the optimized conditions, the model predicted 4,998 IU/g dry content, whereas validation experiments produced an enzymatic activity of xylanase at 5,662 IU/g dry content after 60 h fermentation. This study innovatively developed a fermentation medium and process to utilize inexpensive agro-industrial wastes to produce a high yield of xylanase.     

木聚糖酶生产菌株的筛选及产酶条件的优化

以甘蔗渣半纤维素为碳源,从垃圾场土壤中分离到6株分解半纤维素的菌株。通过固态发酵的木聚糖酶活力比较筛选到1株木聚糖酶活力较高的菌株。该菌株18S rDNA序列与曲霉（Aspergillus sp.）的同源性达97%,根据对菌株形态学分析和18S rDNA序列分析的结果,将该菌株鉴定为曲霉HQ3。HQ3的最佳产酶条件为：甘蔗渣:麸皮为7:3（W/W）,固液比为1:4（W/W）,尿素0.4 %,pH7.0,温度30℃,发酵产酶时间4 d。在最佳产酶条件下,其木聚糖酶活最高可达3421U/g干曲。     

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

Abstract

The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.     

嗜热真菌DSM10635生产耐热木聚糖酶的小试研究

应用嗜热真菌Thermomyces lanuginosus DSM10635,采用固体发酵的方法探索耐热木聚糖酶的优化生产条件。在研究玉米芯,玉米皮,玉米秆,麸皮,松树屑,桦树屑等不同底物,在不同温度、玉米芯颗粒大小以及料水比条件下培养比较酶产量后,发现该嗜热真菌产耐热木聚糖酶的最佳底物为玉米芯或玉米皮,最佳培养温度为50℃--55℃,在加水量为1份玉米芯：2．8份水,玉米芯的颗粒直径大约为1mm时产酶量最高。实验结果显示,嗜热真菌DSM10635在优化后的培养条件下木聚糖酶产量可达到12525．80IU／g玉米芯。