Fed-batch production of cellulases using trichoderma reesei rutgers c-30

In the past ten years there has been considerable attention devoted to improving the microbial production of cellulases for their potential in saccharification of cellulosic materials. The Rut. C-30 strain of the fungus Trichoderma reesei has yielded some of the best enzyme activities and productivities reported to date. The use of fed-batch techniques has also been shown to improve enzyme productivities while maintaining or increasing enzyme activities and reducing problems associated with aeration, mixing and foaming. In this work on fed-batch production of cellulases using Rut. C-30 the effects of both the size of the substrate injection and the time between injections are examined. The results indicate an appropriate feeding policy and provide insight into the mechanism for cellulase production.     

Über die Herstellung von Cellulasen und die enzymatische Spaltung von Cellulose

Production of cellulases and enzymatic cleavage of cellulose . Recent work on production of cellulolytic enzymes and on enzymatic degradation of cellulose is described. For optimum enzyme production by Trichoderma reesei under carefully balanced growth conditions, cellulose levels as high as 2% may be employed. Nitrogen deficit may be economically avoided by adding organic nitrogen in the form of distiller's spent grain and dried ground T. reesei cell mass. Semicontinuous production has been shown to offer further special advantages. Cellulase activity may be directly monitored automatically in a computer-controlled pilot fermentation system. Xylitol and furfural process wastes have been shown to be suitable substrates for cellulase, requiring little or no costly pretreatment. The rate of cellulose hydrolysis is considerably increased by combined use of T. reesei and cellobiase active microorganisms such as Aspergillus phoenicia.     

里氏木霉QM9414尿嘧啶缺陷型转化体系改进和β-葡萄糖苷酶的过量表达

里氏木霉是广泛应用于纤维素酶生产的工业真菌,但高产突变株遗传操作困难,限制了菌种改良。首先利用定点整合策略敲除里氏木霉突变株QM9414的pyr4基因,成功构建尿嘧啶营养缺陷型菌株QP4,其遗传转化效率显著提高,而且产酶能力不受影响。进一步在QP4中过量表达β-葡萄糖苷酶(BGL)基因bgl1,经大量平板显色筛选获得2株BGL活力明显增强的工程菌QPB4和QPB5,其酶活分别提高10.01倍和8.26倍。利用发酵酶液对2种不同预处理的玉米芯底物进行水解糖化实验,结果显示以酸处理玉米芯为底物时QPB4和QPB5的葡萄糖得率比QP4分别提高60.98%和52.44%,而以脱木素处理玉米芯为底物时其葡萄糖得率分别提高80.01%和86.00%。研究表明改进里氏木霉高产突变株遗传转化体系可以显著促进菌株改良,提高糖化应用效果。     

里氏木酶利用预处理小麦秆生产纤维素酶的应用研究

为了降低里氏木霉生产纤维素酶的成本,研究了小麦秸秆经白腐菌和酸处理后主要成分如纤维素、木聚糖、木质素含量的变化,采用正交实验考察了里氏木霉菌发酵小麦秆生产纤维素酶的最佳条件,研究得到的最佳条件为:小麦秆∶麸皮=2∶3,培养温度30℃,起始pH 5.0,发酵时间48 h。通过对正交实验条件的优化,发酵液滤纸酶活(FPA)为6.11 IU.mL-1,羧甲基纤维素酶活(CMCase)为29.11 IU.mL-1,纤维二糖酶活(CBA)为16.11 IU.mL-1。和原工艺相比FPA、CMCase和CBA分别提高了30.78%、26.82%和37.11%。     

低聚木糖生产用里氏木霉木聚糖酶选择性合成的研究现状

对低聚木糖生产用里氏木霉木聚糖酶选择性合成的研究现状进行了较全面的总结和评述。系统地介绍了里氏木霉木聚糖酶的的多样性以及碳源、pH和碳氮比等培养条件对合成内切木聚糖酶和木糖苷酶的影响,提出了调控这些培养条件选择性合成低木糖苷酶活的木聚糖酶的方法。     

柄篮状菌与里氏木霉酶解棕榈纤维效果的比较

以青霉属柄篮状菌和里氏木霉Rut-C30为研究对象,在50 IU/g底物酶用量的条件下,比较研究了这两种菌株各自对棕榈空果串纤维的酶解能力。结果表明,柄篮状菌对纤维素的酶解能好于Rut-C30,96 h后,两者葡萄糖转化率为83.7%、61.7%。Rut-C30对木聚糖的酶解能力相对较好,其木糖转化率为98.3%,而柄篮状菌酶解的木糖转化率仅为33.1%。将两菌株的酶按1∶1酶用量混合后,棕榈纤维的整体水解效果增强,葡萄糖、木糖转化率依次为91.3%和98.1%。该研究为柄篮状菌与木霉混合水解棕榈纤维提供了依据。     

Conversion of Canola Meal into a High-Protein Feed Additive via Solid-State Fungal Incubation Process

The study goal was to determine the optimal fungal culture to reduce glucosinolates (GLS), fiber, and residual sugars while increasing the protein content and nutritional value of canola meal. Solid‐state incubation conditions were used to enhance filamentous growth of the fungi. Flask trials were performed using 50 % moisture content hexane‐extracted (HE) or cold‐pressed (CP) canola meal with incubation for 168 h at 30 °C. Incubation on HE canola meal Trichoderma reesei (NRRL‐3653) achieved the greatest increase in protein content (23 %), while having the lowest residual levels of sugar (8 % w/w) and GLS (0.4 μM/g). Incubation on CP canola meal Trichoderma reesei (NRRL‐3653), A. pullulans (NRRL‐58522), and A. pullulans (NRRL‐Y‐2311‐1) resulted in the greatest improvement in protein content (22.9, 16.9 and 15.4 %, respectively), while reducing total GLS content from 60.6 to 1.0, 3.2 and 10.7 μM/g, respectively. HE and CP canola meal GLS levels were reduced to 65.5 and 50.7 % by thermal treatments while solid‐state microbial conversion further reduced GLS up to 99 and 98 %, respectively. Fiber levels increased due to the concentration effect of removing oligosaccharides and GLS.     

Dry action of Trichoderma reesei cellulases on cotton fabrics

Following treatment with Trichoderma reesei cellulases, the mechanical properties of cotton fabrics were measured to assess the effect of adsorbed enzymes. The ability of adsorbed cellulases to act as anchors for further wet finishing processes was studied. Dried fabric samples, after 105 days under usual storage conditions, showed neither significant strength loss, nor any changes in the degree of polymerisation. After five months of storage, the enzymes were still found to be active. The adsorption of T reesei cellulases produced an increase in staining levels after dyeing with an acid dye, but washing fastness was poor. Washing with soap under alkaline conditions removed proteins almost completely. Adsorption of T reesei cellulases is reversible and desorption increases from pH 5 to pH 10. Because of the protein desorption under usual alkaline washing conditions, no practical application of adsorbed cellulases for further finishing processes is suggested and more work is required.     

Laboratory- and commercial-scale investigations into the action of cellulase enzymes on Tencel

The surface appearance and feel of fabrics produced from the new cellulosic fibre Tencel can be enhanced by cellulase treatment. Laboratory-scale studies have been carried out using purified Trichoderma reesei cellulase components to determine which enzymes provide a benefit and which cause detrimental effects on Tencel fibre. Two enzymes were identified as beneficial and this information was used to produce new commercial cellulase products, using genetic engineering to remove detrimental components and increase amounts of the beneficial enzyme components. The effects of the new cellulases on Tencel fibre (both 100% pure and blends) were then evaluated. The observed advantages included improved hand-feel and reduced garment damage, especially in blends with other cellulosics.     

两级超滤部分纯化里氏木霉纤维素酶

研究了里氏木霉纤维素酶超滤过程中pH值的影响,结果显示适宜的pH为7.0。在pH 7.0的条件下,依次用PVDF100超滤膜和PS30超滤膜对纤维素酶粗酶液进行恒体积洗滤方式超滤,以洗出其中的杂蛋白,纯化之后的里氏木霉纤维素酶系中内切葡聚糖酶、外切葡聚糖酶、β-葡萄糖苷酶分别被纯化了2.1、1.78和1.49倍,而且维持了里氏木霉纤维素酶系构成的稳定性。此时,相应酶组分的回收率分别为75.9%、81.8%和54.0%。     

酶水解木质纤维材料制取可发酵糖研究进展

本文对木质纤维材料的化学组成、结构特点,纤维素酶和半纤维素酶及影响酶水解的关键因素,如产物抑制、酶学特性、木质素的存在、表面活性剂的使用、酶的回收等进行了综述,对高效酶水解制取发酵糖技术的研究进行了展望。     

盐酸-乙二醇-水介质高效预处理山核桃壳及其糖化工艺

为了提高木质纤维素的酶解效率,采用盐酸辅助乙二醇对山核桃壳进行预处理。通过油浴的处理方式优化得出的最佳预处理条件为:处理介质为盐酸-乙二醇-水(1.2%:88.8%:10%,质量分数)的混合物,预处理温度为130℃,预处理时间为30min。为了减小预处理的温度和时间,采用微波辐射的辅助预处理,最佳预处理条件为:微波辐射温度100℃,微波辐射时间5min,微波辐射功率200W。糖化预处理后的山核桃壳经水解72h后,其还原糖产率可达到88.6%(油浴)和74.2%(微波)。利用电镜(SEM)和红外光谱(FT-IR)分析油浴和微波预处理后的山核桃壳,可以发现山核桃壳紧密的结构遭到破坏,变成更加易于酶解的松散、多孔结构,增加了酶可及度,因此很大程度上提高了糖化率。可见,盐酸-乙二醇-水溶液高效预处理可以提高山核桃壳酶解糖化的效率。     

高酸值废弃油脂制备生物柴油的预酯化

为了有效解决现有酯化体系中存在反应慢、时间长、产能低等问题,提出了一种高温下甲醇连续酯化反应的新技术,并采用该技术以对甲苯磺酸为催化剂催化高酸值废弃油脂预酯化试验研究。系统讨论了不同酯化方法对反应的影响,并着重研究了工艺条件对预酯化效果的影响。实验结果表明:高温下甲醇连续酯化的新技术可显著提高酯化反应效率,在反应温度120℃、甲醇流量4.0mL/min、催化剂加入量0.8%的条件下,酯化率达98.8%以上,可将油脂的酸值降至1.0mgKOH/g以下,满足下一步酯交换制备生物柴油的要求;并基于实验研究的基础上将该技术工艺对不同酸值的废弃油脂进行了放大试验研究,皆取得了较好的酯化效果,为产业化、规模化的应用提供理论依据和参数指导。     

Increase in bioethanol production yield from triticale by simultaneous saccharification and fermentation with application of ultrasound

BACKGROUND: Bioethanol produced from renewable biomass, such as sugar, starch or lignocellulosic materials, is one of the alternative energy resources that is environmentally friendly. Triticale crops have a high yield as well as a high starch content and amylolytic enzyme activity and are therefore considered to be ideal for bioethanol production. RESULTS: This study examined the feasibility of ultrasound pretreatment to enhance the release of fermentable sugars from triticale meal during pretreatment and consequently increase bioethanol yield in the simultaneous saccharification and fermentation (SSF) process by Saccharomyces cerevisiae yeast. Ultrasonic pretreatment effectively increased the glucose and maltose content after liquefaction by 15.71% and 52.57%, respectively, compared with the untreated control sample under determined optimal conditions of sonication (5 min, 60 °C). The ultrasound pretreatment consequently improved bioethanol production during SSF processing since the bioethanol content was increased by 10.89%. CONCLUSION: Taking into consideration significant process parameters obtained in the SSF process of triticale meal with ultrasound pretreatment at 60 °C, the process time may be reduced from 72 to 48 h. At that point of the SSF, maximum bioethanol content of 9.55% (w/v), bioethanol yield of 0.43 g g^?1 of triticale starch, and percentage of the theoretical bioethanol yield of 84.56% were achieved. Copyright © 2011 Society of Chemical Industry     

The influence of pH upon the hydrolysis of carboxymethylcellulose with cellulases from Trichoderma reesei

We have studied the enzymatic hydrolysis of carboxymethylcellulose (CMC) with Celluclast, a commercial preparation of cellulases deriving from Trichoderma reesei, by monitoring the variation in the concentrations of glucose and reducing sugars at a constant temperature of 50°C and different pH values. We determined both glucose and overall yield, concluding that the production of glucose directly by endoglucanase is higher than that coming from the hydrolysis of cellobiose by β‐1,4‐glucosidase and that the rate at which cellobiose is formed can be calculated via kinetic parameters that are due in the end mainly to exoglucanase activity. We observed the effect of pH upon the kinetic parameters and found that the ideal value for the hydrolysis of CMC is one of pH 4.9.     

绿液预处理玉米秸秆产纤维素酶的研究

研究了绿液预处理玉米秸秆对里氏木霉产纤维素酶的影响。通过正交试验考察3个预处理条件对玉米秸秆产纤维素酶的影响,从极差和方差分析可知,对绿液预处理玉米秸秆产纤维素酶的影响程度由大到小依次是总碱量、蒸煮温度、硫化度,最大滤纸酶活(FPA)达到2.6 IU/mL。比较在蒸煮温度140 ℃和170 ℃,总碱量4 %,硫化度0、20 %、30 % 与40 %下所产的FPA,经综合评定,在最优条件下蒸煮温度140 ℃,总碱量4 %和硫化度0时可以尽量避免原料损失的前提下,保证FPA的大小,结果表明,里氏木霉利用绿液预处理玉米秸秆来产纤维素酶是可行的。以蒸煮温度140 ℃,总碱量4 %和硫化度0的条件下绿液预处理得到的玉米秸秆为碳源,碳源浓度为12 g/L,FPA和β-葡萄糖苷酶活(β-GA)分别达到2.5 IU/mL和1.3 IU/mL,产酶周期5 d。     

低(无)纤维素酶活的木聚糖酶制备途径与潜在应用

低 (无 )纤维素酶活的木聚糖酶在制浆造纸工业中有着巨大的应用潜力 ,特别是在纸浆漂白中的应用。但大多数微生物在自然条件下会同时产生木聚糖酶和纤维素酶。文章对低 (无 )纤维素酶活的木聚糖酶在制浆造纸工业中的应用潜力及其制备途径进行了综述     

绿色木霉木聚糖酶在燕麦木聚糖上的亲和吸附和解析

研究了液相环境对绿色木霉木聚糖酶在水不溶性燕麦木聚糖上的亲和吸附及木聚糖酶―木聚糖复合体的解吸的影响。结果表明,燕麦木聚糖对木聚糖酶的亲和吸附作用主要依赖于它们之间的静电作用和氢键作用,疏水作用对吸附没有贡献。以pH3.0的柠檬酸―柠檬酸钠缓冲溶液为吸附体系的液相时,每克燕麦木聚糖的最大吸附容量为77IU的木聚糖酶。用含0.025mol/LNaCl、pH为7.0的柠檬酸―磷酸氢二钠缓冲溶液作为洗脱液,能将96%的被吸附的木聚糖酶洗脱下来,此时木聚糖酶活回收率为74.3%。     

木聚糖酶最适pH值的研究

研究了以里氏木霉RutC - 3 0合成的木聚糖酶的最适pH值范围。结果表明 ,内切—木聚糖酶的最适pH值范围在 4.0～ 5 .0之间 ,β—木糖苷酶的最适pH值范围在 3 .0～ 4.0之间。研究还表明 ,与内切—木聚糖酶相比 ,pH值对β—木糖苷酶的影响更大。当pH值为 4.0时 ,酶水解总糖得率最高 ,适于制备木糖 ,而当pH值为 5 .0～ 6 .0时 ,较适于制备木低聚糖。     

Production of enzymes from potato pulp using batch operation of a bioreactor

Trichoderma reesei strains MCG77 and Rut C30 were cultivated in batch operation in a stirred tank reactor on potato pulp (PP) consisting of starch, pectin, cellulose, and hemicellulose; potato pulp residue (PR) without starch and pectin; or enzymatic residue (ER), respectively, as substrate and potato protein liquor (PPL) as supplement. The enzyme mixtures produced by the fungi were characterized by four assays: with avicelase, CMCase, xylanase, and FP-activities. Trichoderma reesei Rut C30 yielded higher activities than Trichoderma reesei MCG77. The enzyme activities and productivities obtained with PR are comparable to those obtained by other groups using pure cellulose and a synthetic nutrient solution in batch culture. An optimal operation strategy was derived to obtain maximum enzyme activity and productivity. After converting PR with the enzymes produced and using the residue, ER, as substrate for enzyme production, 85% (w/w) of the pulp was utilized.