Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Comprehensive studies on optimization of cellulase and xylanase production by a local indigenous fungus strain via solid state fermentation using oil palm frond as substrate

The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 10⁶ spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.     

绿色木霉ZY-1固态发酵产纤维素酶

利用筛选的绿色木霉ZY-1（Trichoderma viride ZY-1）固态发酵产纤维素酶,采用稻草和麸皮为底物,考察稻草与麸皮比例随发酵时间对产酶的影响。结果表明：底物中,在m（稻草）：m（麸皮）为0：5和1：4时,发酵48h,pH保持4．5左右,还原糖量急剧上升,胞外蛋白产量最低;仅以稻草作底物时,整个发酵过程中pH约为7,还原糖量最低,胞外蛋白产量较高而滤纸酶活、羧甲基纤维素酶（CMCase）和β-葡萄糖苷酶（β-Gase）酶活均较低;在m（稻草）：m（麸皮）为3：2时,发酵96h,滤纸酶活达最大值5．01U／g干曲;m（稻草）：m（麸皮）为1：4时,发酵96h,β-Gase酶活达最大值4．6U／g干曲;m（稻草）：m（麸皮）为4：1时,发酵72h,CMCase酶活达最大值6．01U／g干曲。因此,底物中存在适量的稻草和麸皮有利于Trichoderma viride ZY—1产纤维素酶。     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

固态混合发酵提高木聚糖酶和纤维素酶活力的研究

研究了接种比例、接种时间、碳源、氮源等因素对木霉和黑曲霉混合发酵产木聚糖酶和纤维素酶的影响。试验结果表明,当木霉和黑曲霉按4:6同时接种,以玉米芯3．75g、麸皮3．75g、葡萄糖37．5mg为混合碳源,Mandels营养盐11．5mL、添加NH_4NO_37．5mg为氮源,在84h产纤维素酶活力达到230IU/g干物质,木聚糖酶活力达到1308IU/g干物质,与两菌纯培养相比,纤维素酶活力提高163％,木聚糖酶活力提高79．5％。     

利用柑橘皮固体发酵生产复合酶菌株的选育

本试验对14个菌株以柑橘皮为主要原料固体发酵生产复合酶的生产性能进行了比较, 发现宇佐美曲霉Aspergillus usaanii具有较好的复合酶产率,为进一步提高该菌株的产酶能力, 我们利用γ-射线辐射对其进行了诱变育种,选育得到一株纤维素酶活提高26%、酸性蛋白酶提高28%、木聚糖酶提高24.5%的突变菌株AU—C33。采用正交试验方法对该菌株的基础培养基进行了优化,结果表明豆粕和尿素含量对各酶活具有极显著影响。以柑橘粉计,当添加23% 豆粕、8%麸皮、3%尿素,水分含量在60%,28℃培养60h后,CMCase、FPA、β-葡萄糖苷酶、酸性蛋白酶和木聚糖酶分别达到了20.8U/g、7.25U/g、74.7U/g、7248.4U/g和3222.6U/g。     

Streptomyces thermocerradoensis I3 secretes a novel bifunctional xylanase/endoglucanase under solid-state fermentation

Lignocellulosic wastes can be potentially converted into several bioproducts such as glucose, xylo-oligosaccharides, and bioethanol. Certain processes, such as enzymatic hydrolysis, are generally needed to convert biomass into bioproducts. The present study investigated the production of xylanases and cellulases by Streptomyces thermocerradoensis I3 under solid-state fermentation (SSF), using wheat bran as a low-cost medium. The activities of xylanase and carboxymethyl cellulase (CMCase) were evaluated until 96 hr of incubation. The highest enzyme activity was observed after 72 hr of incubation. The crude enzyme extract was sequentially filtered, first using a 50 kDa filter, followed by a 30 kDa filter. Fraction 3 (F3) exhibited activities of both xylanase and CMCase. Xylanase and CMCase showed optimum activity at 70°C and pH 6.0 and 55°C and pH 6.0, respectively. The zymogram analysis showed a single activity band with a molecular mass of approximately 17 kDa. These findings provide strong evidence that the enzyme is a bifunctional xylanase/endoglucanase. This enzyme improved the saccharification of sugarcane bagasse by 1.76 times that of commercial cellulase. This enzyme has potential applications in various biotechnological procedures.     

一株产纤维素酶真菌的筛选、鉴定及酶学性质初步研究

经过初筛和复筛从土样中分离出1株高产纤维素酶真菌SNB9,经形态学和ITS序列分析。鉴定为黑曲霉（Aspergu Uusniger）。生长条件的测定显示该菌生长范围偏酸。发酵后纤维素酶的最适作用pH在4．0—5．0,最适作用温度在45—55℃。滤纸酶活为9．29U／mL,C,酶活为23．69U／mL,CMCase酶活为38．23U／mL,β-葡萄糖苷酶活为65．52U／mL。发酵液中除了纤维素酶,还发现有辅助酶,包括木聚糖酶、淀粉酶、果胶酶、蛋白酶。     

Effects of Tween 80 and rhamnolipid on the extracellular enzymes of Penicillium simplicissimum isolated from compost

Extracellular enzymes of microorganisms play an important role in the decomposition of macromolecules in the composting process. In this study, the effects of Tween 80 and rhamnolipid on the extracellular amylase, carboxymethyl cellulose enzyme (CMCase), xylanase and protease of Penicillium simplicissimum isolated from compost were investigated during solid-state fermentation. The results showed that the enzyme activities of amylase, CMCase and xylanase were increased by Tween 80 and rhamnolipid, which, however, had a negative effect on the protease production. The stimulative effects on the three enzymes were quite different during the whole fermentation process. Tween 80 and rhamnolipid also increased the fungal biomass slightly. As a result of the enhanced enzyme activities, the organic matter were also improved to different extents by both surfactants, and the decomposition rates of hemicellulose and cellulose were increased about 8.0% and 11.6% by Tween 80 at best, respectively, as well as 5% and 5.5% by rhamnolipid.     

Enhanced production of cellulase from a novel strain Trichoderma gamsii M501 through response surface methodology and its application in biomass saccharification

Cellulolytic enzymes produced by Trichoderma sp. have attracted interest in converting the biomass to simple sugars in the production of cellulosic ethanol. In this work, a novel cellulolytic strain M501 was isolated and identified as T. gamsii by sequencing the ITS rDNA region. The production of cellulase (CMCase) by T. gamsii M501 was enhanced by employing statistical methods. The strain grown in the optimized production medium composed of mineral salts, microcrystalline cellulose (13.7 g/l), tryptone (4.8 g/l) and trace elements (2 mL/l) at pH 5.5 and 28 °C for 72 h produced a maximum CMCase of 61.3 U/mL. The optimized production medium also showed the other enzyme activity of FPU (2.6 U/mL), β-glucosidase (2.1 U/mL), xylanase (681 U/mL) and β- xylosidase (0.6 U/mL). The crude cellulase cocktail produced by T. gamsii M501 efficiently hydrolyzed alkali pretreated sugarcane bagasse with glucose and xylose yield of 78 % and 74 % respectively at 10 % solid loading. This study is the first of its kind research on biomass saccharification using T. gamsii cellulase cocktail. Therefore, the novel strain T. gamsii M501 would be useful for further development of an enzyme cocktail for cellulosic ethanol production.     

Cellulase production by Aspergillus niger in biofilm, solid-state, and submerged fermentations

Cellulase production by Aspergillus niger was compared in three different culture systems: biofilm, solid-state, and submerged fermentation. Biofilm and solid-state fermentations were carried out on perlite as inert support, and lactose was used as a carbon source in the three culture systems. In cryo-scanning electron microscopy, biofilm and solid-state cultures gave similar morphological patterns and confirmed that both spore first attachment and hyphal adhered growth are helped by the production of an adhesive extracellular matrix. Biofilm cultures produced higher cellulase activities than those in submerged and solid-state cultures (1,768, 1,165, and 1,174 U l⁻¹, respectively). Although biofilm cultures grew less than the other cultures, they produced significantly higher cellulase yields (370, 212, and 217 U g⁻¹ lactose, respectively) and volumetric productivities (24, 16, and 16 U l⁻¹ h⁻¹, respectively). Likewise, endoglucanase and xylanase activities were higher in biofilm cultures. Under the conditions tested, it seems that fungal attached growth on perlite may favor better enzyme production. Biofilms are efficient systems for cellulase production and may replace solid-state fermentation. Biofilm fermentation holds promise for further optimization and development. The results of this work reveal that fungal biofilms may be used for the commercial production of cellulase employing the technology developed for submerged fermentation at high cell densities.     

Effects of phenolic monomers on the enzymes activities and volatile fatty acids production of Neocallimastix frontalis B9

The effects of phenolic monomers (i.e. rho-coumaric acid, ferulic acid, rho-hydroxybenzaldehyde and vanillin) on the enzymes and fermentation activities of Neocallimastix frontalis B9 grown in ball-milled filter paper and guinea grass media were studied. The enzymes studied were carboxymethylcellulase (CMCase), filterpaperase (FPase), xylanase and beta-glucosidase. At 96 h of incubation, N. frontalis grown in ball-milled filter paper medium produced comparable xylanase and CMCase activities (0.41, 0.5 micromol/min/mg protein) while in guinea grass medium, N. frontalis produced higher xylanase activity than that of CMCase activity (2.35, 0.05 micromol/min/mg protein). The other enzymes activities were low. When N. frontalis was grown in ball-milled filter paper medium, only acetic acid was produced. However, when grown in guinea grass medium, the major end-product was acetate, but propionic, butyric and isovaleric were also produced in lesser amount. Vanillin showed the least inhibitory effects to enzyme activities of N. frontalis B9 grown in both ball-milled filter paper and guinea grass media. For total volatile fatty acid production, all phenolic monomers showed inhibitory effects, but rho-coumaric and ferulic acids were the stronger inhibitors than rho-hydroxybenzaldehyde and vanillin.     

High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation

The production of extracellular xylanase by a newly isolated thermophilic fungus, Paecilomyces themophila J18, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The strain grew well at 50 degrees C and produced a high-level of xylanase activity using the selected lignocellulosic materials, especially wheat straw. Production of xylanase by P. themophila J18 on wheat straw was enhanced by optimizing the particle size of wheat straw, nitrogen source, initial moisture level, growth temperature and initial pH of the culture medium. Under the optimized conditions, yield as high as 18,580 Ug(-1) of carbon source of xylanase was achieved. No CMCase activity was observed. The xylanase exhibited remarkable stability and retained more than 50% of its original activity at 70 degrees C for 4h at pH 7.0-8.0. Therefore, P. themophila J18 could to be a promising microorganism for thermostable, cellulase-free xylanase production in SSF.     

Isolation and characterization of a thermostable cellulase-producing <Emphasis Type="Italic">Fusarium chlamydosporum</Emphasis>

A thermostable cellulase-producing fungus, HML 0278, was identified as Fusarium chlamydosporum by morphological characteristics and ITS rDNA sequence analysis. HML 0278 produced extracellular cellulases in solid-state fermentation using sugar cane bassage as the carbon source. Native-PAGE analysis demonstrated that this fungus strain was capable of producing the three major components of cellulases and xylanase, with a yield of 281.8 IU/g for CMCase, 182.4 IU/g for cellobiohydrolase, 135.2 IU/g for β-glucosidase, 95.2 IU/g for filter paper activity, and 4,720 IU/g for xylanase. More importantly, the CMCase and β-glucosidase produced by HML 0278 showed stable enzymatic activities within pH 4–9 and pH 4–10, and at temperatures below 70 and 60°C, respectively.     

Optimization for xylanase and cellulase production from Aspergillus niger ATTC 6275 in palm oil mill wastes and its application

Optimization of enzyme production from Aspergillus niger ATCC 6275 under both submerged and solid-substrate cultivation was investigated. Results from submerged cultivation using palm oil mill effluent revealed that pretreatment of ground palm cake did not improve enzyme production. Addition of 0.60g NH4NO3/l generated maximum activity of xylanase and cellulase (CMCase). The optimum aeration rate was 1.2 v/v min. Under solid-substrate cultivation, the results indicated that heating and alkali treatment of the ground palm cake gave no further improvement in enzyme production. The optimal N-source was 2% urea. Optimal initial moisture contents for xylanase and CMCase activities were 60% and 50% respectively, with temperature optima of 30°C and 35°C, respectively. The optimal inoculum size was 1× 108 spores/g palm cake with an initial pH of 4.5–5.0. The maximum activities of xylanase (282.9U/g) and CMCase (23.8U/g) were obtained under the optimum conditions. Solid-substrate cultivation was a better method for the production of enzyme, particularly xylanase, from A. niger ATCC 6275. The application of these enzymes to decanter effluent showed the separation of oil and grease and suspended solids from the effluent. This is comparable to the result achieved from using the commercial xylase preparation Meicelase and superior to the effect of Sumyzyme.     

稻草秸秆预处理方法对烟曲霉产纤维素酶的影响

采用机械粉碎、高温、酸碱处理等方法对稻草秸秆进行预处理,以烟曲霉为实验菌株,研究预处理方法对菌株产纤维素酶的影响。结果表明,取机械粉碎后的稻草（30~120目）进行121℃高压蒸汽处理20min（即灭菌处理）,有利于菌株的生长与纤维素酶的产生;与未粉碎的稻草秸秆相比,烟曲霉羧甲基纤维素钠（CMC）酶、微晶纤维素酶、β-葡萄糖苷酶和滤纸（FPA）酶的活力分别提高了63.2%、164.0%、10.2%和14.1%。而采用不同种类、不同浓度的酸碱常温处理稻草秸秆4d或100℃高温处理30min,纤维素酶活力均出现了不同程度的下降。     

表面活性剂对绿色木霉产纤维素酶影响

利用绿色木霉,以稻草为唯一碳源,采用液态发酵的方法,分别加入生物表面活性剂鼠李糖脂和化学表面活性剂Tween 80,重点研究了生物表面活性剂对绿色木霉产纤维素酶的影响。实验分析了加入不同浓度的表面活性剂时滤纸酶活、羧甲基纤维素酶活、微晶纤维素酶活及酶液的表面张力随时间的变化情况。结果表明,添加鼠李糖脂能够促进绿色木霉产酶,分别使滤纸酶活、羧甲基纤维素酶活、微晶纤维素酶活最大提高了1.08倍,1.6倍和1.03倍。与Tween 80相比,鼠李糖脂促进产酶的效果明显优于Tween 80。     

真空微波诱变结合化学诱变选育酸性纤维素酶高产菌株

以酸性纤维素酶产生菌绿色木霉（Trichoderma viride）WL0512作为原始出发菌株,首先经自然分离筛选出一株产酶较稳定的菌株TVN-18,其羧甲基纤维素酶活（CMC酶活）达2765．8U／g,滤纸酶活（FPA酶活）达48．5U／g。再经真空微波和甲基磺酸乙酯（EMS）逐级诱变处理,获得了一株高产、稳产酸性纤维素酶的E6—1菌株,其CMC酶活达4396．6U／g,FPA酶活达126．0U／g,分别是菌株TVN-18的1．59倍和2．60倍。通过对固态发酵培养基麸皮和稻草比例、料水比以及初始pH值的优化,突变株的产酶能力进一步得到提高,其产的CIVIC酶活和FPA酶活分别提高了22．3％和22．4％。     

Optimization of cellulase-free xylanase production by alkalophilic Cellulosimicrobium sp. CKMX1 in solid-state fermentation of apple pomace using central composite design and response surface methodology

Microbial xylanases and associated enzymes degrade the xylans present in lignocellulose in nature. Xylanase production by Cellulosimicrobium sp. CKMX1, isolated from mushroom compost, produced a cellulase-free extracellular endo-1, 4-β-xylanase (EC 3.2.1.8) at 35 °C and pH 8.0. Apple pomace—an inexpensive and abundant source of carbon—supported maximal xylanase activity of 500.10 U/g dry bacterial pomace (DBP) under solid state fermentation. Culture conditions, e.g., type of medium, particle size of carbon source, incubation period, temperature, initial pH, and inoculum size, were optimized and xylanase activity was increased to 535.6 U/g DBP. CMCase, avicelase, FPase and β-glucosidase activities were not detected, highlighting the novelty of the xylanase enzyme produced by CKMX1. Further optimization of enzyme production was carried out using central composite design following response surface methodology with four independent variables (yeast extract, urea, Tween 20 and carboxymethyl cellulose), which resulted in very high levels of xylanase (861.90 U/g DBP). Preliminary identification of the bacterial isolate was made on the basis of morphological and biochemical characters and confirmed by partial 16Sr RNA gene sequencing, which identified CKMX1 as Cellulosimicrobium sp. CKMX1. A phylogenetic analysis based on the 16Sr RNA gene sequence placed the isolate within the genus Cellulosimicrobium, being related most closely to Cellulosimicrobium cellulans strain AMP-11 (97% similarity). The ability of this strain to produce cost-effective xylanase from apple pomace on a large scale will help in the waste management of apple pomace.     

Identification of Mycelium-Associated Cellulase from Streptomyces reticuli

Among 180 Streptomyces strains tested, 25 were capable of hydrolyzing microcrystalline cellulose (Avicel) at 30°C. Streptomyces reticuli was selected for further studies because of its ability to grow at between 30 and 50°C on Avicel. Enzymatic activities degrading Avicel, carboxymethyl cellulose, and cellobiose were found both in the culture supernatant and in association with the mycelium and crystalline substrate. The bound enzymes were efficiently solubilized by repeated washes with buffer of low ionic strength (50 mM Tris hydrochloride [pH 7.5]) and further purified by fast protein liquid chromatography. A high-molecular-weight Avicelase of >300 kilodaltons could be separated from carboxymethyl cellulase (CMCase) and β-glucosidase activities (molecular mass, 40 to 50 kilodaltons) by gel filtration on Superose 12. The CMCase fraction was resolved by Mono Q anion-exchange chromatography into two enzymes designated CMCase 1 and CMCase 2. The β-glucosidase activity was found to copurify with CMCase 2. The purified cellulase components showed optimal activity at around pH 7.0 and temperatures of between 45 and 50°C. Avicelase (but not CMCase) activity was stimulated significantly by the addition of CaCl₂.