Synergistic effects of cellulolytic and pectinolytic enzymes in degrading sugar beet pulp

Three cellulases, one hemicellulase and three pectinases were used, separately or in binary and ternary combinations, to hydrolyze dried beet-pulp, a by-product of the sugar industry. By IE-HPLC the compositions and concentrations of the sugars released were determined. The results obtained by enzymatic saccharification were compared to those obtained by acid hydrolysis. The synergistic action of cellulolytic and pectinolytic enzymes in release of total monosaccharides, and of glucose, arabinose and galacturonic acid was also studied. The combination of cellulase, hemicellulase and pectinase, commercially available, was as effective in degrading the beet pulp as the acid hydrolysis. Pectinase appeared to be the most important enzyme, since by hydrolyzing the pectic surface of the lignocellulosic substrate, it favoured the degradation of cellulose and hemicellulose by the respective enzymes.     

Improvement in the bleaching of kraft pulp with xylanase from Penicillium crustosum FP 11 isolated from the Atlantic forest

Xylanase produced from the newly isolated Penicillium crustosum FP 11 and its potential in the prebleaching of kraft pulp were evaluated using a statistical approach. A Plackett–Burman design (PBD) was carried out to select the significant variables of the medium, these being NaNO₃, KH₂PO₄, MgSO₄, KCl, Fe₂(SO₄)₃, yeast extract, corn stover, and initial pH, in a liquid culture under static conditions for 6 d at 28?°C. Statistical analysis with a central composite design and response surface methodology showed that 0.15% (w/v) KH₂PO₄, 2% (w/v) corn stover, and an initial pH of 6.0 provided the best conditions for xylanase production. Furthermore, xylanase from P. crustosum FP 11 was effective in the bleaching of Eucalyptus kraft pulp, with a significant kappa efficiency of 35.04%. Therefore, the newly isolated P. crustosum FP 11 from the Atlantic Forest biome in Brazil showed two advantages: xylanase production with agricultural residue (corn stover) as a carbon source and an improvement in the bleaching of kraft pulp. Environmental pollution could thus be minimized because of a reduction in the use of chlorine as a bleaching agent.     

Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.     

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).     

Biochemical properties of xylanases from a thermophilic fungus,Melanocarpus albomyces, and their action on plant cell walls

Melanocarpus albomyces, a thermophilic fungus isolated from compost by enrichment culture in a liquid medium containing sugarcane bagasse, produced cellulase-free xylanase in culture medium. The fungus was unusual in that xylanase activity was inducible not only by hemicellulosic material but also by the monomeric pentosan unit of xylan but not by glucose. Concentration of bagasse-grown culture filtrate protein followed by size-exclusion and anion-exchange chromatography separated four xylanase activities. Under identical conditions of protein purification, xylanase I was absent in the xylose-grown culture filtrate. Two xylanase activities, a minor xylanase IA and a major xylanase IIIA, were purified to apparent homogeneity from bagasse-grown cultures. Both xylanases were specific forβ-1,4 xylose-rich polymer, optimally active, respectively, at pH 6.6 and 5.6, and at 65°C. The xylanases were stable between pH 5 to 10 at 50°C for 24 h. Xylanases released xylobiose, xylotriose and higher oligomers from xylans from different sources. Xylanase IA had a M_r of 38 kDa and contained 7% carbohydrate whereas xylanase IIIA had a M_r of 24 kDa and no detectable carbohydrate. The K_m for larchwood xylan (mg ml⁻¹) and V_max (μmol xylose min⁻¹ mg⁻¹ protein) of xylanase IA were 0.33 and 311, and of xylanase IIIA 1.69 and 500, respectively. Xylanases IA, II and IIIA showed no synergism in the hydrolysis of larchwood glucuronoxylan or oat spelt and sugarcane bagasse arabinoxylans. They had different reactivity on untreated and delignified bagasse. The xylanases were more reactive than cellulase on delignified bagasse. Simultaneous treatment of delignified bagasse by xylanase and cellulase released more sugar than individual enzyme treatments. By contrast, the primary cell walls of a plant, particularly from the region of elongation, were more susceptible to the action of cellulase than xylanase. The effects of xylanase and cellulase on plant cell walls were consistent with the view that hemicellulose surrounds cellulose in plant cell walls.     

青霉菌胞外半纤维素酶的固定化研究

青霉菌m8胸外半纤维素酶经过离子交换和分子筛层析两步纯化,纯化的酶固定在戊二醛交联的壳聚精上,残活力为45．6％。固定化酶的最适pH为3．6,最适温度为65℃,且固文化酶在65～75℃活力都较高。该酶的耐热性比较强,固定化酶热稳定性优于原酶;以半纤维素为底物,固定化酶的表观米氏常数为3．58×10－2g／L。     

碳源和氮源对彩绒革盖菌Coriolus versicolor木质纤维素酶和木质素酶分泌的影响

研究了彩绒草盖菌在不同碳源和氮源培养基中生长时,对纤维素酶、半纤维素酶、木质素酶（漆酶、多酚氧化酶、愈创木酚氧化酶）分泌的影响。结果表明不同碳源和氮源对酶类的分泌影响很大,富含淀粉的物质能明显促进木质素酶的分泌,而专一性底物（纤维素和半纤维素）对纤维素酶和半纤维素酶有诱导作用,麸皮也能诱导半纤维素酶的产生。     

Development of an efficient production method for beta-mannosidase by the creation of an overexpression system in Aspergillus aculeatus

AIM: To develop an overexpression system in Aspergillus aculeatus in order to establish an efficient overproduction method of beta-mannosidase (MANB). METHODS AND RESULTS: An overexpression plasmid for the manB gene, encoding A. aculeatus MANB, was constructed and introduced into A. aculeatus cells. The gene was overexpressed under an improved promoter containing 12 copies of Region III cis-elements of Aspergillus oryzae in the transformant, and it secreted 2.56 mg MANB ml(-1) in liquid culture, which obtained a 9.4-fold higher productivity than that achieved in an overexpression system in A. oryzae. Most of the secreted protein in the cultured medium of the transformed A. aculeatus was the overproduced enzyme. CONCLUSIONS: Aspergillus aculeatus with the introduced overexpression plasmid produced 2.56 mg MANB ml(-1) in cultured medium. The improved promoter with A. oryzae Region III functioned in A. aculeatus; thus the strain is an expectant host for recombinant protein productions. SIGNIFICANCE AND IMPACT OF THE STUDY: The overexpression system with the improved promoter in A. aculeatus brought the highest productivity of MANB reported to date. The expression system would be a strong bioindustrial tool for protein production.     

培养基初始pH值对木聚糖酶合成的影响

本文探讨了培养基三种初始ｐＨ值对木聚糖酶合成的影响。试验结果表明,培养基初始ｐＨ值对木聚糖酶的合成有重大影响,低ｐＨ有利于提高木聚糖酶活力。在碳源为７ｇ／Ｌ条件下,初始ｐＨ值为４．０时合成的木聚糖酶活力高达３２．８７ＩＵ／ｍｌ,酶产率和得率分别为６５７４．０ＩＵ／Ｌ·ｄ和４６９５．７ＩＵ／ｇ木聚糖。酶活力和酶产率是初始ＰＨ５．０的１．７倍,分别是初始ＰＨ６．０的３．８倍和２．３倍。进一步研究表明,     

金针菇液体培养特性及胞外酶研究

测定了金针菇在PDY液体培养基中生长时 ,培养液 pH值的变化 ,蛋白质含量和菌丝球重量 ;研究了羧甲基纤维素酶、半纤维素酶、多酚氧化酶、愈创木酚氧化酶和漆酶的酶活性变化。结果表明 ,在整个培养期内 5种酶均有酶活性 ,但不同酶的活性有较大差异 ,产酶高峰也不尽相同