低聚乳果糖酶法制备的研究进展

低聚乳果糖是一种新型功能性低聚糖,是双歧杆菌的有效增殖因子。本文主要介绍了低聚乳果糖国内外研究状况、产品类型、安全性;重点介绍低聚乳果糖酶法制备方法,微生物来源,现有酶的改造修饰,并对制约低聚乳果糖发展的瓶颈问题简单概述。     

一种微生物酶法生产纤维低聚糖的新工艺

一种微生物酶法生产纤维低聚糖的工艺,是以经过预处理的纤维素为原料,真菌纤维素酶为初始酶制剂,采用纤维素“底物原位吸附-固液分离拆分-定向水解法”酶解制备纤维低聚糖,     

多酶级联反应合成能够促进肠道益生菌生长的纤维寡糖

聚合度2–6的可溶性纤维寡糖是一种具有多种生物功能的低聚糖,它能够促进双歧杆菌(Bifidobacteria)、副干酪乳杆菌(Lactobacillus paracei)等肠道益生菌的增殖,因此对人体肠道微生态具有调节作用。本研究通过在大肠杆菌中表达纤维寡糖磷酸化酶(cellodextrin phosphorylase,CDP),构建Cc 01菌株,并与之前构建的COS 01菌株联合使用,建立了基于COS 01、Cc 01的三酶级联反应催化底物葡萄糖和蔗糖合成纤维寡糖反应体系。经过优化后,最终可溶性纤维寡糖的产量达到97g/L,纯度约为97%,其中含有纤维二糖(16.8wt%)、纤维三糖(49.8wt%)、纤维四糖(16.4 wt%)、纤维五糖(11.5 wt%)和纤维六糖(5.5 wt%)。在纤维寡糖对益生菌株生长促进作用的测试中,以菊粉、低聚木糖、低聚果糖为基准,干酪乳杆菌(WSH004)、副干酪乳杆菌(WSH005)以及嗜酸乳杆菌(WSH 006)利用纤维寡糖(聚合度2–6)为碳源进行生长后,益生菌的生物量(OD600)相比对照增加约2倍。该研究证明了三酶级联反应能够高效合成纤维寡糖,并表明聚合度2–6的纤维寡糖是一类具有促进肠道微生物增殖的功能性碳水化合物。     

益生菌体外增殖因子的初步研究

目的研究单糖、pH、温度及时间对青春双歧杆菌、长双歧杆菌和类干酪乳杆菌体外增殖的影响。方法用甘露糖、半乳糖、山梨醇及果糖代替MRS中的葡萄糖,筛选出每种细菌的最适碳源。以此为基础,选择其最佳初始pH、培养温度、碳源添加量及培养时间。结果青春双歧杆菌、长双歧杆菌和类干酪乳杆菌的最适碳源分别为葡萄糖、甘露糖和半乳糖;最佳初始pH为6.0、7.0和6.0;培养温度为42、30和30℃;碳源添加量为20、15和25 g/L;培养时间都为28-48 h。结论益生菌具有不同的最适增殖条件,本文研究结果为优化益生菌的生长条件提供了基础数据。     

酶法合成功能性低聚糖

功能性低聚糖具有无毒、无残留、稳定性强等特点,作为新型绿色添加剂被广泛应用在食品、饲料、医药行业。国际市场上10余种低聚糖产品中除大豆低聚糖、棉籽糖外,主要采用酶法制备。用于合成功能性低聚糖的酶包括糖苷酶、糖基转移酶和磷酸化酶。本文综述了功能性低聚糖种类、性质和制备方法,分析了酶法合成低聚糖的优缺点,阐述了磷酸化酶种类、催化特性和低聚糖产物。多酶法合成策略和目标酶的分子改造将是酶法合成功能性低聚糖的发展方向。     

玉米低聚糖对双歧杆菌增殖与发酵的影响

目的研究玉米低聚糖对双歧杆菌增殖和发酵的影响。方法试验I组以乳糖替代正常TPY培养基中葡萄糖作为碳源,试验II组以玉米低聚糖替代TPY培养基中的葡萄糖作为唯一碳源,分别在接种培养0、12、24、36、48和72h对两组双歧杆菌活菌数量、培养液中还原糖含量和培养液pH值进行测定。同时以不同剂量玉米低聚糖饲喂小鼠,分析对小鼠粪便中双歧杆菌数量的影响。结果培养72h后,试验II组双歧杆菌活菌数显著高于试验I组(t=5.832,P0.05)。试验II组培养液中葡萄糖含量在12、24、36、48和72h时均较试验I组显著偏高(P0.05)。试验II组培养液中pH在24h之后一直显著低于试验I组(P0.05)。试验I组、II组小鼠粪便中双歧杆菌数量显著高于饲喂前(t=20.109、22.982,P0.05);试验III组小鼠粪便中双歧杆菌数量较饲喂前显著降低(t=4.692,P0.05)。结论玉米低聚糖能够显著促进双歧杆菌体外增殖,增强发酵,并能提高小鼠肠道中双歧杆菌的数量。     

新鞘氨醇杆菌属9-1的纤维素酶基因Nspcel8A的克隆表达与酶学特性

木质纤维素转化成燃料酒精是缓解能源和环境危机的途径之一.降低将木质纤维素转化成生物燃料的生产成本,需要提高纤维素酶产量或筛选到具更高酶活性的纤维素酶.新鞘氨醇杆菌(Genus Novosphin-gobium)属于鞘氨醇杆菌科(Sphingomonads),该科的细菌新陈代谢多样化,能够降解有机化合物,也可应用于木质素的降解,但目前新鞘氨醇杆菌属细菌的纤维素酶基因的研究未见报道.本研究对新鞘氨醇杆菌属细菌菌株9-1的纤维素酶基因Nspcel8A进行了克隆表达和酶学特性鉴定.Nspcel8A含有属于糖基水解酶家族8的催化结构域.该酶在大肠杆菌中实现了异源表达并获得了表达产物.Nspcel8A对羧甲基纤维素(car-boxymethylcellulose,CMC)的最适作用pH值和温度分别为4.0和40℃,Nspcel8A具有良好的pH值稳定性,在pH值3.5～11.0范围内放置24 h后能够保持60％以上的酶活力.Nspcel8A对CMC的Km值为10 mg/mL,Vmax为14 μmol·min-1·mg-1.底物特异性测试显示Nspcel8A对CMC有最高的酶活力(8.40 U/mg),但对不可溶纤维维如磷酸膨胀纤维素和Avicel只有较低的酶活力或没有酶活.高效液相色谱法分析显示Nsp-ce18A 不能降解纤维二糖、纤维三糖、纤维四糖,能把纤维五糖部分降解成纤维二糖和纤维三糖,能把纤维六糖降解为纤维二糖、纤维三糖和纤维四糖,并以纤维三糖为主.以上结果显示Nspcel8A是一个内切葡聚糖酶,由于它不能水解结晶纤维素,说明它不是Novosphingobium sp.9-1主要的纤维素降解酶.     

耐消化道逆环境双歧杆菌菌株A04体外促进生长因子的研究

研究了4种低聚糖、8种中药及4种食品原料体外对双歧杆菌菌株A04的增殖作用。结果表明,大豆低聚糖的增殖效果最明显（P〈0．01）,其次是低聚异麦芽糖和低聚果糖,水苏糖则几乎没有增殖效果,而且当改良MRS中低聚糖的浓度达到10％时,对其反而具有一定的抑制作用;8种中药在浓度为2％的时均有增殖效果,以Z3、Z1和Z2效果最显著（P〈0．01）,但其浓度不同对双歧杆菌菌株A04的增殖影响截然不同;本试验采用的4种食品原料对双歧杆菌均有促生长作用,较好的增殖作用的组合分别为：M-5％,CJ-2％,TE-2％和LE-5％。     

耐消化道逆环境双歧杆菌菌株A04体外促进生长因子的研究*

研究了4种低聚糖、8种中药及4种食品原料体外对双歧杆菌菌株A04的增殖作用。结果表明,大豆低聚糖的增殖效果最明显(P<0·01),其次是低聚异麦芽糖和低聚果糖,水苏糖则几乎没有增殖效果,而且当改良MRS中低聚糖的浓度达到10%时,对其反而具有一定的抑制作用;8种中药在浓度为2%的时均有增殖效果,以Z₃、Z₁和Z₂效果最显著(P<0·01),但其浓度不同对双歧杆菌菌株A04的增殖影响截然不同;本试验采用的4种食品原料对双歧杆菌均     

魔芋粉酶解产物与低聚果糖对双歧杆菌的促生长作用比较研究

由枯草芽胞杆菌以摩芋粉为底物经发酵培养获得的β-甘露聚糖酶酶活力达10U／ml,用该酶液水解魔芋粉制备含有低聚糖的魔芋粉酶解产物。在无碳源的GAM基础培养基中,添加不同量的魔芋粉酶解产物对双歧杆菌具有明显的促生长作用,其促生长作用与低聚果糖相当,均以2％的浓度增殖效果最明显,与不加低聚糖的对照组相比,活菌数可以提高几倍到十几倍。     

Reversibility and competition in the adsorption of Trichoderma reesei cellulase components

Adsorption reversibility and competition between fractionated components of the Trichoderma reesei cellulase system were studied. Specific endoglucanase (EGI), nonspecific endoglucanases (EGII, EGIII), and cellobio-hydrolase (CBHI) were previously grouped according to their hydrolytic function. At 5 degrees C, direct evidence of exchange between adsorbed and free enzyme was obtained for each component using [(3)H] and [(14)C] radiolabeled tracers. No release of bound enzymes was detected upon dilution of the free enzyme solution. In simultaneous adsorption of enzyme pairs, CBHI was shown to predominate adsorption. Endoglucanase EGI was preferentially adsorbed over EGII and EGIII. Sequential adsorption studies have shown that interaction between enzyme components largely determines the degree of their adsorption. Evidence suggests that both common and distinct adsorption sites exist and that their occupation depends on which components are involved. Predominance in adsorption by any one of the enzyme components is decreased at 50 degrees C. Light microscopy and monitoring of sugar production during cellulose hydrolysis provided evidence that reduction in the ionic strength decreases the adsorption predominance of CBHI and enhances the synergism between the cellulase components.     

Method for characterization of the enzyme profile and the determination of CBH I (Cel 7a) core protein in Trichoderma reesei cellulase preparations

Summary Fast protein liquid chromatography (FPLC) was used to characterize a commercial cellulase preparation (Celluclast 1.5L, Novozymes) in relation to its protein profile and activity against hydroxyethylcellulose (HEC) and other substrates. Co-elution of CBHII (Cel 6A) with other enzyme components of the cellulase system was characterized by immunochemical assays using monoclonal antibodies, whereas the occurrence of EGII (Cel 5A) was assessed based on its ability to cleave the heterosidic bond of 4-methylumbellyferyl-β-d-cellotrioside (MUmbG₃). The main cellulase constituents of Celluclast 1.5L were pooled into six fractions containing EGII (Cel 5A) and EGIII (Cel 12A) (F1), EGII and CBHII (Cel 6A) (F2), CBHII and EGI (Cel 7B) (F3), EGI (F4), and CBHI (Cel 7A) (F5). The occurrence of CBHI core protein within the CBHI fraction of the FPLC profile was determined by hydrophobic interaction chromatography. Using this method, we were able to demonstrate that the batch of Celluclast 1.5L used in this study contained 10.9–18.8% of CBHI as its corresponding free core protein.     

Cloning, sequencing, and expression of the cellulase genes of Humicola grisea var. thermoidea

We have cloned an endoglucanase (EGI) gene and a cellobiohydrolase (CBHI) gene of Humicola grisea var. thermoidea using a portion of the Trichoderma reesei endoglucanase I gene as a probe, and determined their nucleotide sequences. The deduced amino acid sequence of EGI was 435 amino acids in length and the coding region was interrupted by an intron. The EGI lacks a hinge region and a cellulose-binding domain. The deduced amino acid sequence of CBHI was identical to the H. grisea CBHI previously reported, with the exception of three amino acids. The H. grisea EGI and CBHI show 39.8% and 37.7% identity with the T. Reesei EGI, respectively. In addition to TATA box and CAAT motifs, putative CREA binding sites were observed in the 5′ upstream regions of both genes. The cloned cellulase genes were expressed in Aspergillus oryzae and the gene products were purified. The optimal temperatures of CBHI and EGI were 60 °C and 55–60 °C, respectively. The optimal pHs of these enzymes were 5.0. CBHI and EGI had distinct substrate specificities: CBHI showed high activity toward Avicel, whereas EGI showed high activity toward carboxymethyl cellulose (CMC).     

Enhanced production of cellobiohydrolases in Trichoderma reesei and evaluation of the new preparations in biofinishing of cotton

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).     

Enhanced production of Trichoderma reesei endoglucanases and use of the new cellulase preparations in producing the stonewashed effect on denim fabric

Trichoderma reesei strains were constructed for production of elevated amounts of endoglucanase II (EGII) with or without cellobiohydrolase I (CBHI). The endoglucanase activity produced by the EGII transformants correlated with the copy number of the egl2 expression cassette. One copy of the egl2 expression cassette in which the egl2 was under the cbh1 promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A T. reesei strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the cbh2 locus with the coding region of the egl2 gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.     

Circular Dichroism Studies on Conformation of Cellobiohydrolase and Endoglucanase from Trichoderma pseudokiningii S-38: Effects of pH and Ligand Binding

Effects of pH and ligand binding upon the conformation of Cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from Trichoderma pseudokiningii S-38 have been studied by circular dichroism measurements. In the high-pH range (6–9), increasing pH resulted in a similar conformational change occurring in free CBHI and EGI, while such treatment gave different changes of the two enzyme conformations in the presence of cellobiose. On the other hand, in the low-pH region, with both CBHI an EGI in the active form, decreasing pH resulted in a large conformational change of free EGI compared to that of free CBHI, whereas ligand binding resulted in a similar change of both CBHI and EGI, independent of pH change.     

Circular Dichroism Studies on Conformation of Cellobiohydrolase and Endoglucanase from Trichoderma pseudokiningii S-38: Effects of pH and Ligand Binding

Effects of pH and ligand binding upon the conformation of Cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from Trichoderma pseudokiningii S-38 have been studied by circular dichroism measurements. In the high-pH range (6–9), increasing pH resulted in a similar conformational change occurring in free CBHI and EGI, while such treatment gave different changes of the two enzyme conformations in the presence of cellobiose. On the other hand, in the low-pH region, with both CBHI an EGI in the active form, decreasing pH resulted in a large conformational change of free EGI compared to that of free CBHI, whereas ligand binding resulted in a similar change of both CBHI and EGI, independent of pH change.     

Improvement of cellulose-degrading ability of a yeast strain displaying Trichoderma reesei endoglucanase II by recombination of cellulose-binding domains

To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414, the genes encoding the cellulose-binding domain (CBD) of EGII, cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T. reesei QM9414, were fused with the catalytic domain of EGII and expressed in Saccharomyces cerevisiae. Display of each of the recombinant EGIIs was confirmed using immunofluorescence microscopy. In the case of EGII-displaying yeast strains in which the CBD of EGII was replaced with the CBD of CBHI or CBHII, the binding affinity to Avicel and hydrolytic activity toward phosphoric acid swollen Avicel were similar to that of a yeast strain displaying wild-type EGII. On the other hand, the three yeast strains displaying EGII with two or three tandemly aligned CBDs showed binding affinity and hydrolytic activity higher than that of the yeast strain displaying wild-type EGII. This result indicates that the hydrolytic activity of yeast strains displaying recombinant EGII increases with increased binding ability to cellulose.     

Isolation and purification of isoenzymes of cellobiohydrolase I and II of trichoderma reesei using LPLC methods

Five cellulases were fractionated from a commercial cellulase preparation (Celluclast^TM) Two isoenzymes of cellobiohydrolase I (CBHI)(pI = 4.1) could be proved to be real exo-glucanases due to their activity towards MU (=methylumbelliferyl)-lactoside being inhibited by cellobiose (5 mM) and due to production of cellobiose from carboxymethylcellulose (CMC) as the sole final product.Two isoenzymes of CBHII (pI=6.15, 6.0) were shown to act as endo-glucanases because they produced glucose, cellobiose and cellotetraose from CMC and because they were not inhibited by cellobiose when decomposing MU-lactoside. Results confirm recent reports in the literature classifying CBHI and CBHII as exo-type and endo-type cellulases, respectively. Both the CBHI and the CBHII isoenzymes were shown to be active towards CMC and amorphous cellulose.CBHI and CBHII reactions could be differentiated from one another by the velocities of decomposition of CMC: CBHI acts slowly and linearly whereas CBHII acts strongly and exponentially.The fifth of the purified enzymes must be classed as a conventional endoglucanase which exhibits activity towards CMC but fails to be active towards MU-lactoside and amorphous cellulose.     

Cellulosic ethanol production by combination of cellulase-displaying yeast cells

As an effort to find suitable endoglucanases to generate cellulolytic yeast strains, two fungal endoglucanases, Thermoascus aurantiacus EGI and Trichoderma reesei EGII, and two bacterial endoglucanases, Clostridium thermocellum CelA and CelD, were expressed on the yeast surface, and their surface expression levels, pH- and temperature-dependent enzyme activities, and substrate specificities were analyzed. T. aurantiacus EGI showed similar patterns of pH- and temperature-dependent activities to those of T. reesei EGII which has been widely used due to its high enzyme activity. Although EGII showed higher carboxymethyl cellulose (CMC) degradation activity than EGI, EGI showed better activity toward phosphoric acid swollen cellulose (PASC). For ethanol production from PASC, we combined three types of yeast cells, each displaying T. aurantiacus EGI, T. reesei CBHII (exoglucanase) and Aspergillus aculeatus BGLI (β-glucosidase), instead of co-expressing these enzymes in a single cell. In this system, ethanol production can be easily optimized by adjusting the combination ratio of each cell type. A mixture of cells with the optimized EGI:CBHII:BGLI ratio of 6:2:1 produced 1.3 fold more ethanol (2.1 g/l) than cells composed of an equal amount of each cell type, suggesting the usefulness of this system for cellulosic ethanol production.