水稻秸秆腐解复合菌系RSS-4的选育及其腐解特性

采用限制性培养技术与温度梯度诱导相结合的方法, 从四川成都平原多年还田的土壤中筛选、构建出一组在中温条件下对水稻秸秆具有腐解功能的复合菌系RSS-4。该复合菌系在22°C条件下, 稻秆腐解试验表明: pH先升高后降低, 最后稳定在7.20; 纤维素酶活、半纤维素酶活均经历了先升后降的变化趋势, 最高酶活分别为0.91、3.40 U; 到16 d腐解结束时, RSS-4对稻秆、纤维素及半纤维素的降解率分别达到了45.0%、55.5%和44.1%, 而木质素在整个腐解过程中未发生明显的变化; 说明所筛选构建的这组腐解复合菌系可加速稻秆的腐解。同时发现采用未灭菌的筛选方法筛得的复合菌系RSS-4比灭菌所得的RSS-4￠腐解效果要好。     

复合菌系RSS-4腐解稻秆过程中的菌系动态变化

采用传统平扳分离培养方法和PCR—DGGE技术研究了水稻秸秆腐解复合菌系RSS-4在腐解稻秆过。程中菌种区系变化情况。结果表明：平板分离培养方法显示,在稻秆腐解过程中,微生物的数量呈现出先升后降的变化趋势,在整个腐解过程中细菌的数量占优势;DGGE图谱显示,至少有12种细菌和18种真菌的近缘种参与到稻秆的腐解过程。在其腐解过程中,不同腐解阶段真菌的组成呈现出多样性,数量变化差异也较大：细菌DGGE图谱中的条带1、9、10等以及真菌DGGE图谱中的条带8、9、13等为优势菌株,它们贯穿于稻秤腐解的整个过程;细菌中的条带12以及真菌中的条带4在腐解的前期起作用,而后迅速消失;细菌中的条带3、11等以及真菌中的条带3、10等在腐解的后期才出现而起作用;而细菌中的条带2以夏真菌中的条带1、5等仅出现在腐解的莱一时期。     

一组多功能细菌复合系NSC-7的培养特性及稳定性

本研究探讨了一组具有分解纤维素和农药林丹双重功能的复合菌系NSC-7的培养特性和稳定性。NSC-7在14 d的培养过程中, 使稻秆分解73.6％; 用GC-MS测定结果发酵液中检测到10种化合物成分, 其中峰值较大的依次为乙酸、甘油、丁酸、丙酸; NSC-7在-80℃冷冻和冻干条件下保存4年后仍具有稳定的秸秆分解能力和纤维素内切酶活性; 经90℃高温处理30 min后, 仍能够保持分解能力, 105℃处理30 min后转接2次就能恢复分解能力, 显示出很高的保存稳定性和热稳定性。利用DGGE分析多次继代接种过程的培养物结果条带基本没有变化, 表明NSC-7的菌种组成稳定。     

兼性厌氧复合菌群H纤维素降解和产乙醇能力及生态组成初探

通过限制性培养条件和连续继代培养,筛选获得了一组具有高效稳定降解纤维素能力的复合菌群H。该菌群在传代30代以上仍能保持各项性状稳定,其工作pH为6～9,3 d可以完全降解置于100 mL PCS缓冲液培养基中的滤纸,发酵液中能够检出1.54 g/L乙醇。通过16S rDNA扩增和DGGE的方法,对菌群在不同阶段的微生物组成进行了研究,确定了琥珀酸嗜热梭菌Clostridium thermo succinogene、产气荚膜梭菌Clostridium straminisolvens和紫色板蓝根梭菌Clostridium isatidis等多种可直接实现纤维素到乙醇转化的菌株。菌群通过菌种之间的协同作用,共同维持了体系的稳定及降解能力的稳定。明确菌系的组成,对于进一步研究菌群降解机理、优化菌群和提高乙醇产率意义重大。     

小麦秸秆高效腐解菌复合系WSS-1的选育及其菌群分析

采用限制性培养技术和温度梯度诱导法,从四川成都平原多年还田的土壤中筛选得到了常温条件下对小麦秸秆具有高效腐解功能的复合菌系WSS-1。该复合菌系在28℃、4d可完全崩解滤纸,进一步的腐解麦秆的试验效果表明:接种WSS-1处理在第6天左右达到CMC酶活最高值,这比目前广泛使用的菌剂A提前了4d;经WSS-1腐解20d后的麦秆断裂拉力值下降了73.66%,降低幅度分别比空白对照和菌剂A处理多出33.7%和8.9%。采用16S rDNA克隆文库法对其细菌组成进行解析,结果表明WSS-1的优势菌群为枯草芽孢杆菌(Bacillus subtilis)、球形芽孢杆菌(Bacillus sphaericus)、粪产碱菌(Alcaligenes faecalis)双酶梭菌(Clostridium bifermentans)和粪肠球菌(Enterococcus faecalis)。     

一组多功能细菌复合系NSC-7的培养特性及稳定性

本研究探讨了一组具有分解纤维素和农药林丹双重功能的复合菌系NSC-7的培养特性和稳定性. NSC-7在14d的培养过程中, 使稻秆分解73. 6%;用GC. MS测定结果发酵液中检测到10种化合物成分, 其中峰值较大的依次为乙酸、甘油、丁酸、丙酸:NSC. 7在. 80"C冷冻和冻干条件下保存4年后仍具有稳定的秸秆分解能力和纤维素内切酶活性;经90C高温处理30 min后, 仍能够保持分解能力,105℃处理30 min后转接2次就能恢复分解能力,显示出很高的保存稳定性和热稳定性.利用DGGE分析多次继代接种过程的培养物结果条带基本没有变化,表明NSC.7的菌种组成稳定.     

白腐菌对培养环境pH的调节及其产漆酶的相关性

研究了不同种属白腐菌多孔菌属C1(Polyporussp.)、侧耳属B2(Pleurotussp.)、香菇属A3(Lentinusedodes)对培养环境pH的调节,及其与菌株产漆酶状况的相关性。结果表明:3株白腐菌均可调节培养液酸化,培养环境的pH可分别由初始的4.5~5.0持续下降至培养结束时3.0~3.5水平,相应C1、B2、A3三者的漆酶合成与分泌在pH值较高(pH 4.5以上)时均表现较弱(酶活分别处于约1 500、200、50 U/mL以下的水平),在pH 3.2~4.5范围内的不同偏酸性条件下则得到较好表达(最高酶活分别达5 000、340、144 U/mL)。统计学分析指出,白腐菌调节环境pH状况与其产漆酶状况之间存在显著或极显著相关性,但不同白腐菌调节培养环境pH的能力之间并不具有显著性差异。     

降解水稻秸秆兼抑制水稻纹枯病菌多功能复合菌系的构建

目的针对稻草直接还田需要,构建能够高效降解水稻秸秆同时又能抑带l水稻纹枯病菌的多功能复合菌系。方法通过将具有高效降解纤维素的天然复合菌群与具有抑制水稻纹枯病菌效果的菌株组合,构建多功能复合菌系;采用失重法检测该复合菌系对水稻秸杆的腐解作用,荧光定量PER法检测其对水稻纹枯病菌的抑制效果。结果成功地构建了一组多功能复合菌系,腐解12d后,水稻秸秆干物质总失重率为41．4％,其中半纤维素降解率为59．5％,纤维素降解率为52．5％,木质素降解率为15．3％,腐解过程中平均CMC酶活为8．1IU／g。该复合菌系对水稻纹枯病菌的抑制效果明显,发酵40d后对水稻纹枯病菌的抑制率为27．1％,对照组抑制率为2．7％。结论该复合菌系能高效降解水稻秸秆,同时又能较好地抑制水稻纹枯病菌,适宜在水稻秸秆直接还田过程中使用。     

三种白腐菌及其组合菌种木质素降解酶比较研究

朱红栓菌Trametes cinnabarina、糙皮侧耳Pleurotus ostreatus、黄孢原毛平革菌Phanerochaete chrysosporium是产生木质素降解酶能力强的菌株。对三种白腐菌及其组合菌种产生木质素降解酶能力和行为进行了比较分析和研究。结果表明,最佳培养方式为液体振荡培养;最佳培养基为酵母膏液体培养基。在产漆酶(laccases,lacs)方面,Pleurotus ostreatus和Phanerochaete chrysosporium的组合菌种的酶活最强,在第6天出现峰值,酶活达到450U/L;在产锰过氧化物酶(manganese peroxidases,mnps)方面,Trametes cinnabarina和Pleurotus ostreatus的组合菌种的酶活最强,在第10天出现峰值,酶活达到1050U/L;在产木质素过氧化物酶(lignin peroxidases,lips)方面,Trametes cinnabarina和Phanerochaete chrysosporium的组合菌种的酶活最强,在第8天出现产酶峰值,酶活达到2990U/L。筛选结果表明,组合菌种比单菌种产生的三种主要木质素降解酶的活性强,这为白腐菌高效产酶提供了一条新的途径,并为白腐菌研究领域的后续工作奠定基础。     

白腐菌紫外诱变选育高产漆酶突变株及其产酶条件研究

以白腐菌为出发菌株,利用紫外线(UV)进行诱变,筛选高产漆酶突变菌株。通过测定致死率绘制出发菌株的致死曲线,采用PDA-RBBR平板变色法进行初筛,ABTS检测酶活对突变株进行摇瓶复筛。结果表明:利用15 w紫外灯在照射距离为30 cm,照射时间为120 s,致死率为72.1%的条件下进行诱变处理,获得一株高产菌株,其酶活提高79.54%,经过5代传代培养,未见酶活下降,具有较好的遗传稳定性,进一步研究了初始pH值,接种量和培养基装液量等对诱变菌株产酶的影响,结果表明在最佳的培养条件pH值6.0,15%的装液量于28℃下,酶活达214.9 U/L。     

Production of cellulase-free thermostable xylanases by an isolated strain of Aspergillus niger PPI, utilizing various lignocellulosic wastes

A strain of Aspergillus niger PPI having prolific xylanolytic potential was isolated and the optimum conditions for maximum xylanase production was studied, resulting in the following: 4% substrate concentration, 10% v/v inoculum size, 72 h of incubation and pH 3.5–4.5 at 28 °C. The production profile of xylanase was examined with various lignocellulosics and maximum yield was achieved with oat. The hemicellulose content of wastes was also determined and oatmeal was found to have maximum hemicellulose content followed by wheat straw, sugarcane bagasse, rice husk and gram residue respectively. The enzyme showed maximum activity at pH 4 and temperature 60 °C. However, maximum stability was achieved at pH 3.5 and temperature 55 °C. Cellulase activity was found altogether absent in the enzyme broth.     

Rice bran lipase: extraction, activity, and stability

A simple procedure for the extraction of the lipolytic activity from rice bran has been developed. Various conditions of extraction have been optimized so as to obtain maximum yield of the lipase. It was found that high enzyme activity could be obtained by first defatting the rice bran to remove the lipid component. This was followed by five cycles of aqueous extraction (potassium phosphate buffer, 50 mM and pH 7, containing 0.5 mM of CaCl(2)). The stability of the rice bran lipase under storage and operative conditions was investigated. Further, the influence of glycerol as a stabilizer has been assessed. It was found that further purification using micro- and ultrafiltration yielded an enzyme preparation with higher activity and specific activity and better stability.     

糖化酶酶解米渣纯化米蛋白的工艺

研究了糖化酶酶解米渣纯化米蛋白的实验条件:液固比、酶解时间、pH、温度和酶量。通过正交实验优化了酶解主要条件,得到糖化酶水解米渣最佳条件:液固比5:1,时间3 h,pH 4.0,温度65℃和酶量30 U.g-1。在最佳条件下实验,米蛋白的提取率为81.3%,纯度为76.8%。     

High-level production of yeast (Schwanniomyces occidentalis) phytase in transgenic rice plants by a combination of signal sequence and codon modification of the phytase gene

This study was designed to produce yeast (Schwanniomyces occidentalis) phytase in rice with a view to future applications in the animal feed industry. To achieve high-level production, chimeric genes with the secretory signal sequence of the rice chitinase-3 gene were constructed using either the original full-length or N-truncated yeast phytase gene, or a modified gene whose codon usage was changed to be more similar to that of rice, and then introduced into rice (Oryza sativa L.). When the original phytase genes were used, the phytase activity in the leaves of transgenic rice was of the same level as in wild-type plants, whose mean value was 0.039 U/g fresh weight (g-FW) (1 U of activity was defined as 1 micromol P released per min at 37 degrees C). In contrast, the enzyme activity was increased markedly when codon-modified phytase genes were introduced: up to 4.6 U/g-FW of leaves for full-length codon-modified phytase, and 10.6 U/g-FW for truncated codon-modified phytase. A decrease in the optimum temperature and thermal stability was observed in the truncated heterologous enzyme, suggesting that the N-terminal region plays an important role in enzymatic properties. In contrast, the optimum temperature and pH of full-length heterologous phytase were indistinguishable from those of the benchmark yeast phytase, although the heterologous enzyme was less glycosylated. Full-length heterologous phytase in leaf extract showed extreme stability. These results indicate that codon modification, combined with the use of a secretory signal sequence, can be used to produce substantial amounts of yeast phytase, and possibly any phytases from various organisms, in an active and stable form.     

BEHAVIOUR OF HORSERADISH PEROXIDASE IN AOT REVERSED MICELLES

The activity and stability of horseradish peroxidase (HRP) solubilised in AOT reversed micelles in isooctane and decalin was studied using guaiacol (2-methoxyphenol) as the electron donor.

The activity of the enzyme in both reversed micellar systems increases with the water content until reaching a maximum value that remains fairly constant for water contents higher than 3.05% (v/v) in isooctane and 2.20% in decalin. The effect of pH on the activity profile was studied in the system AOT/isooctane. The enzyme is fully active at pH 7 and 8 for water contents higher than 3.05% (v/v) but it was completely deactivated at pH 9. The effect of surfactant concentration on HRP activity was also investigated. At low water contents a strong dependence was observed, whilst no further activity increase was observed for water content values higher than 2.7% (v/v).

The stability of HRP was found to be strongly dependent on the water content of the system with higher levels of stability obtained for higher values of water content. HRP stability is also affected by the presence of substrates. Whilst the stability increases markedly when the enzyme is incubated with guaiacol, it does not appear to be so strongly affected by the presence of hydrogen peroxide, at the concentrations studied.     

Continuous production of l(+)-tartaric acid from cis-epoxysuccinate using a membrane recycle reactor

The one-step bioconversion of cis-epoxysuccinate (CES) to l(+)-tartaric acid by dried Rhodococcus rhodochrous cells containing CES hydrolase activity was studied by using a continuous bioconversion process. The influence of the pH and the temperature was assessed. A mathematical model was used to quantify the CES hydrolase activity and stability. The optimal pH, which resulted in a maximal CES hydrolase activity and stability, was pH 8.0. A large increase in stability (half-life time) could be obtained when the temperature was decreased from 37 to 14°C during the continuous bioconversion. A total bioconversion was maintained for more than 100 days. This resulted in a large value for the specific productivity since the effect of the large increase in stability was much more important than the decrease of activity at the lower temperature. This continuous bioconversion process was further optimised by calculating the productivity for several continuously stirred tank reactors in series. The specific productivity could be nearly doubled when the number of reactors in the series was increased from 1 to 4.     

家蚕肠道环境对外源纤维素酶活力稳定性影响的研究

纤维素酶对家蚕消化桑叶纤维素起重要生理作用,本试验研究了不同温度、pH对外源纤维素酶活力的影响及纤维素酶的热稳定性与pH稳定性,同时在模拟家蚕肠道环境条件下,研究了纤维素酶活力的稳定性。结果表明：所选的外源纤维素酶在家蚕肠液中的最适催化温度为30℃左右,最适pH为8．0左右;酶在家蚕体温范围内具有较好的稳定性,在pH8．0-10．0范围内酶活力较稳定;模拟家蚕肠道环境条件下酶活力稳定性实验表明,在80min时间内,纤维素酶能够保持较高的活力而发挥生物活性。     

Study of pH and temperature-induced transitions in urate oxidase (Uox-EC1.7.3.3) by microcalorimetry (DSC), size exclusion chromatography (SEC) and enzymatic activity experiments

Purified recombinant urate oxidase (urate oxygen oxidoreductase EC 1.7.3.3. re-Uox) has been studied by means of differential scanning calorimetry (DSC) in correlation with enzymatic activity measurements and size exclusion chromatography. Differential scanning calorimetry curves versus pH show two endothermal effects in the pH range 6-10. The first endotherm reveals a maximum stability between pH 7.25 and pH 9.5 corresponding to a temperature of transition T(m1) of 49.0 degrees C and an enthalpy of transition of 326 kJ mol(-1). This value dramatically decreases below pH 7.25. The behavior of the second endotherm is more complex but the temperature of transition T(m2) is constant between pH 9 and 7.25 and a maximum for the corresponding enthalpy is obtained near pH 8 with DeltaH(2)=272 kJ mol(-1). An optimal pH of 8.0 for the stability of the enzymatic activity at elevated temperature was also found which was in good agreement with calorimetric results. Reversibility of the first endotherm is obtained from 20 to 51.5 degrees C. The calorimetric result is correlated to enzymatic activity, purity by size exclusion chromatography (SEC) and protein concentration measurements. In contrast, for the second endotherm, after heating up to 68.9 degrees C, no reversibility was found. Interaction with structural analogues of urate has been studied by DSC. 8-Azahyooxanthine has only a small effect and caffeine has no effect at all. With 8-azaxanthine, a rapid increase of the T(m1) function of the concentration is obtained. At high concentration T(m1) reached the T(m2) value which remained unaffected.     

Catalytic properties and stability of horseradish peroxidase immobilized in polyacrylamide gel]

Effect of polyacrylamide (PAA) gel on properties of horseradish peroxidase, immobilized by means of the incorporation into PAA gel is studied. Catalytic properties of immobilized enzyme are studied. Km value and pH-dependency of the enzyme activity are found to be close to those of soluble enzyme, kcat value is 3 times lower at pH 7.0. PH-stability of immobilized peroxidase at 20 degrees C and thermostability of soluble and immobilized peroxidases at pH 7.0 within the temperature range from 20 to 81 degrees C are studied. The stability of peroxidase in PAA gel is found to decrease (in 3 times at 20 degrees C, and in 17 times at 56 degrees C). A mechanism of the effect of PAA gel on catalytic properties and stability of peroxidase is discussed.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.