产低温淀粉酶菌株的筛选、鉴定及酶学性质初步研究

对低温淀粉酶资源的开发是功能酶研究的新热点,本研究从东帕米尔高原江布拉克冰川冻土中分离获得多株高产低温淀粉酶菌株,最高产酶菌株D5-2经16S rDNA鉴定为假单胞菌属(Pseudomonas sp.)细菌.对D5-2所产淀粉酶性质进行初步分析,结果表明,其最适作用温度和pH值分别为25 ℃和6.5,酶的热稳定性较差,40 ℃处理1h后酶活急剧下降,至70℃酶活力仅存23％;在pH 5.5～8.0条件下,酶活力相对稳定;Mn2+和Mg2+对淀粉酶有激活作用,而乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)、Fe2+和Na+则抑制酶活.结果提示,菌株D5-2分泌的淀粉酶符合低温酶特性,应用空间较大,值得深入探究开发.     

一株曲霉的一种中温糖化酶的纯化及其部分酶学特性

本研究从广西花坪自然保护区采集的土壤中筛选获得了一株产糖化酶丝状真菌菌株57-45,通过形态学观察和真菌内转录间隔区（internal transcribed spacer,ITS）序列比对分析,将其初步鉴定为曲霉属（Aspergillus sp.）的一个种。纯化真菌57-45所产的一种胞外糖化酶经过硫酸铵分级沉淀、疏水层析和阴离子交换层析三步蛋白质纯化步骤,得到在SDS-PAGE上约60kD的单一蛋白质条带,薄层层析分析表明该纯化的蛋白质水解可溶性淀粉的产物只有葡萄糖,证明该纯化的蛋白质为糖化酶。纯化的糖化酶Km值为1.9mg/mL,Vmax为4148μmol/（min·mg）,最适作用pH5.5,最适作用温度50℃,在同步糖化发酵中有应用的潜力。金属离子Fe3＋、Zn2＋、Cu2＋对酶活有较强的抑制作用,EDTA对酶活有较强的促进作用。本文结果将为进一步研究曲霉糖化酶的酶学特性提供新的材料。     

不同因素对羔羊皱胃酶凝乳活性的影响

研究结果表明，羔羊皱胃酶最适凝乳温度为45℃；35℃以上热处理对酶凝乳活性有不同程度的损失，60℃热处理10 min活性完全丧失；pH值为5～8时凝乳活性随乳pH值的降低而增强，酶在pH2.5～7.5之间处理20h凝乳活性稳定；Ca²⁺具有明显的促凝作用；底物浓度对酶活性的影响符合米氏规律。     

一株烟草秸秆降解菌的分离、鉴定及酶学性质研究

从皖南地区烟稻轮作田土壤中经CMC-Na初筛获得5株纤维素降解菌,经DNS法测定纤维素酶活性复筛得到一株降解活性较高的降解菌YC-2。根据该菌16S r DNA序列比对结果,结合形态和生理生化特征,确定YC-2为枯草芽孢杆菌(Bacillus subtilis)。对YC-2酶学性质进行相关研究,并分析YC-2对烟碱的耐受情况及对烟草秸秆的降解情况,结果表明:在7天内,YC-2对烟草秸秆降解率为10.14%;酶学特性表现为最适反应温度为60℃且在15~60℃之间具有稳定性;最适反应pH为7.0,在pH 4.0~7.5范围内稳定性较好;YC-2在浓度为1~2 g/L烟碱中能快速生长,而在高浓度的烟碱中生长受到抑制。因此,菌株YC-2产纤维素酶活性较高、相对耐热耐碱且对烟杆有一定分解作用,通过进一步诱变选育和发酵条件优化有较好的田间应用潜力。     

玉米挤压淀粉酶法改性制膜的工艺优化

为了提高可降解性玉米淀粉膜的力学性能,并获得玉米挤压淀粉酶法改性制膜的最适工艺参数,该研究以普鲁兰酶为酶制剂来改善玉米挤压淀粉膜,以酶作用温度、pH值、酶添加量、酶解时间及玉米挤压淀粉浓度为试验因子,膜的抗拉强度为响应值,采用中心旋转组合试验设计进行试验。结果表明：5个因素对酶改性挤压淀粉膜抗拉强度的影响大小依次为玉米挤压淀粉浓度＞酶添加量＞酶解时间＞pH值＞酶作用温度;最佳酶解制膜工艺条件为：酶作用温度46.57℃,pH值4.44,酶添加量6.63 u/g,酶解时间9.31 h,玉米挤压淀粉浓度7.00%,在此条件下,膜抗拉强度的预测值为24.3654 MPa,验证试验所得膜抗拉强度为24.2539 MPa,比未改性膜的抗拉强度提高了338.01%。回归方程的预测值和试验值差异不显著,所得回归模型拟合情况良好,达到设计要求。膜的抗拉强度与酶解挤压淀粉中直链淀粉含量之间存在极显著正相关关系,相关系数为0.863。     

马铃薯交联淀粉醋酸酯的制备工艺及特性

马铃薯原淀粉在水相体系、碱性条件下与醋酸酐作用，生成马铃薯淀粉醋酸酯，然后与交联剂进行交联反应，得到马铃薯交联淀粉醋酸酯。通过改变反应温度、反应时间、pH值和交联剂用量等条件，得出制取马铃薯交联淀粉醋酸酯的最佳工艺条件：反应温度35℃，pH值11，反应时间2 h，交联剂用量2%。所制取的马铃薯交联淀粉醋酸酯较原淀粉在糊液黏度的稳定性、抗老化性、抗分离性等方面均有较大的改善。     

玉溪醇化烟叶表面细菌酶制剂对烟叶中淀粉和纤维素的降解作用

合理降解烤后烟叶中的淀粉和纤维素含量已成为提高烟叶质量的关键工艺之一。本研究对分离自烤烟叶面的产淀粉酶细菌菌株解淀粉芽孢杆菌(Bacillus amyloliquefaciens A1)和产纤维素酶细菌菌株短小芽孢杆菌(B.pumilus C1)进行发酵培养,提取粗酶液,检测酶活性并应用于初烤烟叶上。通过设计不同酶量、作用时间、相对湿度和温度的方法考察这两种酶制剂对烟叶(Nicotiana tabacum K326)中淀粉和纤维素的降解效果。结果表明,菌株A1主要产α-淀粉酶,酶活力为7×105U/mL,菌株C1以产内切酶为主,酶活力为6×103U/mL;在温度40℃、湿度70%条件下作用96h,用菌株A1所产淀粉酶制剂4×107U/kg处理后的烟叶总糖增加率为12.78%,还原糖增加率为12.03%,菌株C1所产纤维素酶制剂4×105U/kg处理后的烟叶总糖增加率为13.87%,还原糖增加率为18.07%。研究结果提示,利用从烟叶表面分离的产酶菌株进行发酵生产的酶制剂可降解初烤烟叶中淀粉和纤维素,提高还原糖含量,可望在烟叶加工过程中得到应用。     

一株降解生木薯淀粉的曲霉菌株的筛选、鉴定及其淀粉降解特性

本研究利用以生木薯淀粉为唯一碳源的筛选培养基,从腐烂木薯渣中分离筛选出一株可以降解生木薯淀粉的真菌菌株RSDF-7。根据RSDF-7形态和18SrDNA与28SrDNA之间的内转录间隔区（internal transcribed spacer,ITS）序列分析的结果,初步认定该菌株为曲霉属。菌株RSDF-7的粗酶液对多种不同的生淀粉底物均有水解效果;在以大米和玉米淀粉为底物时,其生淀粉分解活力比较高,分别为42%和40%。菌株RSDF-7的粗酶液具有良好的低pH稳定性,对生木薯淀粉的最适作用温度为50℃,最适作用pH为4.5。在30min的吸附后,RSDF-7的粗酶液对生木薯淀粉的吸附力高达60%。使用HPLC对粗酶液的酶解产物进行检测,结果发现酶解产物中仅存在葡萄糖,表明菌株RSDF-7所产的生淀粉降解酶主要为糖化酶。扫描电镜观察结果发现,经RSDF-7粗酶液酶解后的生木薯粉颗粒破裂,形成空洞,说明RSDF-7粗酶液对生淀粉有较强的水解作用。可以预见,经纯化后的曲霉菌株RSDF-7生淀粉酶将来可以用于基于酶解的木薯淀粉转化。     

黑胸散白蚁体内产纤维素酶细菌的筛选及其纤维素酶基因的鉴定与原核表达

白蚁(Isoptera)常以富含纤维素的木材为食,体内存在纤维素酶产生菌。从源于陕西佛坪的白蚁(经鉴定为黑胸散白蚁(Reticulitermes chinensis))体内筛选出了4株产纤维素酶菌株,采用单因素实验设计对酶活最高的菌株进行了产酶条件优化,并对其所产纤维素酶进行了酶学性质研究;同时,依据NCBI数据库设计的引物扩增出了该菌株的内切-β-1,4-葡聚糖酶基因Cbs和β-葡萄糖苷酶基因Ba G,之后在大肠杆菌(Escherichia coli)中进行了表达实验。结果表明:从黑胸散白蚁体内筛选出4株菌分别是阿氏肠杆菌(Enterobacter asburiae)、炭疽芽胞杆菌(Bacillus cereus)、枯草芽胞杆菌(Bacillus subtilis)、解淀粉芽胞杆菌(Bacillus amyloliquefaciens),其中枯草芽胞杆菌所产纤维素酶的活力最高,该菌发酵产酶的最适温度和pH分别是42℃、6.5,酶促反应的最适温度和pH分别是70℃、4.5,在此条件下该菌株酶活力为2.36 U/mL。该菌株所产的纤维素酶在50℃、pH 6.5的条件下热稳定性和pH稳定性最佳,使用该菌株所产的粗酶液发酵粗饲料,结果发现其对玉米(Zea mays)秸秆、小麦(Triticum aestivum)秸秆、苜蓿(Medicago polymorpha)干草和青贮饲料的粗纤维降解率分别为12.21%、1.3%、3.24%和17.42%。基因克隆结果表明该菌株具有Ba G、Cbs 2个纤维素酶基因,且大肠杆菌表达结果显示Ba G基因的粗酶液pro Ba G无纤维素酶活性,而Cbs基因的粗酶液pro Cbs在60℃、pH 5.0时酶活可达4.14 U/mL,将Ba G和Cbs基因进行原核融合表达,产生的蛋白约35 k D,其在最适条件70℃、pH 4.5时的酶活为4.57 U/mL,说明无纤维素酶活性的Ba G基因与Cbs基因融合后可能表达出了一个活性更高的纤维素酶蛋白。本研究对后期构建可降解木质纤维素的人工重组菌具有重要的理论价值。     

土壤中淀粉酶高产菌株的分离及产酶条件的优化

从土壤中分离到淀粉酶产生菌,获得产酶量相对较高的菌株,并从pH和培养温度两方面对产酶条件进行优化。实验结果显示,土壤中分离的菌株有较高的淀粉酶活力,产酶条件适用于工农业生产。     

酸雨胁迫对大豆萌发种子糖代谢动态的影响

为了探索酸雨胁迫对大豆萌发种子糖代谢动态的影响,试验采用蒸馏水浸泡大豆种子,再以pH2.5、4.5模拟酸雨(AR)处理大豆种子,考察不同强度AR胁迫对大豆萌发种子可溶性糖、还原性糖、蔗糖、淀粉及α、β-淀粉酶含量的影响。结果表明,第1天AR组的可溶性糖含量上升,第2天明显降低,随后稳定降低;AR胁迫下的第1天-第4天,还原糖含量维持较高水平,第3天-第5天呈下降趋势;pH2.5组在第2天-第5天蔗糖含量减少;AR胁迫的第2天-第4天淀粉含量降低;总体上,AR组的α-淀粉酶活性CK,但从第5天起pH2.5组的α-淀粉酶活性CK;β-淀粉酶活性在第1天-第6天高于或接近CK,pH2.5组在第7天低于CK。糖代谢各项指标和淀粉酶活性之间的关系表明,可溶性糖、还原糖、蔗糖含量变化幅度随AR胁迫强度增大而增加,淀粉含量变化与之相反;AR组对淀粉酶活性影响是导致萌发改变的内在因素之一。     

Influence of H+ Ca++, and Mn++ on the development of amylase in the endosperm of several sorghum cultivars 1

Sorghum [Sorghum bicolor (L.) Moench] seeds (Funks G522DR) were planted in ‘white quartz flintshot’ and watered with 0.01 M NaAc buffer at pH 4.0, 4.5, 5.0, 5.5, or 6.0 ± Ca⁺⁺ (0, 10, 100 ppmw) or Mn⁺⁺ (0, 1.4, 140.0 ppmw) and grown for seven days in a growth chamber. Endosperm amylase contents, quantitatively and qualitatively, were dependent upon the environmental conditions during germination. H⁺ concentration at pH 4.0 strongly induced a decrease in amylase activity which was relieved as the pH was raised to pH 6.0. Ca⁺⁺ greatly increased endosperm amylase contents. Mn⁺⁺ (1.4 ppmw) tended to increase endosperm amylase contents while Mn⁺⁺ (140.0 ppmw) strongly decreased endosperm amylase content. In the cultivar SC283, endosperm amylase contents were nearly double those found in Funks G522DR while SC574 had, at most, one half the endosperm amylase content of Funks G522DR endosperm. Endosperm amylase contents of SC283 were pH tolerant while those of Funks G522DR and SC574 were highly pH sensitive. These data further demonstrate the plasticity of plants growing under adverse environmental conditions.     

土壤中淀粉酶高产菌株的分离及产酶条件的优化

从土壤中分离到淀粉酶产生菌，获得产酶量相对较高的菌株，并从pH和培养温度两方面对产酶条件进行优化。实验结果显示，土壤中分离的菌株有较高的淀粉酶活力，产酶条件适用于工农业生产。     

中温和高温环境下乙酸和丙酸厌氧发酵产甲烷动力学特征

采用不同浓度的乙酸和丙酸在中高温下进行厌氧发酵批次试验,采用修正的Gompertz模型和产甲烷的一级动力学模型分析,研究酸浓度和温度对发酵产气动力学的影响。研究表明,当乙酸和丙酸浓度较低时降解较快,高浓度酸抑制产气。乙酸在中温条件下降解较快,质量浓度为5 000 mg/L时中温反应有最大产甲烷速率101 mL/d;质量浓度为10 000 mg/L时高温条件下有最大产甲烷速率77 mL/d,随酸浓度增加,最大产甲烷速率减小,高温反应器对酸的耐受度较高。丙酸在高温条件下更易降解,浓度为4 000 mg/L时,中高温反应均有最大产气速率：78 mL/d（中温）和96 mL/d（高温）。另外,高浓度乙酸和丙酸厌氧降解产气具有滞后性,且随酸浓度的增加滞后期延长,降解过程受到抑制,一级动力学常数减小。温度对厌氧降解的影响大于酸浓度对厌氧降解的影响。     

酶与茶皂素组合液对重金属Cd和Pb复合污染土壤的生态修复作用

[目的]评价α-淀粉酶与茶皂素对污染土壤中的Cd,Pb洗脱效果,为重金属污染土壤的修复提供更准确的科学依据。[方法]以重金属Cd,Pb污染的耕作层土壤为研究对象,利用α-淀粉酶和茶皂素复合淋洗对Cd,Pb污染土壤进行生态修复,并测定土壤淋洗前后Cd,Pb形态的变化。[结果]在反应时间12h,pH 4.0,反应温度30℃,茶皂素溶液和酶溶液配比4∶1条件下,Cd,Pb去除率分别为88.87%,43.97%。通过对修复前后重金属的形态分析,发现酸提取态、可还原物态的重金属较容易去除,淋洗后土壤中Cd和Pb均达到土壤环境质量Ⅱ级标准。[结论]α-淀粉酶和茶皂素组合对土壤中Cd,Pb有明显的去除效果,起到协同、增强作用。表明α-淀粉酶和茶皂素组合在生态修复重金属污染的土壤方面具有一定的应用前景。     

Some enzyme and respiratory activities of tropical soils from new hebrides

Activities of invertase and amylase and respiratory activities of samples of 11 soils from New Hebrides were determined. The soils mostly were under forest and were acid with medium to low C/N ratios. Invertase activities were rather low but amylase activities were similar to those found in New Zealand soils. The ratios of invertase to amylase activities were mostly low.O₂ uptakes mostly responded markedly to glucose. Most values of respiratory quotients were about 1.0. Most, but not all, dehydrogenase activities were strongly related to O₂ uptakes. On an organic C basis, these respiratory activities declined with the depths to which the soils were sampled.Biochemical activities were mostly similar in forest soils derived from basalt and from andesite. Invertase activities were lower in soils under forest than under grassland covers.All biochemical activities were correlated significantly with contents of soil moisture and organic C, less with numbers of aerobic bacteria, and negatively with soil pH. On an organic C basis, none of the biochemical activities was significantly correlated with either soil moisture content or pH.     

Effects of artificial acidification and liming on biomass and on the activity of digestive enzymes in Enchytraeidae (Oligochaeta): Results of an ongoing study

Summary The effects of simulated acid rain and acidification combined with liming on enzymatic activities in the gut of the enchytraied Fridericia sp. were studied under laboratory conditions. Simulated mild (pH 4.4) and strong (pH 3.1) acid rain was applied throughout a 52-day experiment. Liming, at rates of 1500 and 4000 kg CaCO₃ powder ha^-1, was applied once on the 27th day of acid rain. After 52 days, the treatment effects were determined by analysing changes in the fresh body weight of enchytraeids and the activities of amylase (EC 3.2.1.1), xylanase (EC 3.2.1.8), trehalase (EC 3.2.1.28) and C₁-cellulase (EC 3.2.1.91) in the gut. The effects were significant in only a few instances. After acidification, xylanase and trehalase activities decreased. The changes in fresh body biomass were not significant. Amylase and cellulase activities increased slightly, possibly because the acidification had a stimulatory effect on soil amylolytic and cellulolytic microorganisms. After liming, both xylanase activity and the enchytraeid body biomass decreased. This was the only marked evidence of a negative effect on the enchytraeids. The high amylase, trehalase and cellulase activities that were observed might have been caused by intensive digestion of dead acidophilous microflora.     

Wet‐Milling and Dry‐Milling Properties of Dent Corn with Addition of Amylase Corn

A transgenic corn (amylase corn) has been developed that produces an endogenous α‐amylase that is activated in the presence of water and elevated temperature (>70°C). Wet‐ and dry‐milling characteristics of amylase corn were evaluated using laboratory wet‐ and dry‐milling procedures. Different amounts of amylase corn (0.1–10%) were added to dent corn (with the same genetic background as the amylase corn) as treatments. Samples were evaluated for wet‐ and dry‐milling fraction yields using 1‐kg laboratory procedures. Milling yields for all amylase corn treatments were compared with the control treatment (0% amylase corn or 100% dent corn). No significant differences were observed in wet‐ and dry‐milling yields between the control and the 0.1, 1, and 10% amylase corn treatments. Most of the amylase activity (77%) in wet‐milling fractions was detected in the protein fraction. In dry‐milling, amylase activity (68.8%) was detected in endosperm fractions (fines, small grits, and large grits).     

α‐Amylase Inhibitors from Wheat Against Development and Toxigenic Potential of Fusarium verticillioides

Fusarium verticillioides is one of the most important pathogens in maize and is a producer of fumonisin B₁ (FB₁). Although reports of its presence in wheat are scarce, the susceptibility of this cereal to fungus of the same genus motivates interest in investigating compounds present in the grain with inhibitory activity against this species. The aim of this study was to extract α‐amylase inhibitors from wheat and apply them in vitro to evaluate its effect on the development and expression of toxigenic potential of F. verticillioides. The α‐amylase inhibitors, both crude (P₀) and purified (P₁), were applied to in vitro culture containing a pathogen mycelium disc. Mycelial growth of the pathogen, glucosamine content, α‐amylase activity, and production of FB₁ were investigated. All protein extracts of wheat showed the ability to inhibit pathogen growth, especially the extract P₀ from cultivar Quartzo, which resulted in a reduction of glucosamine content (66%) and α‐amylase activity (84%). Furthermore, the protein inhibitors showed antifumonisin effect, reducing by 33 and 47% the mycotoxin production when applied as P₀ and P₁, respectively. These results suggest that α‐amylase inhibitor contributed to resistance against pathogen attack, acting in a diversified manner for each fungal species.