球孢白僵菌Bb174固态发酵产几丁质酶产酶及酶学特征研究

对球孢白僵菌(Beauveria bassiana)Bb174产几丁质酶进行了固态发酵条件及酶学特征的研究.结果表明,以4:1麸皮:蚕蛹粉、蛋白胨1g·L^-1作为产酶最适培养基,在75g培养基中接种3ml液态种子,自然pH下28℃培养2d,酶活可达最高,为126U·g^-1(干培养基).粗酶液的最适反应温度为40℃,最适反应pH5.0,在30～70℃保温1h,得半失活温度48℃.在30～40℃、pH4～6范围内,酶的性质最稳定.根据Lineweaver-Burk作图法,得到该酶的动力学参数K_m为0.52mg·ml^-1,V_m为0.7△E₆₈₀·h^-1.     

球孢白僵菌固态罐发酵产孢培养的研究

建立了一套两升圆柱体玻璃发酵罐的固态通风培养装置及通风系统。以球孢白僵菌Bb371为生产菌,培养基220g(82％麸皮 4．5％黄豆粉 4．5％玉米粉 9％稻壳)作为生产原料,接种30ml,23℃培养温度下,分阶段调节通风量和空气湿度,培养5d,产孢量可达到116亿／g干培养基。     

球孢白僵菌的紫外诱变及几丁质酶高产菌株的筛选

以球孢白僵菌（Ｂｅａｕｖｅｒｉａｂａｓｓｉａｎａ）１３１６为出发菌株,通过２０ｍｉｎ和３０ｍｉｎ的紫外线交替诱变分生孢子,采用透明圈法初筛和摇瓶培养复筛的方法,得到一突变株ＣＨ－１３１６。和原始菌株相比,该突变株的几丁质酶活力提高了近３倍,经传代培养,该菌株的几丁质酶高产特性能稳定遗传。     

球孢白僵菌胞内几丁质酶的分离纯化及性质

球孢白僵菌(Beauveria bassiana)突变株CH-1316细胞裂解液经(NH_4)_2SO_4沉淀,DEAE-纤维素层析及凝胶过滤,分离出一种几丁质酶,该酶的分子量为32000;最适pH为5.0;最适温度为40℃;最适离子强度为0.2mol/L NaCl;Hg²⁺、Fe²⁺是该酶的强抑制剂;该几丁质酶完全不水解纯的片状几丁质,脱矿几丁质也不是该酶的良好底物;该几丁质酶水解几丁寡糖,但不水解几丁二糖;对几丁五糖以上的寡糖水解速度较快,而对几丁三糖和四糖水解速度则慢得多。     

球孢白僵菌两种胞外几丁质酶的诱导和纯化

球孢白僵菌突变株CH-1316在完全培养基中培养至对数前期后转入以胶体几丁质为唯一碳、氮源的液体诱导培养基中继续培养20～25h,几丁质酶被诱导产生;在对数生长期胞外几丁质酶活力最高。发酵液经(NH_4)_2SO_4沉淀、DEAE-纤维素层析和凝胶过滤分离出二种几丁质酶组分,在聚丙烯酰胺凝胶电泳图上显示出两条均一的带,并且每条带都具有几丁质酶活力。几丁质酶1既是外切酶又是内切酶,而几丁质酶2只表现内切酶活力。分子排阻法测得这两种酶的分子量分别为52000和39000。     

分阶段调控关键因素提高白僵菌固态发酵产孢量

采用分阶段调控关键因素策略,对白僵菌固态发酵过程进行优化.优化后最佳发酵模式为:初始含水量为65％,发酵至48 h将含水量调为68％;发酵至20 h和40h分别适当翻料;0～48h(适应期)发酵温度为25℃,48 ～108 h(快速生长期)发酵温度为24℃,108 ～168 h(稳定期)发酵温度为28℃;同时,48 ～108 h按通气比1∶2通气,0～48 h和108 ～168 h不通气.采用上述关键因素调控策略后,白僵菌固态发酵产孢量达18亿孢子/g(干培养基),与优化前相比产孢量提高约360％,效果显著.     

白僵菌固态发酵试验研究*

以产孢量为指标,筛选出白僵菌固态发酵的培养基为:90%麦麸+5%玉米粉+5%黄豆粉,原孢粉的产孢量可达244.7亿/g。在25m³发酵罐内进行浅盘固态发酵,培养基的最适起始含水率为60%~65%;最佳通气条件为:前12h不通气,12~48h通气量为1:2,48~144h的通气量为1:1;而温度只需在12~48±8h内进行控制。常温干燥能最大限度地维持白僵菌的活孢率。     

球孢白僵菌几丁质酶诱发突变的探讨

从球孢白僵菌Beauveria bassiana 017菌株获得了几丁质水解活性较高的突变株。诱变原为紫外线及高能电子束。诱变体为分生孢子及芽生孢子。通过测定在几丁质琼脂平板上透明圈的大小、透明圈直径与菌落直径之比值,选出酶高产菌株,并通过摇瓶培养选育出EU-120高酶活性突变株,其几丁质酶活性比原始菌株提高了近3倍。并发现以上诱变原均能获得较好的诱变效果,在选育过程中加入猩红液可提高透明圈的清晰度。并通过比较几丁质酶对胶体几丁质及“晶体”几丁质的活性,探索酶调节的可能机理。     

Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation

Prawn waste, a chitinous solid waste of the shellfish processing industry, was used as a substrate for chitinase production by the marine fungus Beauveria bassiana BTMF S10, in a solid state fermentation (SSF) culture. Theprocess parameters influencing SSF were optimized. A maximum chitinase yield of 248.0 units/g initial dry substrate (U/gIDS) was obtained in a medium containing a 5:1 ratio (w/v) of prawn waste/sea water, 1% (w/w) NaCl,2.5% (w/w) KH2PO4, 425–600m substrate particle size at 27°C, initial pH 9.5, and after 5 days of incubation. The presence of yeast extract reduced chitinase yield. The results indicate scope for the utilization of shellfish processing (prawn) waste for the industrial production of chitinase by using solid state fermentation.     

白僵菌固态发酵试验研究

以产孢量为指标,筛选出白僵菌固态发酵的培养基为：90％麦麸＋5％玉米粉＋5％黄豆粉,原孢粉的产孢量可达244．7亿／g。在25m^3发酵罐内进行浅盘固态发酵,培养基的最适起始含水率为60％～65％;最佳通气条件为：前12h不通气,12～48h通气量为1：2,48～144h的通气量为1：1;而温度只需在12～48＋8h内进行控制。常温干燥能最大限度地维持白僵菌的活孢率。     

Enzymatic production of N-acetyl-D-glucosamine by solid state fermentation of chitinase by Penicillium ochrochloron MTCC 517 using agricultural residues

The optimization studies for production of chitinase were carried out by response surface methodology (RSM) based on statistics experimental design using three substrates, which were wheat, rice and red gram bran. 2⁴ full factorial central composite design was applied to evaluate optimal combinations of variables. These variables were chitin concentration, initial moisture content, inoculum level, and incubation time. The results of second order polynomial showed that all four variables had significant effect on chitinase production. Maximum chitinase activity was recorded for wheat bran (2443.23 U g⁻¹) than rice (1216.65 U g⁻¹) and red gram bran (961.32 U g⁻¹). An overall 3-fold increase in chitinase activity was achieved using optimized strategies of RSM. Growth of the fungus on all bran particles have been visualized by scanning electron microscopy. These results indicated the potential of Penicillium ochrochloron for economical production of chitinase using agricultural residues. TLC and HPLC analysis of colloidal chitin hydrolysate with partially purified chitinases revealed that the major reaction product was monomeric GlcNAc indicating the potential of these enzymes for efficient production of GlcNAc.     

Extracellular production of L-glutaminase by alkalophilic Beauveria bassiana BTMF S10 isolated from marine sediment

Beauveria sp. BTMF S10 isolated from marine sediment produced extracellular L-glutaminase. Maximal L-glutaminase yield (46.9U/ml) was obtained in a medium supplemented with 1% (w/v) yeast extract and sorbitol, 9% (w/v) sodium chloride and 0.2% (w/v) methionine, initial pH 9.0 and at 27 ^°C after 108h. This enzyme was inducible and growth-associated.     

变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响

[目的]评价球孢白僵菌固体发酵产物的干燥温度对产后分生孢子性能的影响.[方法]采用28℃2和35℃组合的7种恒温或变温处理干燥发酵产物,分析收获的分生孢子质量.[结果]变温干燥可显著降低产后孢子粉的杂菌污染.干燥温度对活孢率和孢子萌发速度影响不一致.35℃恒温干燥5 h后活孢率与新鲜孢子无明显差异,但萌发中时缩短了9.3%.干燥处理提高了孢子对高温和紫外辐射的耐受性.适当的变温干燥比恒温干燥有利于增强孢子抗逆性.干燥温度影响分生孢子胞内海藻糖积累,但其含量与抗逆性无直接相关性.优化干燥温度可提高产后分生孢子毒力.在370～450孢子/mm2剂量下,经28℃ 24 h后升至35℃干燥2 h或35℃恒温干燥5 h的分生孢子对桃蚜的致死中时分别比新鲜孢子缩短了10.6 h和7.5 h.[结论]球孢白僵菌固体发酵产物的干燥温度是影响产后孢子粉杂菌污染、孢子活力、抗逆性和毒力的重要因素.     

施菌方式和寄主状态对白僵菌萌发与侵染的影响

在白僵菌侵染的适宜温度下,菌液处理的侵染进程和寄主死亡速率都较菌粉处理的快,这是由于不同湿度引起的。在0～10℃的自然变温下,孢子不能萌发引起侵染。白僵菌在死虫体上的发展速度比在活虫体上快得多;经冷处理的活虫与新采活虫同样具有抵御侵染的能力。因此,林间不宜选择冬季放菌防治马尾松毛虫.     

球孢白僵菌Mars发酵牛奶产生紫红色物质的初步研究

球孢白僵菌Mars菌株发酵牛乳能使牛乳呈紫红色。但在查氏培养基、马铃薯葡萄糖培养基和牛乳固态培养基中没有紫红色物质产生。培养温度和pH对Mars菌株在牛乳培养基中产生的紫红色物质有明显的影响。28℃、pH在6．0～7．0的范围内Mars菌株能够产生大量的紫红色物质;对牛乳发酵液进行离心处理后,用不同萃取剂对沉淀进行萃取,最后用紫外分光光度计进行扫描,结果表明：沉淀萃取液在330nm左右有吸收峰且丙酮为最佳萃取刹;上清液在500nm左右有吸收峰。     

Kinetic resolutions with novel, highly enantioselective fungal lipases produced by solid state fermentation

Thirty-eight filamentous fungi cultivated under solid state fermentation (SSF) conditions were screened for lipase activity and enantioselectivity in kinetic resolutions of racemic secondary alcohols (rac-1a–c) by acetylation with vinyl acetate performed in organic solvents. Many of the target fungi have not been studied previously for lipase/esterase activity and enantioselectivity. Without special enzyme isolation processes, the room temperature (25 °C) dried SSF cultures as such were tested in the enantiomer selective biotransformations. The majority of these SSF preparations proved to be effective as enantiomer selective biocatalysts exhibiting high but usual enantioselectivities according to the Kazlauskas rule. However, the SSF preparation of Mucor hiemalis origin acted as a selective anti-Kazlauskas catalyst. The best SSF products were successfully applied in preparative scale resolutions.