层生镰刀菌深层发酵产甲壳素脱乙酰酶的初步研究

壳聚糖在医药、农业、食品等领域有广泛用途。甲壳素脱乙酰酶(CDA)是生物法生产壳聚糖的关键酶。本文首次报道层生镰刀菌深层发酵生产CDA,并研究了层生镰刀菌发酵产CDA的关键培养条件。通过单因素试验确定层生镰刀菌发酵产CDA的4个关键基质参数为:酵母膏(A)、乳糖(B)、硫酸亚铁(C)和甲壳素(D)。进一步采用Box-Behnken设计及响应面分析法对各参数及其交互作用进行了研究。结果显示,A、B、C 3因素及BD的交互作用对CDA得率的影响均为极显著水平(P0.01),得到预测CDA酶活的回归模型。经响应面最优分析,对应4因素的最佳水平为:酵母膏10.57g/L、乳糖10.63g/L、硫酸亚铁5.48g/L、甲壳素10.22/L。在该条件下,CDA酶活可达17.61/mL。     

甲壳素脱乙酰酶产生菌产酶条件的优化

采用斜面培养和液体发酵培养产甲壳素脱乙酰酶的真菌构巢曲霉,并且研究了产酶条件。结果表明,构巢曲霉的最适产酶条件为：发酵培养基初始pH值为6.5、发酵时间为96h、培养温度为31℃、碳源浓度为2%、氮源浓度为2%、金属离子浓度为0.01mol/L、接种量为6%。     

甲壳素脱乙酰酶的研究概况及展望

甲壳素脱乙酰酶(chitin deacetylase,CDA,E.C.3.5.1.41)是一种能催化脱去甲壳素分子中N-乙酰葡糖胺链上的乙酰基,使之变成壳聚糖的酶。而壳聚糖因其独特的性质被广泛应用于医药、食品、化工、化妆品等行业。对CDA的来源、分离纯化和酶学性质、结构和催化机制、基因的克隆表达及应用前景等方面的研究进行了综述,并分析出今后的主要研究方向应在CDA基因的克隆表达、CDA底物的改造及CDA的结构和催化机制等方面。     

枯草芽孢杆菌甲壳素脱乙酰酶的酶学性质

研究了甲壳素脱乙酰酶的热稳定性及酶的反应体系作用条件:酶(干重)添加量为40 mg.L-1,甲壳素底物(干重)质量浓度为75 mg.L-1,反应时间为90 m in,金属离子Mg2+对酶活有激活作用,在最适宜反应条件下的酶活为2250 U.L-1。甲壳素脱乙酰酶的酶解方式为外切酶型,酶降解终产物对酶活力有抑制作用,酶对甲壳素有一定的降解作用。     

湘江河岸土壤中高产甲壳素脱乙酰酶菌株的筛选及鉴定

【目的】甲壳素脱乙酰酶(CDA)是将天然甲壳素生物转化为可商品化利用的壳聚糖的关键酶。本文旨在从湘江河岸的土壤中筛选可高产CDA的新菌株。【方法】以甲壳素为唯一碳源,利用4'-硝基乙酰苯胺为显色剂,通过变色圈法进行产CDA菌株初筛,产酶活性分析复筛;通过形态学和ITS区序列特征对菌株进行鉴定。【结果】从湘江(长沙段)河岸边的土壤中分离出的117株菌株中筛选到可产CDA的菌株30株,其中4株具有较强产CDA的能力。进一步经发酵产酶分析验证,菌株A1具有较强的产CDA能力,其胞外CDA酶活高达13.21 U/m L。结合形态学和ITS区序列特征,菌株A1初步鉴定为层生镰孢菌。【结论】从湘江河岸边的土壤中筛选到可高产CDA的菌株A1,具有较好的开发应用前景。     

枯草芽胞杆菌甲壳素脱乙酰酶的筛选及酶学性质*

从海洋泥土中分离出产甲壳素脱乙酰酶菌株,确定该菌株为产碱属芽孢杆菌,其产酶适宜培养条件为：pH4．0,添加金属离子Ca^2 ,培养时间为80h,温度为350℃。所得甲壳素脱乙酰酶作用的最适温度为40℃～50℃,最适pH为4．5-5．0之间。     

蚧镰孢菌的生长及产孢研究

蚧镰孢菌孢子萌发及产孢的最适温度是 2 8℃ ,菌生长的适温是 2 4℃～ 2 6℃。孢子萌发受 pH值影响较小 ,菌生长以 pH6.5～ 8.5为最适 ,而 pH8.5时产孢最多。菌的生长也可以调节培养液的pH值 ,使其达到最适生长的 pH范围。光照对该菌的生长及产孢也有一定影响。蚧镰孢菌能利用多种碳源和氮源 ,也能以几丁质为唯一碳源和氮源生长 ,但生长很差。Ca2 、Fe2 等金属离子以及硫胺素、核黄素、叶酸等维生素的加入可使产孢量增加 ,而维生素的作用更明显。该菌的产孢高峰期一般在 2 1天左右 ,而且在相同条件下 ,接种量越大 ,产生最大量孢子所需的时间越短。     

甘肃大葱贮藏期镰孢菌腐烂病病原鉴定

【目的】大葱在贮藏期频繁发生镰孢菌腐烂病,损失严重。明确该病害病原种类对病害防治具有重要意义。【方法】利用组织分离法对采集自甘肃省兰州市(区)蔬菜市场的16份大葱贮藏期镰孢菌腐烂病病样进行病原物的分离、纯化培养,经单孢分离后根据形态学特征,再结合r DNA-ITS、EF-1a(tef)基因序列分析的方法进行鉴定。【结果】共分离得到80株镰孢菌,经鉴定分属3个种,即层出镰孢菌(Fusarium proliferatum)、尖孢镰孢菌(F.oxysporum)和燕麦镰孢菌(F.avenaceum),其中层出镰孢菌为大葱镰孢菌腐烂病的优势致病菌,分离频率为52.50%。对兰州白葱不同部位进行致病性测定,结果表明层出镰孢菌对大葱鳞茎的致病力最强,而燕麦镰孢菌对大葱鳞茎的致病力最弱。【结论】3种镰孢菌作为该病害的病原,属国内首次报道。     

中国的膨孢组镰孢菌

膨孢组镰孢菌Fusarium在自然界中广泛分布。该组包括4个种:木贼镰孢菌F.equiseti,藨草镰孢菌F.scirpi,长脚镰孢菌F.longipes和紧致镰孢菌F.compactum。这4个种均产生腹背不平行弯曲的大孢子。分离获得并描述了其中的3个种。木贼镰孢菌F.equiseti是镰孢菌中最常见的种之一,它产生典型的腹背不平行弯曲的大孢子,大孢子的顶细胞和足跟状基细胞明显伸长,菌落因缺乏红色素而呈黄褐色。藨草镰孢菌F.scirpi是比较少见的种类,它的典型特征是在典型的十字形产孢细胞上产生大量的小型分生孢子。由于它在PDA培养基上容易发生小孢子缺乏型的变异,因此,常常被错误地鉴定为木贼镰孢菌F.equiseti。长脚镰孢菌F.longipes的大孢子最容易与其他种的大孢子区分,它的顶细胞和基细胞均极度延长。当长脚镰孢菌F.longipes菌落因为变异而失去产生红色素的能力时,也容易与木贼镰孢菌F.equiseti混淆。基于该组大孢子的典型特征,作者将锐顶镰孢菌F.acuminatum排除在该组之外。     

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.     

Purification and Characterization of Extracellular Chitin Deacetylase from Colletotrichum lindemuthianum

Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mm sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with the blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS–PAGE and gel filtration, respectively, suggesting that the enzyme is a single polypeptide. The optimum temperature was 60°C and the optimum pH was 11.5–12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer and dimer, and inactive with N-acetylglucosamine. The K_m and k_cat for glycol chitin were 2.55 mm and 27.1s^?1, respectively, and those for chitin pentamer were 414 μm and 83.2s^?1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis–Menten kinetics.     

甲壳质脱乙酰基酶的研究概况及进展

甲壳质脱乙酰基酶（ｃｈｉｔｉｎｄｅａｃｅｔｙｌａｓｅ）最初是从真菌毛霉（Ｍｕｃｏｒ．ｒｏｕｘｉ）分离纯化的一种乙酰基转移酶。这种酶可以催化脱去甲壳质分子中Ｎ－乙酰葡糖胺链上的乙酰基,而使之变成壳多糖［１］。除几种真菌外,在昆虫中也发现了这种酶的存在［２］。真菌的甲壳质脱乙酰基酶主要参与真菌细胞壁的形成［３］,还与真菌自溶的过程中的细胞壁裂解有关［４］。最近又发现它参与植物和病原微生物的相互作...     

Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on calmodulin gene sequences

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534 bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants.     

Natural mycosis of mango leafhoppers (Cicadellidae: Hemiptera) by Fusarium sp.

Mango leafhoppers that feed on inflorescences and young shoots of mango (Mangifera indica L.) were found mycotized under natural conditions in Bangalore, India. Isolation and characterisation of the etiological agents by sequencing of the Translation Elongation Factor-1α gene, revealed 99% identity with the plant pathogenic fungus Fusarium proliferatum. This is an early report on the Fusarium associated entomopathogenicity in different mango leafhopper species.     

Factors affecting the growth of Fusarium proliferatum and the production of fumonisin B1: oxygen and pH

Fumonisins are mycotoxins produced primarily by Fusarium moniliforme and Fusarium proliferatum in corn. In liquid culture, production of fumonisin B₁ (FB₁), the most common moiety of the family of fumonisins, can be obtained using a defined medium that is nitrogen-limited. Under nitrogen-limited conditions both growth and the production of FB₁ were greatly influenced by pH and aeration. At pH above 5.0, F. proliferatum grew normally but produced little FB₁ (<100 μg m⁻¹). At pH below 5.0, there was less growth but substantially more FB₁. Below a pH of 2.5, both growth and metabolism were slower with very little FB₁ produced. When the optimal pH range of between 3.0 and 4.0 under well-aerated conditions was used, both growth and FB₁ production were high. However, under oxygen-limited conditions, less growth occurred, glucose consumption was increased, and no FB₁ was produced. Received 16 May 1997/ Accepted in revised form 03 September 1997     

甲壳素酶学研究现状

本文介绍了甲壳素在生物合成和分解代谢过程中所涉及的相关酶,如甲壳素合成酶、甲壳素水解酶和其它相关酶,讨论了它们在分离纯化、结构鉴定、作用机制与模型、酶的固定化、基因工程以及应用等方面的研究现状和进展,对甲壳素的研究开发以及相关领域具有理论和实际意义 。     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.