辐射诱变筛选高生长速率的渐狭蜡蚧菌及其生物学特性研究

本试验利用电子束及γ射线两种射线对渐狭蜡蚧菌(Lecanicillium attenuatum)3166进行诱变处理,研究适于渐狭蜡蚧菌诱变选育的吸收剂量以及诱变菌株生物学特性。结果表明：电子束及γ射线诱变渐狭蜡蚧菌的最适剂量均为200 Gy,渐狭蜡蚧菌的电子束辐射敏感性D10值为191 Gy、γ射线D₁₀值为366 Gy;以产孢速率为指标筛选获得突变菌株8株,其中5株的菌丝生长及产孢速率较出发菌株显著提高,均为电子束诱变所得,产孢速率分别较出发菌株提高了30.45%、31.55%、23.66%、64.83%、68.77%;诱变菌株的胞外几丁质酶比活在培养5 d时达到峰值,诱变株Ⅱ111、Ⅱ164、Ⅱ181的几丁质酶比活分别较出发菌株显著提高29.29%、54.45%、19.18%,其中Ⅱ164酶比活可达1 707.41 U/mg prot;胞外蛋白酶比活在培养7 d时达到峰值,Ⅱ111、Ⅱ164、Ⅱ181的蛋白酶比活分别较出发菌株显著提高17.98%、13.17%、16.50%,其中Ⅱ111酶比活达到30.71 U/mg prot,且渐狭蜡蚧菌诱变株生长速率与胞...     

枯草杆菌BS-98分泌的抗真菌蛋白的分离纯化及其部分性质的研究

拮抗菌（Bacillussubtilis）BS-98菌株在BPY液体培养基中产生的蛋白质经硫酸铵分级盐析、SephsdexG-100柱层析、DEAE-纤维素柱层析和SephadexG－100柱再层析后,分离纯化得到的抗菌蛋白在电泳中显现出三条带。经初步纯化的抗菌蛋白对热稳定,对蛋白酶部分敏感。抑菌谱测定表明,抗菌蛋白对芦笋茎枯病菌、小麦赤霉病菌、棉花枯萎病菌、棉花黄萎病菌、灰霉菌、立枯丝核菌等病原真菌及黄瓜角斑病菌都具有强烈的抑制作用。     

拮抗菌BS—98分泌抗菌蛋白的条件及其发酵液特性

由本室分离得到一株强烈抑制芦笋茎枯病等植物病原真菌的拮抗菌ＢＳ—９８菌株（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）。用环柱法检测该菌株的抗菌活性表明,该菌株除抑制芦笋茎枯病菌ＰｈｏｍａａｓｐａｒａｇｉＳａｃｃ外,对小麦赤霉病菌（Ｆｕｓａｒｉｕｍｇｒａｍｉｎｅａｒｕｍ）,棉花枯萎病菌（Ｆｕｓａｒｉｕｍｏｘｙｓｐｏｒｕｍｆｓｐ．Ｖａｓｉｎｆｅｃｔｕｍ）、棉花黄萎病菌（Ｖｅｒｔｉｃｉｌｌｕｍａｌｂｏ—ａｔｒｕｍ）、黄瓜灰霉病菌（Ｂｏｔｒｙｔｉ     

不同基质多代培养对蚧虫病原真菌蜡蚧霉致病力的影响

【目的】研究昆虫病原真菌蜡蚧霉Lecanicilliurn lecanii(Zimmermann.)菌株No.V3.4504在不同培养基上继代培养,对菌种的菌落生长特性、胞外酶活力和对蚧虫致病力的影响。【方法】试验菌种蜡蚧霉菌株No.V3.4504是从染病蚧虫上分离的。试验蚧虫是沙里院褐球蚧Rhodococcus sariuoni Borchsenius和日本龟蜡蚧Ceroplastes japonicus Green。采用7种培养基继代培养多代。观察菌落形态特征、测定生长速率、产孢量、胞外蛋白酶和几丁质酶活性及对蚧虫的致死率。【结果】在PDA培养基上,菌落生长速率最快,但产孢量最低,胞外蛋白酶和几丁质酶的活性均呈逐代下降趋势,对两种蚧虫致死率也最低;增加蛋白胨对改善菌种致病力没有明显效果;在增加蚧虫尸体的D、E、F培养基上,菌落生长速率虽然较慢,但产孢量上升为8.83×106-9.13×106孢子/cm2。蛋白酶和几丁质酶的活性平均达到2.16-2.13 U/g和1.01-1.03 U/g,对两种蚧虫的致死率分别在55%-58%和39%-42%;在活蚧虫上连续培养3代,蛋白酶和几丁质酶的活性最高,为3.08-2.92 U/g和1.45-1.42 U/g,是PDA培养基上的1.6倍。对两种蚧虫的致死率也最高,分别达到71.30%和58.89%。蛋白酶和几丁质酶的活性与蚧虫死亡率呈正直线相关关系。【结论】采用PDA培养基连续多代培养会引起菌株No.V3.4504明显退化;在培养基中加入蚧虫尸体,对于保持菌种活力有明显效果;在活蚧虫体上继代培养对复壮菌种,提高菌种毒力的效果最佳。     

类芽孢杆菌BD3526抑菌活性物质的初步分离及性质测定北大核心CSCD

本实验以一株类芽孢杆菌BD3526为研究对象,采用丙酮浸提菌体得到抑菌活性粗品,通过Sephadex LH-20对样品进行初步纯化,结果表明粗品对藤黄微球菌CGMCC1.1848的最低抑菌浓度(MIC)为12.5 mg/m L,对金黄色葡萄球菌CGMCC1.879和单增李斯特菌CGMCC1.9136的MIC为25 mg/m L;Sephadex LH-20纯化样品对藤黄微球CGMCC1.1848的MIC为1 mg/m L,对金黄色葡萄球菌CGMCC1.879和单增李斯特菌CGMCC1.9136的MIC为2 mg/m L。样品经链霉蛋白酶处理后,活性基本消失。经加热、酸碱、胰蛋白酶、胃蛋白酶、脂肪酶、蛋白酶K处理后,活性没有明显变化。因此,类芽孢杆菌BD3526产生的是一种细菌素类的抑菌物质,该抑菌物质在食品和医疗行业有广阔的应用前景。     

珍珠柴粗提物对13种植物病原菌的抑制活性

以生长速率法测定了珍珠柴7种溶剂粗提物对棉花黄萎病菌V991、棉花黄萎病菌V43-1、棉花枯萎病菌、茄子黄萎病菌、稻曲病病菌、苹果黑星病菌、番茄枯萎病菌、番茄叶霉病菌、黄瓜黑星病菌、西瓜炭疽病菌、芦笋茎枯病菌、扩展青霉和梨黑斑病菌13种植物病原菌的抑制活性。结果表明,不同溶剂粗提物的抑菌活性差异很大,某些溶剂粗提物对个别病菌表现出较强的抑制作用,如水粗提物对棉花黄萎病菌V991,石油醚粗提物对苹果黑星病菌的抑制率分别达100%和91.89%;有的溶剂粗提物的病菌抑制作用较弱,如二氯甲烷粗提物对13种病菌的抑菌率均较低;有些还表现出一定的促进生长作用,如甲醇粗提物对梨黑斑病菌表现为明显的促进生长作用。     

虫生真菌LL-01鉴定及对两种检疫性粉蚧的致病性

【目的】为丰富南洋臀纹粉蚧Planococcus lilacinus和石蒜绵粉蚧Phenacoccus solani的生防菌资源。【方法】本研究从感病南洋臀纹粉蚧上分离生防菌,采用基因序列分析方法进行种类鉴定,并在室内优化其培养条件,评估其对这2种检疫性粉蚧的致病性。【结果】分离得到1株编号为LL-01的虫生真菌,经r DNA-ITS、18S r DNA和Nad1序列分析确定为蜡蚧轮枝菌;该菌的生长和产孢最适的温度为26℃,光周期为6L︰18D,碳源为果糖,氮源为干酪素,此条件下培养10 d的蜡蚧轮枝菌菌落直径和产孢量分别可达4.66 cm和3.16×10⁸孢子/cm8孢子/cm²;其侵染不同虫龄南洋臀纹粉蚧和石蒜绵粉蚧10 d后的LC_(50)分别为9.80×102;其侵染不同虫龄南洋臀纹粉蚧和石蒜绵粉蚧10 d后的LC_(50)分别为9.80×10⁴-9.17×104-9.17×10⁵孢子/m L和5.00×105孢子/m L和5.00×10⁴-5.30×104-5.30×10⁵孢子/m L。浓度为1.00×105孢子/m L。浓度为1.00×10⁸孢子/m L的蜡蚧轮枝菌侵染南洋臀纹粉蚧和石蒜绵粉蚧后第10天,其累计致死率分别为84.09%-97.62%和89.89%-98.85%,LT_(50)分别为3.70-5.84 d和3.48-5.14 d;在侵染第5天时,蛋白酶和几丁质酶活性分别达峰值19.44 U/m L和15.01 U/m L,脂肪酶活性在侵染第6天时达到峰值7.68 U/m L。【结论】蜡蚧轮枝菌LL-01生长速度快、产孢量高,对这2种检疫性粉蚧的致病性强。     

桑氏链霉菌几丁质酶ChiKJ40基因的克隆表达及其抑菌作用

【背景】目前关于桑氏链霉菌(Streptomyces sampsonii)生防基因的研究不多,仅从其基因组中克隆了2个几丁质酶基因片段,其单个几丁质酶的完整基因序列相关研究未见报道。【目的】克隆S.sampsonii KJ40的几丁质酶基因Chi KJ40并进行原核表达,纯化重组蛋白并研究其抑菌作用。【方法】采用PCR扩增法从S.sampsonii KJ40中克隆几丁质酶基因Chi KJ40,连接到表达载体p ET-32a,导入Escherichia coli BL21(DE3)进行诱导表达。使用His标记蛋白质微量纯化试剂盒对重组几丁质酶进行纯化,Bradford蛋白浓度测定试剂盒测定粗酶液和纯化酶液的浓度,几丁质酶试剂盒测定粗酶液和纯化酶液的几丁质酶活性。观察重组几丁质酶对桉树焦枯病菌(Cylindrocladium scoparium)、栗疫病菌(Cryphonectria parasitica)、链格孢菌(Alternaria alternate)、紫丝核菌(Rhizoctonia violacea)几种致病真菌的抑菌作用。【结果】Chi KJ40基因(登录号为MF434484)在E.coli中经IPTG诱导表达,获得42 k D的重组几丁质酶,不同浓度IPTG在37°C诱导3 h,蛋白产量无明显变化。0.2 mmol/L IPTG 16°C诱导过夜,重组几丁质酶主要以可溶性形式存在于上清,小部分以包涵体存在于沉淀中。粗酶液几丁质酶活性为0.080 U/m L,酶比活力为0.041 U/mg,纯化酶液几丁质酶活性为0.046 U/m L,酶比活力为0.115 U/mg,纯化倍数为2.8,酶活回收率为57.5%。重组几丁质酶处理后,C.scoparium、C.parasitica和A.alternata菌丝细胞出现分节、膨胀,R.violacea菌丝溶解且部分被破坏成碎片。【结论】Chi KJ40基因的研究补充了S.sampsonii的生防背景,为几丁质酶基因找到了新的来源,并为其应用奠定了理论基础。     

抑制西瓜蔓枯病菌的生防真菌筛选、鉴定及发酵条件优化

筛选对西瓜蔓枯病菌具有抑制作用的生防真菌,优化其发酵条件以提升发酵液抑菌效果。以平板对峙试验和摇瓶培养抑菌试验筛选拮抗真菌;根据形态学特征和18S rDNA序列分析进行菌株鉴定;通过扫描电镜观察对西瓜蔓枯病菌的寄生作用,以单因素试验和响应曲面法确定其最适发酵条件,在此基础上,通过盆栽试验研究其防病效果。结果表明:在所筛选到的9株生防真菌中M1菌株的抑菌率最高,其形态学特征与烟管菌(Bjerkandera adusta)相符,18S rDNA序列分析显示其与烟管菌在系统发育树上聚在一支,因此将菌株M1鉴定为黑管菌属(Bjerkandera)、烟管菌。在扫描电镜下观察到烟管菌能够直接穿透西瓜蔓枯病菌菌丝,对其发酵条件优化后确定了最佳条件组合:C/N为7.1,pH为7.4,装瓶量为44%,时间为19d,转速为180r/min,温度为29℃,在此条件下抑菌率为52.64%,高于未经优化的抑菌率。温室盆栽中对西瓜蔓枯病菌的防病效果达到74.3%,高于多菌灵处理。烟管菌作为生防真菌对西瓜蔓枯病菌具有较好的抑制作用,条件优化后进一步提升了抑菌效果,在生物防治中具有巨大应用潜力。     

金属离子对蜡蚧菌几丁质酶活力的影响

以蜡蚧菌(Ll)发酵液为材料,经分离纯化获得Ll几丁质酶(EC3.2.1.14)制剂.研究了金属离子对Ll几丁质酶活力的影响.结果表明,K+、Mg2+、Zn2+、Ca2+和Fe3+对几丁质酶活性有明显的促进作用,而Na+和Cu2+完全抑制几丁质酶的活性;Mn2+在低浓度时对酶有激活作用,随着浓度的升高表现出抑制作用;Fe2+和Ba2+的浓度低于0.5 mmol/L时对酶起抑制作用,而高于该浓度时则对酶有激活作用.     

Chitinolytic enzymes from<Emphasis Type="Italic">Clostridium aminovalericum</Emphasis>: Activity screening and purification

A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.     

Characterization of a chitinolytic enzyme from Serratia sp. KCK isolated from kimchi juice

The novel chitinolytic bacterium Serratia sp. KCK, which was isolated from kimchi juice, produced chitinase A. The gene coding for the chitinolytic enzyme was cloned on the basis of sequencing of internal peptides, homology search, and design of degenerated primers. The cloned open reading frame of chiA encodes for deduced polypeptide of 563 amino acid residues with a calculated molecular mass of 61 kDa and appears to correspond to a molecular mass of about 57 kDa, which excluded the signal sequence. The deduced amino acid sequence showed high similarity to those of bacterial chitinases classified as family 18 of glycosyl hydrolases. The chitinase A is an exochitinase and exhibits a greater pH range (5.0-10.0), thermostability with a temperature optimum of 40 degrees C, and substrate range other than Serratia chitinases thus far described. These results suggested that Serratia sp. KCK chitinase A can be used for biotechnological applications with good potential.     

Deficiency of current methods in assaying endochitinase activity

Since chitin is degraded by a combination of both endo- and exochitinases, it is likely that both enzymes will be present in a crude extract. Currently used substrates for detecting endochitinase activity suffer from the fact that they could easily be digested by the contaminating exochitinase, thus giving either a false-positive or an inaccurate reading of the endochitinase activity. Using Photorhabdus luminescens, a bacterium symbiotically associated with insect-parasitic nematode Heterorhabditis bacteriophora as an exemplary system, we have identified these two chitinases by a simple "fluorimetric zymography" procedure. The exochitinase is a metalloenzyme and its activity is inhibited by 1,10-phenanthroline. Once the exochitinase activity is detected in a crude extract, its contribution must be eliminated before accurate determination of the endochitinase activity can be carried out. Specific properties of these enzymes including the pH activity profile, the requirement of metal ions for activity, and the molecular weight of the enzymes are among the factors to be considered in developing assaying procedures for endochitinase.     

Chitinolytic enzymes produced by ovine rumen bacteria

Two strains of clostridia, isolated from the rumen fluid of sheep as potential antagonists toward anaerobic fungi showed a complete array of chitinolytic enzymes. Enzyme tests in cultures demonstrated endochitinase, exochitinase,N-acetylglucosaminidase, chitosanase and chitin deacetylase activities mainly in the extracellular fractions. In all samples, the highest was the activity of exochitinase (600–1100 nmol mL⁻¹ h⁻¹); the activity of endochitinase (280–500 nmol mL⁻¹ h⁻¹) was also significant. Chitinases were stimulated in the presence of reducing compounds and no dependence on cations was observed. In both strains different isoforms of chitinases of molar mass 36–96 kDa were detected. The chitinases from our isolates lyzed cell walls of anaerobic fungiin vitro and inhibited the activity of fungal β-1,4-endoglucanases. Of the two bacteria examined, one was more effective in both antifungal effects.     

Enhanced degradation of α-chitin materials prepared from shrimp processing byproduct and production of N-acetyl-D-glucosamine by thermoactive chitinases from soil mesophilic fungi

Soil isolates of mesophilic Penicillium monoverticillium CFR 2, Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 were cultivated in solid state fermentation (SSF) using wheat bran solid medium supplemented with α-chitin in order to produce chitinolytic enzyme. Under SSF cultivation, maximum enzymes (U/g IDS) production was 41.0 (endo-chitinase) and 195.4 (β-N-acetylhexosaminidase) by P. monoverticillium, 26.8 (endo-chitinase) and 222.1 (β-N-acetylhexosaminidase) by A. flavus and 13.3 (endo-chitinase) and 168.3 (β-N-acetylhexosaminidase) by F. oxysporum after 166?h of incubation. The crude endo-chitinase and β-N-acetylhexosaminidase derived from A. flavus and F. oxysporum revealed optimum temperature at 62?±?1°C, but the enzymes from P. monoverticillium showed optimum temperature at 52?±?1°C for maximum activity. Several fold increase in endo-chitinase and β-N-acetylhexosaminidase activities in the crude enzymes preparation was achieved after concentrating with polyethylene glycol. The concentrated crude chitinases from P. monoverticillium, A. flavus and F. oxysporum, respectively yielded 95.6, 96.6 and 96.1?mmol/l of N-acetyl-D: -glucosamine (GlcNAc) in 48?h of reaction from colloidal chitin. While, the crude enzyme preparations of P. monoverticillium, A. flavus and F. oxysporum produced 10.11, 6.85 and 10.7?mmol/l of GlcNAc respectively, in 48?h of reaction from crystalline α-chitin. HPLC analysis of colloidal chitin hydrolysates prepared with crude chitinases derived from P. monoverticillium, A. flavus and F. oxysporum revealed that the major reaction product was monomeric GlcNAc (~80%) and a small amount of (GlcNAc)(4) (~20%), indicating the potential of these enzymes for efficient production of GlcNAc from α-chitin.     

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus chitonophagus</Emphasis>

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80°C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.     

Chitinase from Bacillus thuringiensis subsp. pakistani

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Studies on the Cloning and Expression of Bbchit1 Gene of Beauveria bassiana NCIM 1216

Beauveria bassiana is a biocontrol agent which shows entomopathogenecity on insect pests especially the Lepidopterons invading the agriculturally important crops. The mode of infection is through the cuticle by degrading the chitin present on it which is enabled by the exochitinase enzyme of Bbchit1 gene. A good quality genomic DNA was isolated from Beauveria bassiana NCIM 1216 and amplified with specific primers to isolate the gene corresponding to Bbchit1 which codes for the exochitinase enzyme that is responsible for pathogenesis. The Bbchit1 gene of B. bassiana was transformed with the binary plasmid pBANF-bar-pAN-Bbchit1, in which the Bbchit1 gene was placed downstream of the constitutive gpd promoter, which was mediated by A. tumefaciens, and transformants were selected on the basis of herbicide resistance. Fifty herbicide resistant colonies were obtained and analyzed. The exochitinase produced by these transformants was observed maximum on the 7th day of inoculation in both which was 0.09 μmol/ml/min for the purified fraction and 0.06 μmol/ml/min for the crude extract. The chitinolytic activity was observed maximum at pH 5 and at temperature of 40°C. The genetically modified pure form can be used in the production of transgenic plants and in bringing out commercial formulation for the control of Lepidopteran pests.