短小芽孢杆菌HZbp脂肪酶基因的克隆表达

以短小芽孢杆菌HZbp总DNA为模板以PCR的方式获得512 bp的脂肪酶基因,并在该基因的两端引入了EcoR1和Sal1的酶切位点,将该基因与大肠杆菌表达质粒pSE380连接,获得重组质粒pSE380-BPL。重组质粒转入大肠杆菌表达细胞株BL21,获得工程菌株BL21-BPL。序列分析显示所克隆的基因具有脂肪酶的保守G-X-S-X-G序列,SDS-PAGE电泳显示该脂脂肪酶的分子质量约为20 kDa。在LB培养基中,IPTG诱导浓度为1.0 mmol/L,33℃诱导培养10 h后,发酵液酶活达到8 U/mL。     

Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non‐covalent immobilization to poly(vinylimidazole)/clay hydrogel

The characterization of the hydrogel was performed using Fourier‐transform infrared spectroscopy, X‐ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7‐derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K_m, V_m, and k_cat of immobilized enzyme were 4.4, 1.7, and 7.5‐fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16^th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG^≠ (activation free energy) and ΔG^≠_E‐T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG^≠_ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2‐fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS^≠ value implied that the immobilized form of the enzyme was more ordered than its free form.     

Glucuronidation of Lipophilic Substrates: Preparation of 3-Benzo[a]pyrenyl-β-D-glucopyranosiduronic Acid in Multimilligram Quantities by Microsomal UDP-Glucuronyl Transferase

convenient method for the enzyraic conversion of multimilli-gram quantities of 3-hydroxybenzo[a]pyrene to 3-benzo[a]pyrenyl-β-D-glucopyranosiduronic acid in 90% yield is described. Commer-cially available freeze-dried rabbit liver microsomes were incubated in the presence of UDPGA, 3-hydroxybenzo[a]pyrene, and Triton X-100 detergent (Figure 1). The course of the biosynthetic reaction was followed by fluorimetry. The glucuronide product was extracted from the acidified incubation supernate with ethyl acetate and the acid function of the glucuronide was utilized in an acid-base extraction procedure to purify the glucuronide from biological and unreacted starting material. The glucuronide pre-cipitated from ethyl acetate and was collected by centrifugation.

High pressure liquid chromatography and spectroscopic techniques were used to verify the structure and purity of 3-benzo[a]-pyrenyl-β-D-glucopyranosiduronic acid.     

Roost preferences and foraging ranges of the eastern forest bat Vespadelus pumilus under two disturbance histories in northern New South Wales,Australia

Little is known about the habitat requirements of Australian bats; however, this information is needed to make better‐informed decisions when systems are disturbed. This study contrasts the roosting and foraging ecology of the eastern forest bat Vespadelus pumilus (Vespertilionidae), one of Australia’s smallest bats, between two sites of differing disturbance history on the mid‐north coast of New South Wales. Lorne Flora Reserve (182 ha) is primarily old‐growth forest surrounded by regrowth forest and eucalypt plantations, while Swans Crossing is dominated by regrowth and eucalypt plantations established on part of an old dairy farm. A total of 38 bats were tracked during the maternity and mating seasons at the two sites. Roost preferences were determined by comparing trees used as roosts with those randomly available, while foraging bats were triangulated from fixed stations at night. Bats tracked at Lorne Flora Reserve typically roosted in hollows within large, mature trees and showed a strong preference for roosting and foraging (females only) within the Reserve. Lactating females at Swans Crossing roosted in hollows of remnant rainforest trees within a gully and dead eucalypts, while males often roosted in understorey trees (such as Acacia). Dead trees were frequently used as roosts at both sites. Under both disturbance histories, the mean distance of female maternity roosts from creeks was 20 m, indicating that riparian zones provide important roosting habitat for V. pumilus. However, roosts shifted to the mid‐slope prior to winter when bats mate. Retention of mature trees in a variety of topographic locations may allow behavioural adjustments with the seasons. Bats caught in the regrowth forest also foraged there, with foraging ranges averaging just 5.3 ha (n = 10), indicating that regrowth is used by this bat for both foraging and roosting.     

短小芽孢杆菌degQ基因的克隆与鉴定

degQ基因编码一个由46个氨基酸组成的多肽,能增强许多芽孢杆菌胞外酶基因的表达．以pMK4作克隆载体构建短小芽孢杆菌基因文库,并用DNA探针原位杂交法从中钓出degQ基因．对克隆基因的DNA序列进行了分析并证明克隆的短小芽孢杆菌degQ基因具有增强枯草杆菌蛋白酶和果聚糖蔗糖酶基因表达的能力．degQ基因克隆有助于研究芽孢杆菌的正调控机理并可望提高外源基因在芽孢杆菌中表达．     

短小芽孢杆菌作为芽孢杆菌属基因工程受体菌的研究

以质粒pUB110 DNA转化B. pumilus 289原生质体,转化频率为10~(-3)—10~(-9)与B.tubtilis 168系统相当;但B.pumilus 289原生质体的再生频率(0.3—12.0％)略低于B.subtilis 168(1.53—24.16％);在无选择压力条件下质粒pUB110在B.pumilus 289中经过45个世代周期,自发丢失率小于3％,同于B.subtilis 168系统。外源基因在B.pumilus 289中经25个世代周期丢失率低于5％,而在B.subtilis 168系统中则高达24％;外源基因的表达水平亦高于B.subtilis 168系统。因此,B.pumilus 289是一个值得进一步开发的基因工程受体系统。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

Factors affecting the production of prednisolone by immobilization of Bacillus pumilus E601 cells in poly(vinyl alcohol) cryogels produced by radiation polymerization

To increase the yield percent of prednisolone from hydrocortisone (cortisol), Bacillus pumilus E601 (a radioresistant microorganism) was incorporated into poly(vinyl alcohol) (PVA) cryogel grafted with hydroxyethyl-methacrylate (HEMA) as a crosslinking agent. The polymer was prepared by a radiation polymerization technique at 20 kGy from Co-60 source. The optimum temperature for the biotransformation of hydrocortisone by free cells, poly(PVA)/HEMA, and poly(PVA)/HEMA /N-isopropylacrylamide (N-IPAAm) was 30 °C. The highest yield % of prednisolone was obtained by immobilization of the cells on poly(PVA/HEMA), the addition of N-IPAAm to poly(PVA/HEMA) protected the immobilized cells from temperatures above 35 °C during the fermentation process. The optimal pH (buffered pH) of the biotransformation of hydrocortisone by immobilized and free cells was 7.0, but the maximum yield of prednisolone (60%) was obtained by immobilized cells in comparison with free cells (42%) also at pH 7.0. The prednisolone yield reached 60–65% with 1,2-propanediol cosolvent containing media and 60–62% in the case of ethanediol cosolvent containing media at 1% (v/v) of both cosolvents. 10 mg/50 ml Tween 80 the medium increased the prednisolone yield by only 1.1-fold compared with the control. The maximum bioconversion efficiency was obtained at a substrate concentration of 20 mg/50 ml medium. Stability studies showed that the immobilized cells can be used for seven times without any significant decrease in activity.     

l-tryptophan-assisted PGPR-mediated induction of drought tolerance in maize (Zea mays L.)

Drought is an important abiotic stress that limits the plant growth and productivity. Present investigation was aimed that plant growth-promoting rhizobacteria (PGPR) isolated from moisture-stressed area impart drought tolerance in plants and tryptophan may improve their efficiency. Pseudomonas sp. (1), Bacillus cereus and Bacillus pumilus (B. pumilus) were isolated from maize rhizosphere grown in irrigated fields, semi-arid region and arid region, respectively. Proteus sp. and Pseudomonas sp. (2) were isolated from rice rhizosphere grown in irrigated fields and raised bed. B. pumilus produced 5× more abscisic acid (ABA) in culture media than Pseudomonas sp. (1) by the addition of l-tryptophan. These inoculants also modulated the phytohormone content of maize leaves in a pot experiment. Higher ABA was produced by the application of B. pumilus and Pseudomonas sp. (2), while indole 3-acetic acid and gibberellic acid were found higher in Pseudomonas sp. (1) and Proteus sp. treated plants. Addition of l-tryptophan increased the concentration of all phytohormones in soil and leaves of maize. Maximum increase in relative water content, osmotic potential, protein content and photosynthetic pigments was recorded in B. pumilus treated maize plants. Under irrigated condition, response of Pseudomonas sp. co-inoculated with B. pumilus from arid field superseded while under drought stress the effect of later predominated. Bacillus pumilus can be used in the formulation of biofertilizer to alleviate drought stress in arid and semi-arid regions.     

In vivo enzymatic digestion, in vitro xylanase digestion, metabolic analogues, surfactants and polyethylene glycol ameliorate laccase production from Ganoderma sp. kk-02

AIMS: The effect of in vivo enzymatic digestion (IVED), in vitro xylanase digestion (IVXD), metabolic analogues, surfactants and polyethylene glycol (PEG) on laccase production from Ganoderma sp. kk-02 was studied. METHODS AND RESULTS: An acidic laccase producing Ganoderma sp. kk-02 produced 16.0 U ml(-1) and 365.0 U g(-1) of laccase, when grown under submerged (SmF) and solid state (SSF) fermentation conditions, respectively. Modification of the substrate (wheat bran) molecular architecture by IVED and IVXD increased subsequent laccase production from Ganoderma sp. kk-02 by 1.31-fold (21.0 U ml(-1)) (SmF); 2.21-fold (810.0 U g(-1)) (SSF) and 1.10-fold (18.0 U ml(-1)) (SmF); 1.78-fold (650.0 U g(-1)) (SSF) when compared with untreated wheat bran. Further enhancement in laccase yield under SmF and SSF was obtained when IVED treated wheat bran was used in conjunction with amino acids [DL-tryptophan, 2.66-fold (56.0 U ml(-1)) SmF; 2.86-fold (2324.0 U g(-1)) SSF], vitamins [biotin, 1.71-fold (36.0 U ml(-1)) SmF; 3.06-fold (2483.0 U g(-1)) SSF], surfactants [Tween-40, 1.85-fold (39.0 U ml(-1)) SmF; 2.25-fold (1828.0 U g(-1)) SSF], and PEG [PEG 6000, 1.93-fold (40.0 U ml(-1)) SmF; 1.58-fold (1284.0 U g(-1)) SSF]. CONCLUSIONS: The IVED of substrate (wheat bran) facilitated hyper laccase production in presence of additives from Ganoderma sp. kk-02. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights a new methodology viz. IVED for concomitant and economic production of diverse enzymes using the same substrate. The hyper laccase levels obtained could improve the economic competitiveness of environmentally benign processes applied in varied industries. The work also provides an insight into the regulation of complex metabolic pathways governing the expression of extra cellular proteins from white-rot fungi.