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木聚糖是自然界中储量第二大的自然多糖.木聚糖酶是水解木聚糖的一系列酶的统称,其中内切木聚糖酶在木聚糖的水解中起至关重要的作用.内切木聚糖酶的用途十分广泛,并且研究者以不同用途为导向对该酶进行了大量研究.本文介绍了内切木聚糖酶的各种用途,以及酶的发现、生产和改性概况,为内切木聚糖酶的应用相关研究提供参考.

丙酮酸是一种重要的有机酸,在聚合物、化妆品、食品添加剂、医药等领域具有广泛应用.酿酒酵母被认为是丙酮酸生产的潜在最适微生物,但其糖酵解过程产生的丙酮酸会在胞质内的丙酮酸脱羧酶催化作用下降解为CO2和乙醛,造成碳代谢流的损失.为将更多的碳代谢流引向丙酮酸合成,该研究通过敲除丙酮酸脱羧酶的3个结构基因(pdc1、pdc5和pdc6),显著地促进了丙酮酸积累,但也造成突变菌株XY-156生长缓慢.与进化之前相比,采用适应性进化策略驯化得到的菌株XY-156A,在细胞生长、葡萄糖消耗以及丙酮酸积累方面均获得显著提高;进一步利用2 L发酵罐进行批次补料发酵试验,发酵76 h丙酮酸产量可达105 g/L,生产强度为1.38 g/(L·h),得率为0.5 g/g葡萄糖.结果表明,采用失活丙酮酸脱羧酶与适应性定向进化相结合的策略改造酿酒酵母,能够实现其高效积累丙酮酸,可为生物基丙酮酸工业化生产奠定坚实的基础.

作为酶工程、有机化学和合成生物学领域的研究热点,细胞色素P450酶以其独特的催化特性以及广泛的存在性,吸引了大量研究人员的兴趣.近年来,随着全基因组测序技术、合成生物学和结构生物学的迅猛发展,在抗生素的生物合成过程中P450酶的功能研究得到了进一步解析.同时,细胞色素P450酶的蛋白质改造和新催化反应的研发不断取得新突破.本文就链霉菌属来源的细胞色素P450酶的特点以及最近的研究进展进行了总结与论述.

[Objective]Optimizing the auto-induction of the fermentation medium to increase li-pase enzyme production.[Methods]In this research,through the construction of a transposon-integrated lipase recombinant bacterial strain,using automatic induction fermentation expres-sion method combined with CCD response surface experimental design method,the carbon source in the auto-induction medium was optimized,and a quadratic model was obtained,which was used to describe the effect of the carbon source in the fermentation medium on lipase pro-duction.[Results]The optimized fermentation medium(W/V)contained glycerol 2.596,glu-cose 0.035,lactose 1.289,tryptone 1.0,yeast extract 0.5,Na2HPO4·12H2O 1.79,KH2PO4 0.68,NH4Cl 0.2675,Na2SO40.071,MgSO4·7H2O 0.05.[Conclusion]The results of the fermentation test confirmed that the lipase enzyme specific activity in the optimized fermen-tation medium was 6.35 U/mg,which was near-ly three times higher than that in the initial fer-mentation medium.

Cytochrome P450 enzymes(P450s or CYPs)are a widespread large super family of mono-oxygenase that catalyzes the different substrates via heme in life beings.On the one hand,P450s involves in the key step of high-active natural product biosynthetic pathway,its biotransformation studies will help to increase the yield and activity of active molecules.On the other hand,its catalysis involves in metal elements and cofactors,new breakthroughs have been made in the protein modification and new catalytic reaction of P450s.In this article the latest advances in these two aspects of cytochrome P450 enzymes have been summarized and dis-cussed.

[Objective]Studies were conducted on the metabolic network affecting the localomycin E(laE)production of Streptomyces globisporus can increase the yield of secondary metabo-lites.[Methods]By constructing a transposon based on a transposon Tn7 containing a strong promoter and a resistance marker,a chromosome-generated mutation library was integrated in-to S.globisporus,a high-yielding mutant strain was screened and its metabolic network was studied and analyzed.[Results]Using the constructed Tn7 transposon for continuous transfor-mation of S.globisporus,several rounds of mutations and screening were used to obtain six mutants with large changes in yield.Subcloning and sequencing of the integration site showed that the site integration resulted in the inactiva-tion of genes encoding certain regulatory factors such as the TetR and GntR family of proteins.[Conclusion]The constructed and modified micro Tn7 transposon contains not only antibiotic resistance marker and but also strong promoter which can be inserted into the chromosome of Streptomyces to produce mutations,thereby in-creasing the yield of secondary metabolite LaE.It also demonstrates that the transposon-based vector can be applied to non-model Streptomyces sp.

[Objective]Screening thermostable α-amylase and expressed in Escherichia coli,and analyzing the recombinant enzyme characterization.[Methods]The strain Anoxybacillus sp.GXS-3 is isolated from soil samples collected from Tengchong hot spring,with M9 medium and soluble starch as the sole carbon source.According to the amino acid conserved sequence of amylase,degenerate primers are designed for PCR amplification.and then the target sequence is amplified and amplified to obtain the amylase gene AmyGX.AmyGX is ligated with the expression vector pQE30, introduced into E.coli M15, and the recombinantα-amylase is isolated and purified by NTA-affinity chromatography and its enzymological properties are analyzed.[Results]The gene of AmyGX is 1 515 bp in length and encoded a protein of 505 amino acids,of which,the first 23 amino acid residues are signal peptide sequences.The molecular mass of the recombinant AmyGX is 58.04 kDa.Assayed with soluble starch as substrate,this recombinant enzyme displays optimal activity at pH 8.0 and 60℃ with an apparent Km value of 3.14 mg/ml and Vmax of 0.19 U/mg respectively.The temperature at which 50% of the enzyme activity is lost after 30 min heat treatment (T3050) is 65.2℃.Zn2+、Cu2+、Co2+、Fe3+ 、Ba2+has significantly inhibitory effect on the activity of the recombinant enzyme,whereas Na+、K+could increase the activity and Mg2+、Ca2+ has slightly effect on it.[Conclusion]AmyGX is a moderate enzyme,it could be applied in the field of paper,detergent production and the removal of waste material in environment.

[Objective]Screening,cloning,heterologous expression of the pullulanase encoding gene from Tumebacillus flagellatus GST4 in order to obtain efficient expression of a new pullulanase and enhance its activity and thermosatbility.[Methods]Cloning,construction of recombinant and heterologous expression of pullulanase gene in Escherichia coli,purification by Ni-chelating affinity chromatography from cell free culture supernatant and characterization of pullulanase were carried out.[Results]The pullulanase gene pulB was cloned and expressed successfully in E.coli,and the activity of cultur-al supernatant can reach 78 U/mL.And PulB was purified to homogeneity and the specific ac-tivity was 258 U/mg.The optimal temperature of purified PulB is 50℃,its optimal pH value is 5.0 and activity remains stable within the a-cidic range of pH 4.5~5.5.PulB displayed typical Michaelis-Menten kinetics,where its Km is(16.28±0.03)mg/mL andVmax is(22.05±0.02)μmol·min-1·mg-1,respectively,when used pullulan as substrate.GenBank blast results show that there is no homologous DNA se-quences with pulB,and the encoding protein of PulB had the highest identity (54%)with cy-clomaltodextrinase from Thermicanus aegyptius.We find that it has amylase structure do-main by online SMART searching.The substrate specificity analysis shows that it typically hydrolyze pullulan and amylopectin to produce liner oligosaccharides or maltotriose.[Conclu-sion]PulB is a new starch/pullulan hydrolase,which has not yet been reported.It can hydro-lyze pullulan or amylopectin and belong to type I pullulanase.

[目的]研究生淀粉结合域SBD及糖化酶基因在毕赤酵母中的融合表达,提高酶的表达量和水解生淀粉的能力.[方法]利用In-fusionTM PCR克隆技术将淀粉结合域SBD无缝隙插入到黑曲霉糖化酶基因glu的5′端构建融合表达质粒pPIC9K-psg,实现融合酶基因psg在毕赤酵母GS115中的高效表达,并进行酶学性质研究.[结果]融合酶的最适作用条件及热稳定性均与原始酶无明显差别,但反应温度及pH值的范围更为宽泛,在反应温度60～70℃,pH值为4.0～7.0时均较稳定;融合酶PSG降解生淀粉的能力较原始酶PG提高29.6％,比原始菌株提高86.5％.[结论]淀粉结合域SBD的融合提高了糖化酶水解生淀粉的能力.

【Objective】Very high gravity fuel ethanol production was studied with the fermentation of uncooked cassava flour.【Metheds】First,the important parameters were screened by single factor tests.Second,the optimum values of the parameters were obtained by response sur-face methodology. And finally,a gradient temperature control strategy was used with the optimized conditions,all of which were used to improve the ethanol concentration.【Results】The conditions optimized by single factor tests were starch hydrolyzing enzyme dosage 0.8 GAU/g cassava flour,substrate concentration 36%(W/V),and initial pH value 4.2.The optimal concentrations were determined by response surfacemethodology as follows:Starch hydrolyzing enzyme dosage 0.82 GAU/g cassava flour,substrate concentration 37%,and initial pH value 4.3.A gradient temperature control strategy could reduce the reducing sugar and imporve the fermentation efficience.Under such optimum conditions,the ethanol concentration could reach up to 16.24%(V/V),while the reducing sugar and total sugar were 0.29%(W/V)and 1.81%(W/V)correspondingly,after 96 hfermentation.The ethanol content of fermentation mashing increased 25% compared with the original condition.【Conclusion】On the basis of integration technology,very high gravity fuel ethanol production and low production cost with the fermentation of uncooked cassava flour may facilitate industrial scale application in future.

【Objective】The industrial scale of ethanol production from cassava by high ethanol producing strain of Saccharomyces cerevisiae Ygxas-49 was studied in this paper.【Methods】First,the batch fermentation tests were performed in 200 tfermentor to assess the variousfermentation indicators of the strain.Then a viable option was further enlarge in the annual120,000 tethanol production line to investigate the stability of the strain.Finally,the highconcentration ethanol production were performed in 200 tfermentor to assess the potential industrialization of the strain.【Results】Compared with control strain in 200 tfermentor,ethanol concentration of Ygxas-49 increased 8.92% with over 12 hdecrease on the fermentation time and same other fermented index.If the strain was used in the ethanol production,it can increase the ethanol concentration,reduce the fermentation time and increase the equipment utilization.Thereby,the cost of ethanol production could significantly reduce.The strain has stable operation of 30 din 120,000 tof ethanol production line.The results show that the ethanol concentration increased 5.35% and the fermentation time reduced more than 10 h.The ethanol concentration could reach 14.7%(V/V)after 52 hfermentation in 200 tfermenor.However,the current highest ethanol production from cassava reported in the literature was 13.5%(V/V)with 69 hof fermentation time.The ethanol concentration increased8.89% and the fermentation time reduced 17 hthan the report.【Conclusion】The strain of Ygxas-49 has the ability to be applied to industrial production,the fermented index was significantly better than the current domestic fermentation level.So it has a good prospects for industrial application in future.

[目的]通过优化产琥珀酸放线杆菌GXAS137同步糖化发酵木薯粉产丁二酸的发酵培养基,提高丁二酸产量,降低生产成本.[方法]在单因素试验的基础上,先利用Plackett-Burman 试验设计筛选出影响丁二酸发酵的重要参数,再采用正交试验确定重要参数的最佳水平.[结果]价格低廉玉米浆可用作氮源,影响丁二酸产量的重要参数是木薯粉、玉米浆、碱式碳酸镁和糖化酶浓度.最佳条件为(g/L):木薯粉100,玉米浆14,糖化酶2.0 AGU/g底物,碱式碳酸镁75.优化后丁二酸产量达到69.31 g/L,丁二酸得率为90.01％,生产强度为1.44 g/(L·h).与初始条件(52.34 g/L)相比,丁二酸浓度提高了32.42％.并利用1.3L发酵罐对SSF与SHF两种发酵工艺进行了比较,SSF丁二酸产量(72.21 g/L)远高于SHF (56.86 g/L).[结论]产琥珀酸放线杆菌同步糖化发酵木薯粉丁二酸产量高,生产成本低,具有较好的工业化应用前景.

A pullulanase producing bacterium,designated strain GXAS 38,was isolated from samples collected from the waste lagoon of a starch factor in Nanning,Guangxi.Strain GXAS 38 was identified as Klebsiella pneumoniae based on morphological,physiological characterization and 16S rDNA sequences analysis.The optimum temperature and pH for the enzyme were determined as 60℃ and 5.5,respectively.The stable temperature and pH range were 35~50℃ and 5.0~7.5,respectively.The enzyme could be stimulated by Ca2+,Na+ and Li+,but inhibited by Cu2+,Zn2+,Co2+,Mn2+,Fe2+,Fe3+,Ba2+ and EDTA.The pullulanase gene was amplified by PCR and sequenced.The full length of this gene was 3291 bp and presumably encoded 1096 amino acids.

The conditions of mixed fermentation of very high gravity ethanol with cassava flour and sugarcane molasses were optimized.The important parameters were screened by P-B(Plackeet-Burman) design.Then,the optimum values of the important parameters were obtained by the orthogonal experiments.The ferment conditions were initial total sugar as 29.14%,the optimum time of adding sugarcane time as 16.5 h,initial pH of fermentation as 4.7.Under these conditions,the ethanol concentration of mature mashing reached 16.04%(V/V).The content of ethanol fermentation mashing was enhanced 20% than that of the original condition.

Two forms of Ca2+ independent α-amylases(Amy7B and Amy7S),from Bacillus subtilis CN7,were purified and characterized.The enzymatic starch hydrolysis products were analyzed by HPLC.The results showed that the Amy7B was purified by 36 fold and Amy7S was purified by 75 fold simultaneously after thermal denaturation,ultrafiltration,ammonium sulfate precipitation,and molecular sieve chromatography.Both forms had the the same optimum pH 6.5,the same optimum temperature of 65 ℃ and very similar amylolytic pattern.Calcium ions and EDTA had no significant effects on their activities.However,the Amy7S(4.7×104) was approximately 2.0×104,shorter than the Amy7B(6.7×104),the half inactivation temperature and the specific activity of Amy7S were 62.3 ℃ and(1 853.87±75.61) U/mg,they were higher than that at 59.6 ℃ and(905.99±96.52) U/mg of Amy7B.

Response surface analysis was conducted on Aspergillus oryzae ASPE0485 to determine the optimal culture medium for β-glucosidase fermentation.The Box-Behnken and Plackett-Burman design was utilized to the response surface analysis to determine the optimal culture medium consistents(%,w/v)were as follow:corncob 3.8%、soya peptone 0.5%、KH2PO4 0.5%、MgSO4·7H2O 0.05%、CaCl2 0.05%、tween-80 0.27% and inoculum size 5.3%.The β-glucosidase activity was 21.1U/mL under the optimal conditions,which was more 19.55% than the original strain activity(17.65U/mL).

In this experiment,Saccharomyces cerevisiae strain Ygx-5 was used as original strain and its protoplast was treated by UltraViolet(UV)-nitrosoguanidine(NTG)composite mutation to obtain thermotolerant and high ethanol-producing mutants.Through primary screening and confirmed screening,one strain designated as U-N2 was obtained.As the fermentation temperature was at 37 ℃,the final ethanol concentration produced by U-N2 strain could reach to 16.32 %vol,20.44 % higher than original strain,8.04 % higher than mutant underwent UV mutation only,and 6.27 % higher than mutant underwent NTG mutation only.The stable genotype with high ethanol yield had been verified through twenty times of transferring of culture.

The protoplasts of Aspergillus fumigatus was prepared by dissolving it with mixed-enzyme system.After the protoplasts were induced by ultraviolet ray and LiCl,a high-yield strain UV-26 was obtained by first screening of flat plate and second screening of shake flask.The enzymatic ability of the strain was increased from the initial 1.1 U/mL to 3.4 U/mL.And it can descend stably after six generation.

A mutant from Aspergillus oryzae which can synthesize β-glucosidase when high concentration glucose exists,was obtained by using agar plate screening techniques,after mutation by UV-60Co.The mutant strain was isolated from the agar plate,which contains 4% glucose.In liquid state fermentation,the mutant strain produced high β-glucosidase activities,which were 17.65IU after 4 days’ culturing.And these were 2.78 times that of the starting strain.The activity of the expressed enzyme was measured by the assay of transglucosidation reaction and the transglucosidation product was identified by TLC and HPLC.At 60% glucose,50℃,pH3.0,and 72 h reaction time,the gentiooligosacharide reached 44.16 g/L.