Isolation and Identification of an Acidophilic Fungus and Analysis on the Secreted Glycoside Hydrolases

[Objective] This study aimed to isolate an acidophilic fungus and analyze the acidophilic enzymes secreted by this fungus. [Method] A heterotrophic fungus was isolated from the leaching solution of a uranium ore in Jiangxi Province using oligotrophic acid selective medium （pH 2.5）, and was named RBS-6. This strain was then identified according to its colony morphology and molecular indicator rDNA-ITS. Finally, the glycoside hydrolases secreted by RBS-6 were analyzed. [Result] This fungus RBS-6 was acidophilic, and grew best at pH4.0. Its rDNA-ITS sequence shared the highest homology （98%） with that of Phialophora sp. CGMCC 3329 （GU 082377）. So it was identified as a fungus of Phialophora sp., and was temporarily named as Phialophora sp. RBS-6. It can produce six glycoside hydrolases, in cluding α-galactosidase glucosidase, β-glucosidase, β-mannanase and β-glucanase. All the enzymes were acidophilic, for which the optimum reaction pH was 3.0-4.0. Among them, β-glucanase exhibited the highest activity at pH 3.5 and 50 ℃; in addition, it was heat-stable as 58% of the enzyme activity was remained after incubation at 50 ℃ for 60 min. [Conclusion] The isolated fungus which was identified as an acidophilic member of Phialophora sp., was a new strain producing acidophilic enzymes. This study supplied new data for the research on Phialophora fungi.

Microbe-derived non-necrotic glycoside hydrolase family 12 proteins act as immunogenic signatures triggering plant defenses

Plant pattern recognition receptors(PRRs)are sentinels at the cell surface sensing microbial invasion and activating innate immune responses.During infection,certain microbial apoplastic effectors can be recognized by plant PRRs,culminating in immune responses accompanied by cell death.However,the intricated relationships between the activation of immune responses and cell death are unclear.Here,we studied the glycoside hydrolase family12(GH12)protein,Ps109281,secreted by Phytophthora sojae into the plant apoplast during infection.Ps109281 exhibits xyloglucanase activity,and promotes P.sojae infection in a manner dependent on the enzyme activity.Ps109281 is recognized by the membranelocalized receptor-like protein RXEG1 and triggers immune responses in various plant species.Unlike other characterized GH12 members,Ps109281 fails to trigger cell death in plants.The loss of cell death induction activity is closely linked to a sequence polymorphism at the Nterminus.This sequence polymorphism does not affect the in planta interaction of Ps109281 with the recognition receptor RXEG1,indicating that cell death and immune response activation are determined using different regions of the GH12 proteins.Such GH12 protein also exists in other Phytophthora and fungal pathogens.Taken together,these results unravel the evolution of effector sequences underpinning different immune outputs.

MECE:基于深度神经网络及进化分析提高糖苷水解酶的催化效率

糖苷水解酶(glycoside hydrolases,GHs)在各个行业广泛应用,其需求量不断增加.然而,如何提高酶的催化效率仍然是一个挑战.本文开发了基于深度神经网络和分子进化的策略(MECE)预测提高糖苷水解酶催化活性的突变体.作者首先从CAZy数据库中收集整理了119个糖苷水解酶家族的蛋白序列,建立了能够识别糖苷水解酶家族和功能残基的深度学习模型DeepGH,通过10倍交叉验证结果显示DeepGH模型的预测准确率为96.73%.随后利用梯度加权类激活图谱(Grad-CAM)方法提取分类相关特征,结合序列进化信息对突变体进行设计最后获得了具有7个氨基酸突变位点的壳聚糖酶突变体CHIS1754-MUT7.实验结果表明,CHIS1754-MUT7的k_(cat)/K_m是野生型的23.53倍.该策略计算效率高,实验成本低,具有显著的优势,为酶催化效率的智能设计提供了一种新的途径,具有广泛的应用前景.

A revisiting of transition metal phosphide(Cu_(3)P and FeP)nanozymes for two sugar-related reactions

Transition metal phosphides(TMPs)are essential catalysts for some general catalytic reactions.However,their potentials for biological catalysis have seldom been explored.Herein,we investigated the enzyme-like properties of four TMPs(FeP,CoP,Ni_(2)P,and Cu_(3)P)towards two sugar-related reactions.Among the four TMPs,Cu_(3)P nanoparticles(NPs)efficiently catalyzed the hydrolysis of glycosidic bonds as glycoside hydrolase mimics,and FeP NPs possessed both glucose oxidase-like(GOx-like)and peroxidase-like activities,which combined into a cascade reaction for glucose’s simple and one-step colorimetric biosensor without GOx.Cu_(3)P and FeP NPs with distinctive enzyme-like activities have shown unique biological catalysis potentials for further applications with an attractive and challenging goal of developing nanomaterials to mimic natural enzymes,which encourages more efforts to reveal TMP’s capabilities towards biocatalysis.

Cloning, expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis

Chitinase catalyzes β,4-glycosidic linkages in chitin and has attracted re- search interest due to it being a potential pesticide target and an enzymatic tool for preparation of N-acetyl-β D-glucosamine. An individual insect contains multiple genes encoding chitinases, which vary in domain architectures, expression patterns, physiological roles and biochemical properties. Herein, Of ChtS, the glycoside hydrolase family 18 chiti- nase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation. Complementary DNA sequence analysis indicated that Of CHT5 consisted of an open reading frame of 1 665-bp nucleotides. Phy- logenic analysis suggested Of Cht5 belongs to the Group I insect chitinases. Expression of Of Cht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate pre- cipitation and metal chelating chromatography, about 7 mg of the recombinant Of Cht5 was purified to homogeneity from 1 L culture supernatant. Of Cht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward a-chitin. When chi- tooligosaccharides [（GlcNAc）n, n = 3-6] were used as substrates, Of Cht5 was observed to possess the highest catalytic efficiency parameter toward （GlcNAc）4 and predominan- tely hydrolyzed the second glycosidic bond from the non-reducing end. Together with fl-N-acetyl-D-hexosaminidase OfHexl, Of Cht5 achieved its highest efficiency in chitin degradation that yielded N-acetyl-β D-glucosamine, a valuable pharmacological reagent and food supplement, within a molar concentration ratio of Of Cht5 versus Of Hexl in the range of 9 ： 1-15 ： 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.

A Novel Thermostable β-Galactosidase from Geobacillus kaustophilus HTA42

A novel thermostable β-galactosidase gene, designated as GkGallA, from the thermophilic bacterium Geobacillus kaustophilus HTA426 was cloned and heterologously overexpressed in Escherichia coli（E, coli）. Based on the sequence analysis, GkGallA belongs to the glycosyl hydrolase family 1 that was the first β-galactosidase of bacterial origins expressed by us in this family. The apparent molecular weight of GkGallA determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis is 52000. It exhibited the highest activity toward p-nitrophenyl-β-D-galactopyranoside at pH 7.8 and 70℃ and displayed high thermal stability, Divalent cations are prerequisite for the activity of GKGallA, with the highest activity in the presence of Mn2＋. Moreover, the three-dimensional structure of GkGaI1A was modeled to speculate the structure of the catalytic residues and the reac- tion mechanism. The catalytic residues consisting of Glu166 and Glu355 were verified by site-directed mutagenesis.