Kinetics of prolyl hydroxylation,intracellular transport and C-terminal processing of the tobacco vacuolar chitinase

The dynamics of intracellular transport and processing of one of the vacuolar chitinases of tobacco (Nic-otiana tabacum L.), chitinase A (CHN A; EC 3.2.1.14), was investigated with pulse-chase experiments in conjunction with cell fractionation and immunoprecipitation. Mature CHN A is composed of two domains, the N-terminal cysteine-rich chitin-binding domain and the catalytic domain, linked by a short peptide spacer containing several hydroxyprolines. It is synthetized as a preproprotein with a signal peptide for cotranslational transport into the endoplasmic reticulum (ER) and a C-terminal, vacuolar targeting peptide (VTP) required for targeting to the vacuole, which is removed by proteolytic cleavage. We investigated transformed N. sylvestris plants constitutively expressing CHN A or a mutant CHN A lacking the chitin-binding domain and spacer (CS CHN A), as well as N. plumbaginifolia protoplasts transiently expressing the same constructs. Processing and transport in the two systems was very similar. A shift in the apparent molecular weight of chitinase, indicative of prolyl hydroxylation, was detectable only 30 min after appearance of newly synthesized prochitinase, indicating that it might occur in a post-ER compartment. In total, labelled chitinase was detected in the microsomal fraction for up to 90–120 min as a prochitinase, bearing the VTP. Later, it appeared only in the soluble fraction (comprising the vacuolar sap) as the mature CHN A without the VTP. In both systems, intracellular transport and processing of CS CHN A was faster than that of the wildtype form, indicating that correct folding of the cysteine-rich chitin-binding domain and/or prolyl hydroxylation of the spacer delays transport to the vacuole.Abbbreviations CBD chitin-binding domain - CHN A chitinase A - PBS phosphate-buffered saline - S proline-rich spacer - VTP vacuolar targeting peptide - CS deletion of CBD and S; - VTP deletion of VTP We thank M. Müller and T. Hohn, Friedrich Miescher-Institute, Basel, for the preparation of the protoplasts and F. Fischer, Friedrich Miescher-Institute, Basel, for the synthesis of the peptide. This work was supported by the Swiss National Science Foundation, Grants 31-26402.89 and 3100-037434.93.     

棉铃虫单核衣壳核多角体病毒几丁质酶基因的克隆与表达

根据棉铃虫单核衣壳核多角体病毒(HaSNPV)几丁质酶基因的序列,设计引物,引入适当的酶切位点,利用PCR扩增出不含N末端信号肽以及C末端内质网定位肽的几丁质酶基因片段.将该基因片断克隆至原核表达载体pProEXHTb,经IPTG诱导,在大肠杆菌DH5α中获得了高效表达,表达产物的大小为60kD,含量占菌体总蛋白量的40%.利用来源于AcMNPV 几丁质酶的抗体对表达蛋白进行检测,获得特异性的显色信号,证实所获原核表达产物与杆状病毒的几丁质酶具有同源性.     

转几丁质酶和葡聚糖酶基因水稻对土壤三种弹尾目昆虫和一种环节动物的影响

采用 3个试验研究转几丁质酶和苜蓿 1,3- β -葡聚糖酶双价基因水稻对土壤弹尾目昆虫和环节动物线蚓的影响。①平皿实验 :观察弹尾目与转基因水稻组织间的营养关系及其在无土壤情况下对弹尾目昆虫繁殖率的短期影响 ;②土壤 -植物残留物单物种微宇宙试验 :观察土壤中不同转基因水稻组织含量对土壤动物繁殖率的短期影响 ;③土壤 -植物残留物多物种微宇宙试验 :模拟大田状况 ,观察多物种共存较长时间后转基因水稻对土壤动物密度的影响。结果表明 :Folsomiacandida取食新鲜转基因水稻叶片和根组织 2 1d后 ,繁殖率比野生型水稻组分别降低 4 5 1% (F1,8=9 9,P <0 0 5 )和 30 5 % (F1,8=5 8,P <0 0 5 ) ;在掺有转基因水稻组织 (30g/kg)的土壤中 ,F .candida 2 1d后的繁殖率比野生型水稻组下降 19 3% (F3 ,16=3 6 ,P <0 0 5 ) ;在掺有转基因水稻组织土壤 (30g/kg)的多物种实验中 ,转基因水稻组中的F .candida、Enchytraeuscrypticus16周后的密度比野生型水稻组分别降低 19 4 % (F1,48=6 8,P <0 0 5 )和 38 0 % (F1,48=6 4 2 ,P <0 0 0 1) ,而Sinellacurviseta的密度升高 84 8% (F1,48=6 4 2 ,P <0 0 0 1)。因此 ,转几丁质酶和葡聚糖酶双价基因水稻对土壤弹尾目和线蚓都是有影响的 ,对某些种类表现     

The 92-kDa chitinase from Streptomyces olivaceoviridis contains a lysine-C endoproteinase at its N-terminus

Abstract Serine proteinases of 42, 22 and 14 kDa were purified from the culture fluid of Streptomyces olivaceoviridis by FPLC. The first 14 amino acids at their N-termini were identical and coincide with the N-terminal amino acid sequence of 92-kDa chitinase, which was found to hydrolyse casein. The four proteins hydrolyse synthetic substrates at the carboxyl group of lysine and (more slowly) arginine. The 14-kDa endoproteinase releases only two fragments of 42 and 43 kDa from β-galactosidase. When the pure 92-kDa chitinase was incubated at 37°C in Tris·HCl buffer, it was cleaved into a 70-kDa chitinase and a 22-kDa proteinase which in its part is rapidly degraded to a 14-kDa proteinase.     

Directed evolution of a Bacillus chitinase

Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito-oligosaccharide-based value-added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error-prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose at various pH in microtiter plates. From a medium-throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose as substrate, the overall catalytic efficiency, k_cat/K_m of the improved chitinase was 2.7- and 2.3-fold higher than the average k_cat/K_m of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications.     

Enzymatic characterization of the Plasmodium vivax chitinase, a potential malaria transmission-blocking target

The chitinase (EC 3.2.1.14) of the human malaria parasite Plasmodium falciparum, PfCHT1, has been validated as a malaria transmission-blocking vaccine (TBV). The present study aimed to delineate functional characteristics of the P. vivax chitinase PvCHT1, whose primary structure differs from that of PfCHT1 by having proenzyme and chitin-binding domains. The recombinant protein rPvCHT1 expressed with a wheat germ cell-free system hydrolyzed 4-methylumbelliferone (4MU) derivatives of chitin oligosaccharides (β-1,4-poly-N-acetyl glucosamine (GlcNAc)). An anti-rPvCHT1 polyclonal antiserum reacted with in vitro-obtained P. vivax ookinetes in anterior cytoplasm, showing uneven patchy distribution. Enzymatic activity of rPvCHT1 shared the exclusive endochitinase property with parallelly expressed rPfCHT1 as demonstrated by a marked substrate preference for 4MU-GlcNAc₃ compared to shorter GlcNAc substrates. While rPvCHT1 was found to be sensitive to the general family-18 chitinase inhibitor, allosamidin, its pH (maximal in neutral environment) and temperature (max. at ~ 25 °C) activity profiles and sensitivity to allosamidin (IC₅₀ = 6 µM) were different from rPfCHT1. The results in this first report of functional rPvCHT1 synthesis indicate that the P. vivax chitinase is enzymatically close to long form Plasmodium chitinases represented by P. gallinaceum PgCHT1.     

渗压震扰法释放Aeromonas sp.2016菌株外周间质中几丁质酶的研究

气单胞菌Aeromonassp．2016菌株能产生多种几丁质酶,其中的胞外酶C可能聚集于细胞外周胞质。为了避免破碎菌体而产生过多的杂蛋白,探索了用渗压震扰法(osmoticshock)来释放这部分酶。主要步骤是:先将菌体悬浮在20％蔗糖-0．03mol／LTris-HCI(pH8．0)高渗透压的溶液中,再快速转移到纯水低渗透压溶液中,产生瞬间渗压震荡,释放细胞外周胞质中的酶。结果表明,通过渗压震扰法释放出的酶纯度最高,比活力达到142．79U／g,比培养液上清液的54．46U／g和菌体破碎样品的14．66U／g分别高1．6倍和8．7倍,可用于纯化目的蛋白。     

Isolation of <Emphasis Type="Italic">Serratia marcescens</Emphasis> SR1 as a Source of Chitinase Having Potentiality of Using as a Biocontrol Agent

Serratia marcescens, strain SR₁ was isolated from the local soil of a cultivated farm and it was screened as potent strain for chitinase production. Maximum chitinase production (77.3 u Mh⁻¹ 100⁻¹) was observed after 96 h of incubation period with pH 5.5 at 30°C under shake conditions (120 rpm). Compare to still flasks, shake culture with prawn fish colloidal chitin of 0.5% (w/v) concentration, showed a better enzyme yield. Crude enzyme showed antifungal activity against plant pathogens.     

Structure of chitinase D from Serratia proteamaculans reveals the structural basis of its dual action of hydrolysis and transglycosylation

Chitinases are known to hydrolyze chitin polymers into smaller chitooligosaccharides. Chitinase from bacterium Serratia proteamaculans (SpChiD) is found to exhibit both hydrolysis and transglycosylation activities. SpChiD belongs to family 18 of glycosyl hydrolases (GH-18). The recombinant SpChiD was crystallized and its three-dimensional structure was determined at 1.49 Å resolution. The structure was refined to an R-factor of 16.2%. SpChiD consists of 406 amino acid residues. The polypeptide chain of SpChiD adopts a (β/α)₈ triosephosphate isomerase (TIM) barrel structure. SpChiD contains three acidic residues, Asp149, Asp151 and Glu153 as part of its catalytic scheme. While both Asp149 and Glu153 adopt single conformations, Asp151 is observed in two conformations. The substrate binding cleft is partially obstructed by a protruding loop, Asn30 - Asp42 causing a considerable reduction in the number of available subsites in the substrate binding site. The positioning of loop, Asn30 - Asp42 appears to be responsible for the transglycosylation activity. The structure determination indicated the presence of sulfone Met89 (SMet89). The sulfone methionine residue is located on the surface of the protein at a site where extra domain is attached in other chitinases. This is the first structure of a single domain chitinase with hydrolytic and transglycosylation activities.