HpYPS1 and HpYPS7 encode functional aspartyl proteases localized at the cell surface in the thermotolerant methylotrophic yeast Hansenula polymorpha

In the present study, we functionally analysed two yapsin genes of the thermotolerant methylotrophic yeast Hansenula polymorpha, HpYPS1 and HpYPS7, for their roles in maintaining cell wall integrity and proteolytic processing. Both HpYPS1 and HpYPS7 proteins were shown to largely localize on the cell wall via glycosylphosphatidylinositol anchor. Heterologous expression of HpYPS1 completely restored all of the growth defects of the Saccharomyces cerevisiae yps1-deletion strains, while HpYPS7 expression exhibited a limited complementation effect on the S. cerevisiae yps7-deletion strain. However, different from S. cerevisiae, deletion of the HpYPS genes generated only minor influence on the sensitivity to cell wall stress. Likewise, HpYPS1 expression was significantly induced only by a subset of stressor agents, such as sodium dodecyl sulphate and tunicamycin. HpYps1p was shown to consist of two subunits, whereas HpYps7p comprises a single long polypeptide chain. Biochemical analysis revealed that HpYps1p has much stronger proteolytic cleavage activity at basic amino acids, compared to HpYps7p. Consistent with the much higher proteolytic activity and expression level of HpYps1p compared to HpYps7p, the sole disruption of HpYPS1 was sufficient in eliminating the aberrant proteolytic cleavage of recombinant proteins secreted by H. polymorpha. The results indicate that, although their roles in the maintenance of cell wall integrity are not critical, HpYps1p and HpYps7p are functional aspartic proteases at the cell surface of H. polymorpha. Furthermore, our data present the high biotechnological potential of H. polymorpha yps1-mutant strains as hosts useful for the production of secretory recombinant proteins.     

章鱼肠道产蛋白酶菌的筛选、产酶条件及酶学性质

采用检测水解透明圈和测定蛋白酶活力的方法,从章鱼肠道中分离筛选出一株高产蛋白酶的菌株QDV-3,并对其产酶条件及酶学性质进行研究。结果表明:QDV-3菌株在以果糖为碳源,蛋白胨为氮源,培养基初始pH8.0,培养温度30℃的条件下振荡培养3.5d时,所产蛋白酶活力最高,Mn2+和Ba2+对菌株产蛋白酶有促进作用。所产蛋白酶在SDS-PAGE电泳上显示至少有5种蛋白酶,分子质量范围为32.4～124.2kD,蛋白酶的最适作用温度为50～60℃,最适作用pH值为9～11,在50℃条件下保温1h,剩余酶活力为95%,热稳定性较好。     

羊毛蛋白酶减量细化的研究

用不同的方法对羊毛进行细化处理，考查了不同蛋白酶制剂、不同处理条件对羊毛细度、减量率、强度的影响。实验表明：碱性蛋白酶对羊毛纤维的减量效果比酸性蛋白酶要好，用蛋白酶与氧化剂同时对羊毛纤维进行处理，在取得较好减量效果时，也对羊毛纤维造成较大损伤，在进行酶处理前，用保护剂对羊毛纤维进行预处理，可以降低羊毛纤维的强力损失。     

Hepatic metabolism of 2-hydroxy-4-methylthiobutyrate in growing lambs

This study was undertaken to determine how, and where, 2-hydroxy-4-methylthiobutyrate (HMTBA) can augment Met metabolism in lambs. Four lambs (initial body weight of 50 kg, SE = 2, and 6 mo of age) prepared with catheters in the mesenteric, portal, hepatic, and jugular veins plus the aorta, were fed at 1.5× maintenance on a grass hay, barley, fish meal, molasses/pre-mix (5:3:1:1, as fed) diet, supplied as hourly meals. Lambs were infused for 10 h with [methyl-²H₃]Met (0.11 mmol/h) in a jugular vein and p-aminohippurate into the mesenteric vein. From 1 h onwards, successive 3-h infusions of saline (control), 0.55 mg/min (3.67 μmol/min), and 4.44 mg/min (29.6 μmol/min) of HMTBA were also infused into the mesenteric vein. Plasma, sampled continuously, was collected every 20 min during the last 60 min of each infusion. All infused HMTBA was recovered at the portal vein with 25% extracted subsequently by the liver. Portal appearance of total Cys and Met was unaltered by HMTBA infusion, but net splanchnic appearance of Cys increased (0.04, 0.08, 0.23 mmol/h, SEM = 0.05), whereas Met decreased (0.14, −0.01, −0.21 mmol/h, SED = 0.05). Despite this, arterial Met increased (27.0, 30.7, 51.5 μM, SEM = 2.1) as did Met irreversible loss rate (27.6, 28.7, 40.1 μmol/h, SEM = 0.51), equivalent to 40% of the HMTBA reentering the plasma after conversion to Met. These data indicate that, in ruminants, HMTBA is probably converted to Met within peripheral tissues; that is, where the metabolic need for Met exists.     

Purification and Properties of an Insecticidal Metalloprotease Produced by Photorhabdus luminescens Strain 0805-P5G,the Entomopathogenic Nematode Symbiont

A total of 13 Photorhabdus luminescens strains were screened for proteolytic activity. The P. luminescens strain 0805-P5G had the highest activity on both skim milk and gelatin plates. The protease was purified to electrophoretical homogeneity by using a two-step column chromatographic procedure. It had a molecular weight of 51.8 kDa, as determined by MALDI-TOF mass spectrometry. The optimum pH, temperature, as well as pH and thermal stabilities were 8, 60 °C, 5–10, and 14–60 °C, respectively. It was completely inhibited by EDTA and 1,10-phenanthroline. Bioassay of the purified protease against Galleria mellonella by injection showed high insecticidal activity. The protease also showed high oral toxicity to the diamondback moth (Plutella xylostella) of a Taiwan field-collected strain, but low toxicity to an American strain. To our knowledge, this is the first report to demonstrate that the purified protease of P. luminescens has direct toxicity to P. xylostella and biopesticide potentiality.     

Characterization of a novel protease from Bacillus cereus and evaluation of an eco‐friendly hydrolysis of a brewery byproduct

Proteases and proteolytic enzymes constitute one of the most important groups of enzymes and are attracting worldwide attention in attempts to exploit their physiological and biotechnological applications. In this study, partial purifications and biochemical and antimicrobial characterizations of a protease from Bacillus cereus spp., originally isolated from fermented cabbage, were carried out. The crude extract obtained after purification, involving ammonium sulphate precipitation and dialysis, was designated as a partially purified protease (PPP). The obtained PPP had a specific activity of 0.395–2.539 U/g at 32 °C, with maximum activities for the fractions precipitated at 60 and 80% ammonium sulphate. The PPP activity ranged between 20 and 55 °C, with an optimum temperature at 40 °C. At 60 °C, the PPP retained more than 30% of its activity. The optimum pH for the PPP was achieved at pH 9, indicating the alkaline source of the enzyme. Protease production was specifically dependent on the calcium concentration in the culture medium. Also the robustness of the protease on brewer's spent grain hydrolysis was demonstrated. This suggests a potential eco‐friendly application of the enzyme. Finally, it was found that the PPP inhibited the growth of Escherichia coli O157:H7. This novel property of the PPP liberated by the B. cereus spp. could provide important future benefits to industry. Copyright © 2015 The Institute of Brewing & Distilling     

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome

The A+T-rich genome of the human malaria parasite Plasmodiumfalciparum encodes genes of biological importance that cannotbe expressed efficiently in heterologous eukaryotic systems,owing to an extremely biased codon usage and the presence ofnumerous cryptic polyadenylation sites. In this work we haveoptimized an assembly polymerase chain reaction (PCR) methodfor the fast and extremely accurate synthesis of a 2.1 kb Plasmodiumfalciparum gene (pfsub-1) encoding a subtilisin-like protease.A total of 104 oligonucleotides, designed with the aid of dedicatedcomputer software, were assembled in a single-step PCR. Theassembly was then further amplified by PCR to produce a syntheticgene which has been cloned and successfully expressed in bothPichia pastoris and recombinant baculovirus-infected High Five^TMcells. We believe this strategy to be of special interest asit is simple, accessible and has no limitation with respectto the size of the gene to be synthesized. Used as a systematicapproach for the malarial genome or any other A + T-rich organism,the method allows the rapid synthesis of a nucleotide sequenceoptimized for expression in the system of choice and productionof sufficiently large amounts of biological material for completemolecular and structural characterization.